http://www2.uah.es/farmamol/New_Science_Press/nsp-protein-4.pdf
Protein structure and function summary
Showing posts with label protein structure. Show all posts
Showing posts with label protein structure. Show all posts
Monday, February 4, 2013
Tuesday, August 7, 2012
Molecular architecture of the 26S proteasome holocomplex determined by an integrative approach
http://www.pnas.org/content/early/2012/01/19/1120559109
Keren Laskera,b,1, Friedrich Försterc,1, Stefan Bohnc, Thomas Walzthoenid,e, Elizabeth Villac, Pia Unverdorbenc, Florian Beckc, Ruedi Aebersoldd,f, Andrej Salia,2, and Wolfgang Baumeisterc,2
The 26S proteasome is at the executive end of the ubiquitin-proteasome pathway for the controlled degradation of intracellular proteins. While the structure of its 20S core particle (CP) has been determined by X-ray crystallography, the structure of the 19S regulatory particle (RP), which recruits substrates, unfolds them, and translocates them to the CP for degradation, has remained elusive. Here, we describe the molecular architecture of the 26S holocomplex determined by an integrative approach based on data from cryoelectron microscopy, X-ray crystallography, residue-specific chemical cross-linking, and several proteomics techniques. The “lid” of the RP (consisting of Rpn3/5/6/7/8/9/11/12) is organized in a modular fashion. Rpn3/5/6/7/9/12 form a horseshoe-shaped heterohexamer, which connects to the CP and roofs the AAA-ATPase module, positioning the Rpn8/Rpn11 heterodimer close to its mouth. Rpn2 is rigid, supporting the lid, while Rpn1 is conformationally variable, positioned at the periphery of the ATPase ring. The ubiquitin receptors Rpn10 and Rpn13 are located in the distal part of the RP, indicating that they were recruited to the complex late in its evolution. The modular structure of the 26S proteasome provides insights into the sequence of events prior to the degradation of ubiquitylated substrates.
Keren Laskera,b,1, Friedrich Försterc,1, Stefan Bohnc, Thomas Walzthoenid,e, Elizabeth Villac, Pia Unverdorbenc, Florian Beckc, Ruedi Aebersoldd,f, Andrej Salia,2, and Wolfgang Baumeisterc,2
The 26S proteasome is at the executive end of the ubiquitin-proteasome pathway for the controlled degradation of intracellular proteins. While the structure of its 20S core particle (CP) has been determined by X-ray crystallography, the structure of the 19S regulatory particle (RP), which recruits substrates, unfolds them, and translocates them to the CP for degradation, has remained elusive. Here, we describe the molecular architecture of the 26S holocomplex determined by an integrative approach based on data from cryoelectron microscopy, X-ray crystallography, residue-specific chemical cross-linking, and several proteomics techniques. The “lid” of the RP (consisting of Rpn3/5/6/7/8/9/11/12) is organized in a modular fashion. Rpn3/5/6/7/9/12 form a horseshoe-shaped heterohexamer, which connects to the CP and roofs the AAA-ATPase module, positioning the Rpn8/Rpn11 heterodimer close to its mouth. Rpn2 is rigid, supporting the lid, while Rpn1 is conformationally variable, positioned at the periphery of the ATPase ring. The ubiquitin receptors Rpn10 and Rpn13 are located in the distal part of the RP, indicating that they were recruited to the complex late in its evolution. The modular structure of the 26S proteasome provides insights into the sequence of events prior to the degradation of ubiquitylated substrates.
Saturday, April 14, 2012
Ab initio
http://www.ncbi.nlm.nih.gov/pubmed/22163331
The evolutionary trajectory of a protein through sequence space is constrained by its function. Collections of sequence homologs record the outcomes of millions of evolutionary experiments in which the protein evolves according to these constraints. Deciphering the evolutionary record held in these sequences and exploiting it for predictive and engineering purposes presents a formidable challenge. The potential benefit of solving this challenge is amplified by the advent of inexpensive high-throughput genomic sequencing.In this paper we ask whether we can infer evolutionary constraints from a set of sequence homologs of a protein. The challenge is to distinguish true co-evolution couplings from the noisy set of observed correlations. We address this challenge using a maximum entropy model of the protein sequence, constrained by the statistics of the multiple sequence alignment, to infer residue pair couplings. Surprisingly, we find that the strength of these inferred couplings is an excellent predictor of residue-residue proximity in folded structures. Indeed, the top-scoring residue couplings are sufficiently accurate and well-distributed to define the 3D protein fold with remarkable accuracy.We quantify this observation by computing, from sequence alone, all-atom 3D structures of fifteen test proteins from different fold classes, ranging in size from 50 to 260 residues, including a G-protein coupled receptor. These blinded inferences are de novo, i.e., they do not use homology modeling or sequence-similar fragments from known structures. The co-evolution signals provide sufficient information to determine accurate 3D protein structure to 2.7-4.8 Å C(α)-RMSD error relative to the observed structure, over at least two-thirds of the protein (method called EVfold, details at http://EVfold.org). This discovery provides insight into essential interactions constraining protein evolution and will facilitate a comprehensive survey of the universe of protein structures, new strategies in protein and drug design, and the identification of functional genetic variants in normal and disease genomes.
Protein 3D structure computed from evolutionary sequence variation
The evolutionary trajectory of a protein through sequence space is constrained by its function. Collections of sequence homologs record the outcomes of millions of evolutionary experiments in which the protein evolves according to these constraints. Deciphering the evolutionary record held in these sequences and exploiting it for predictive and engineering purposes presents a formidable challenge. The potential benefit of solving this challenge is amplified by the advent of inexpensive high-throughput genomic sequencing.In this paper we ask whether we can infer evolutionary constraints from a set of sequence homologs of a protein. The challenge is to distinguish true co-evolution couplings from the noisy set of observed correlations. We address this challenge using a maximum entropy model of the protein sequence, constrained by the statistics of the multiple sequence alignment, to infer residue pair couplings. Surprisingly, we find that the strength of these inferred couplings is an excellent predictor of residue-residue proximity in folded structures. Indeed, the top-scoring residue couplings are sufficiently accurate and well-distributed to define the 3D protein fold with remarkable accuracy.We quantify this observation by computing, from sequence alone, all-atom 3D structures of fifteen test proteins from different fold classes, ranging in size from 50 to 260 residues, including a G-protein coupled receptor. These blinded inferences are de novo, i.e., they do not use homology modeling or sequence-similar fragments from known structures. The co-evolution signals provide sufficient information to determine accurate 3D protein structure to 2.7-4.8 Å C(α)-RMSD error relative to the observed structure, over at least two-thirds of the protein (method called EVfold, details at http://EVfold.org). This discovery provides insight into essential interactions constraining protein evolution and will facilitate a comprehensive survey of the universe of protein structures, new strategies in protein and drug design, and the identification of functional genetic variants in normal and disease genomes.
Thursday, March 22, 2012
Crystallography tools
CCP4 exists to produce and support a world-leading, integrated suite of programs that allows researchers to determine macromolecular structures by X-ray crystallography, and other biophysical techniques. CCP4 aims to develop and support the development of cutting edge approaches to experimental determination and analysis of protein structure, and integrate these approaches into the suite. CCP4 is a community based resource that supports the widest possible researcher community, embracing academic, not for profit, and for profit research. CCP4 aims to play a key role in the education and training of scientists in experimental structural biology. It encourages the wide dissemination of new ideas, techniques and practice.
http://www.ccp4.ac.uk/
Crystallographic Object-Oriented Toolkit
Coot is for macromolecular model building, model completion and validation, particularly suitable for protein modelling using X-ray data.
Coot displays maps and models and allows model manipulations such as idealization, real space refinement, manual rotation/translation, rigid-body fitting, ligand search, solvation, mutations, rotamers, Ramachandran plots, skeletonization, non-crystallographic symmetry and more.
http://www.biop.ox.ac.uk/coot/
http://www.ccp4.ac.uk/
Crystallographic Object-Oriented Toolkit
Coot is for macromolecular model building, model completion and validation, particularly suitable for protein modelling using X-ray data.
Coot displays maps and models and allows model manipulations such as idealization, real space refinement, manual rotation/translation, rigid-body fitting, ligand search, solvation, mutations, rotamers, Ramachandran plots, skeletonization, non-crystallographic symmetry and more.
http://www.biop.ox.ac.uk/coot/
Monday, February 6, 2012
Statistical Approaches to RNA Secondary Structure Prediction and Applications
Title: Statistical Approaches to RNA Secondary Structure Prediction and Applications
Speaker: Ye Ding
Wadsworth Center, New York State Department of Health
Abstract
Abstract: RNAs are versatile regulators of gene expression. RNA secondary structures are known to be important for regulatory functions by various types of RNAs. An RNA molecule, particularly a long-chain mRNA, may have a population of structures in the cell. Furthermore, multiple structures have been demonstrated to play important functional roles. Thus a representation of the ensemble of probable structures is of interest. We developed a statistical algorithm to sample rigorously and exactly from the Boltzmann ensemble of secondary structures, and introduced the notion of centroid structures as a new class of structure predictors. These approaches can overcome inherent limitations in conventional algorithms and are the bases for our Sfold RNA folding program (http://sfold.wadsworth.org).
MicroRNAs are small non-coding RNAs that repress protein synthesis by binding to target mRNAs in multicellular eukaryotes. Target identification of microRNA targets is essential to fully understand this new dimension of the complex gene regulatory networks. By employing a two-step model for modeling microRNA:target hybridization, we found that target secondary structure has a major impact on target recognition by microRNAs. Based on analyses of large microRNA targeting data using the model parameters and other sequence and conservation features, we have recently developed a novel computational framework that offers major improvement over established algorithms for prediction of microRNA targets. Computational tools are available through Sfold web server.
http://www.cs.ubc.ca/labs/beta/Courses/BioinfoReadingGroup/winter2011/07Feb2012.html
not really protein structure but mRNA structure ...
Speaker: Ye Ding
Wadsworth Center, New York State Department of Health
Abstract
Abstract: RNAs are versatile regulators of gene expression. RNA secondary structures are known to be important for regulatory functions by various types of RNAs. An RNA molecule, particularly a long-chain mRNA, may have a population of structures in the cell. Furthermore, multiple structures have been demonstrated to play important functional roles. Thus a representation of the ensemble of probable structures is of interest. We developed a statistical algorithm to sample rigorously and exactly from the Boltzmann ensemble of secondary structures, and introduced the notion of centroid structures as a new class of structure predictors. These approaches can overcome inherent limitations in conventional algorithms and are the bases for our Sfold RNA folding program (http://sfold.wadsworth.org).
MicroRNAs are small non-coding RNAs that repress protein synthesis by binding to target mRNAs in multicellular eukaryotes. Target identification of microRNA targets is essential to fully understand this new dimension of the complex gene regulatory networks. By employing a two-step model for modeling microRNA:target hybridization, we found that target secondary structure has a major impact on target recognition by microRNAs. Based on analyses of large microRNA targeting data using the model parameters and other sequence and conservation features, we have recently developed a novel computational framework that offers major improvement over established algorithms for prediction of microRNA targets. Computational tools are available through Sfold web server.
http://www.cs.ubc.ca/labs/beta/Courses/BioinfoReadingGroup/winter2011/07Feb2012.html
not really protein structure but mRNA structure ...
Saturday, February 4, 2012
Conditional Random Fields applied to protein fold recognition
http://online.liebertpub.com/doi/pdf/10.1089/cmb.2006.13.394
CRFs are “undirected” graphical models (also known as random fields, as opposed to directed graphical models such as HMMs) to compute the conditional likelihood P (y|x) directly.
Protein folds are frequent arrangement pattern of several secondary structure elements: some elements
are quite conserved in sequences or prefer a specific length, while others might form hydrogen bonds with
each other, such as two β-strands in a parallel β-sheet. To model the protein fold better, it would be natural
to think of each secondary structure element as one observation, corresponding to one node in the graph,
and the edges between elements as indicating their interactions in 3-D. Then, given a protein sequence,
we can search for the best segmentation defined by the graph and determine whether the protein adopts
the fold or not.
CRFs are “undirected” graphical models (also known as random fields, as opposed to directed graphical models such as HMMs) to compute the conditional likelihood P (y|x) directly.
Protein folds are frequent arrangement pattern of several secondary structure elements: some elements
are quite conserved in sequences or prefer a specific length, while others might form hydrogen bonds with
each other, such as two β-strands in a parallel β-sheet. To model the protein fold better, it would be natural
to think of each secondary structure element as one observation, corresponding to one node in the graph,
and the edges between elements as indicating their interactions in 3-D. Then, given a protein sequence,
we can search for the best segmentation defined by the graph and determine whether the protein adopts
the fold or not.
Tuesday, January 3, 2012
Cell signalling caught in the act
http://www.nature.com/news/2011/110719/full/475273a.html
The structure of this complex could finally reveal how one of biology's most important signalling mechanisms, G-protein-coupled receptors (GPCRs), do their job. This structure, published online in Nature1 by a team led by Kobilka at Stanford University in California and Roger Sunahara at the University of Michigan in Ann Arbor, now reveals the complete three-dimensional atomic structure of an activated GPCR — the β2 adrenergic receptor (β2AR) — in a complex with its G protein.
GPCRs sit in the membranes of cells throughout the body, where they detect signals from the outside world — such as light, odours and flavours — and signals from within the body, such as hormones and neurotransmitters. These signals are transmitted to the inside of the cell where they activate intracellular G proteins, which then trigger a variety of biochemical pathways.
The β2AR is activated by the hormones adrenaline and noradrenaline, and kicks off the body's fight-or-flight response by speeding up the heart and opening airways. It is a key target for anti-asthma drugs. Kobilka's X-ray crystallographic snapshot of β2AR associated with its G protein reveals some surprises, and could help in the design of more effective medicines — GPCRs are targeted by between one-third and one-half of all drugs on the market, including most of the best-sellers.
The structure of this complex could finally reveal how one of biology's most important signalling mechanisms, G-protein-coupled receptors (GPCRs), do their job. This structure, published online in Nature1 by a team led by Kobilka at Stanford University in California and Roger Sunahara at the University of Michigan in Ann Arbor, now reveals the complete three-dimensional atomic structure of an activated GPCR — the β2 adrenergic receptor (β2AR) — in a complex with its G protein.
GPCRs sit in the membranes of cells throughout the body, where they detect signals from the outside world — such as light, odours and flavours — and signals from within the body, such as hormones and neurotransmitters. These signals are transmitted to the inside of the cell where they activate intracellular G proteins, which then trigger a variety of biochemical pathways.
The β2AR is activated by the hormones adrenaline and noradrenaline, and kicks off the body's fight-or-flight response by speeding up the heart and opening airways. It is a key target for anti-asthma drugs. Kobilka's X-ray crystallographic snapshot of β2AR associated with its G protein reveals some surprises, and could help in the design of more effective medicines — GPCRs are targeted by between one-third and one-half of all drugs on the market, including most of the best-sellers.
Friday, May 7, 2010
A WHAT IF check report: what does it mean?
http://swift.cmbi.kun.nl/gv/pdbreport/checkhelp/explain.html
Some of the 20 amino acid types in proteins have atoms that "look" the same. So, for instance, the C-delta-1 and C-delta-2 atoms in phenylalanine look the same, and thus all PHE residues would have two ways of naming the atoms:
Naming in Phenylalanine
This would result in infinite confusion. So, a committee designed a standard: the torsion angle Chi-2 (defined by C-alpha, C-beta, C-gamma, C-delta-1) should always be between -90 and 90 degrees.
Similar considerations hold for ASP, GLU, PHE and TYR.
Some of the 20 amino acid types in proteins have atoms that "look" the same. So, for instance, the C-delta-1 and C-delta-2 atoms in phenylalanine look the same, and thus all PHE residues would have two ways of naming the atoms:
Naming in Phenylalanine
This would result in infinite confusion. So, a committee designed a standard: the torsion angle Chi-2 (defined by C-alpha, C-beta, C-gamma, C-delta-1) should always be between -90 and 90 degrees.
Similar considerations hold for ASP, GLU, PHE and TYR.
Wednesday, April 1, 2009
Immunology
- b-cells (bone marrow) has antibodies that bind antigens, then it makes clones of these b-cell types and the clones secrete antibodies into the blood stream, eg IgG, IgM (pentamer) (Y structure)
- t-cells 'killer' (thymus) has t-cell receptors which also has immnuglobulin (antibody) folds that recognizes antigens that are bound to mhc complex of infected cells (U structure only)
IgG (humoral, B-cell antibodies):
- immunoglobulin fold has 4 chains, 2 light and 2 heavy chains, 2 anti-parallel beta sheets packed against each other forming a barrel
- fc = crystalizable fragment, fab - antigen binding fragment
- fc and fab connected together by hinge region / flexible linker
- constant domain (ch1, cl) - 4+3 beta strand connected by short loops, linked by disulfide bonds, sheets are 90 degrees, hydrophobic interactions, 4 strands (longest sheet) in the middle, 3 strands in the outside
- variable domain (vh and vl) - 4+3+2 beta strand, long loops for antigen binding, cdr1-3 (complementarityy determining region) or hypervariable region on the same side, c' and c'' forms cdr2, cdr1 connects sheet 1 and 2 (crossover turn), cdr3 has the most variability, 5 strands in the middle (longest sheet) and 4 strands on the outside, c" not involved in packing (outside the barrel)
- epitope (Antigenic determinants) - part of antigen recognized by antibody (vs paratope - part of antibody recognize epitope)
T-Cell Receptors:
- t-cells 'killer' (thymus) has t-cell receptors which also has immnuglobulin (antibody) folds that recognizes antigens that are bound to mhc complex of infected cells (U structure only)
IgG (humoral, B-cell antibodies):
- immunoglobulin fold has 4 chains, 2 light and 2 heavy chains, 2 anti-parallel beta sheets packed against each other forming a barrel
- fc = crystalizable fragment, fab - antigen binding fragment
- fc and fab connected together by hinge region / flexible linker
- constant domain (ch1, cl) - 4+3 beta strand connected by short loops, linked by disulfide bonds, sheets are 90 degrees, hydrophobic interactions, 4 strands (longest sheet) in the middle, 3 strands in the outside
- variable domain (vh and vl) - 4+3+2 beta strand, long loops for antigen binding, cdr1-3 (complementarityy determining region) or hypervariable region on the same side, c' and c'' forms cdr2, cdr1 connects sheet 1 and 2 (crossover turn), cdr3 has the most variability, 5 strands in the middle (longest sheet) and 4 strands on the outside, c" not involved in packing (outside the barrel)
- epitope (Antigenic determinants) - part of antigen recognized by antibody (vs paratope - part of antibody recognize epitope)
T-Cell Receptors:
Protein crystallography
crystallography
- shine x-ray (1.5A = 1x10^-10 wavelength) on crystals (protein + h2o)
- treat reflections as summation waves (constructive interference = intensity)
- Bragg's Law: n(x-ray wavelength) = 2dsin(theta), d=lattice plane separation distance=resolution, theta is the incidence=reflection angle bouncing off the lattice plane, n=integral number of wavelengths in path length (extra distance travelled by beam on the 2nd lattice plane)
- structure factors (F) - wave equations that describe the reflections
- recombine structure factors to get an image of the molecular structure in the unit cell
- fourier transform - transforms reflections (diffraction patterns in reciprocal space) to electron density function in real space (and vice-versa)
- need to measure intensity and position (+ angle of reflection) of dots (miller indices hkl - name of reflections) in diffraction patterns, not errors (sigma)
- large unit crystals (proteins) produce reflections closer together (inverse relationship of theta and d in Bragg's Law)
- For a unit cell edge of 40 Å, and data collected to 2 Å resolution (d spacing)
40/2 = 20 planes = 20 spots along that direction
- For a unit cell edge of 80 Å, you now have 40 spots in the"same space"
so the reflections are twice as close together
- "as cell gets bigger, diffraction info is more + more compressed"
- phase problem - phase information is lost in the experiment, work arounds:
- MAD (multiple anomalous dispersion) - use SeMet, estimate Se atom phase from difference in Friedel mates, need to collect lots of data to find extent of error
- MIR (multiple isomorphous replacement) - soak crystals in heavy atoms (HA) while keeping the protein (P) isomorphic (same form), then we take the difference in structure factors, F(HA+P) - F(HA) = F(P), distance shown in Patterson map
- MR (molecular replacement) - homology, need at least 35% sequence identity, performs rotational and translational operations to match model with unknown
Methods for phase improvement:
- solvent flattening - ignore solvent envelope from e- density and mask it out
- histogram matching
- non-crystallographic symmetry (NCS) averaging
- shine x-ray (1.5A = 1x10^-10 wavelength) on crystals (protein + h2o)
- treat reflections as summation waves (constructive interference = intensity)
- Bragg's Law: n(x-ray wavelength) = 2dsin(theta), d=lattice plane separation distance=resolution, theta is the incidence=reflection angle bouncing off the lattice plane, n=integral number of wavelengths in path length (extra distance travelled by beam on the 2nd lattice plane)
- structure factors (F) - wave equations that describe the reflections
- recombine structure factors to get an image of the molecular structure in the unit cell
- fourier transform - transforms reflections (diffraction patterns in reciprocal space) to electron density function in real space (and vice-versa)
- need to measure intensity and position (+ angle of reflection) of dots (miller indices hkl - name of reflections) in diffraction patterns, not errors (sigma)
- large unit crystals (proteins) produce reflections closer together (inverse relationship of theta and d in Bragg's Law)
- For a unit cell edge of 40 Å, and data collected to 2 Å resolution (d spacing)
40/2 = 20 planes = 20 spots along that direction
- For a unit cell edge of 80 Å, you now have 40 spots in the"same space"
so the reflections are twice as close together
- "as cell gets bigger, diffraction info is more + more compressed"
- phase problem - phase information is lost in the experiment, work arounds:
- MAD (multiple anomalous dispersion) - use SeMet, estimate Se atom phase from difference in Friedel mates, need to collect lots of data to find extent of error
- MIR (multiple isomorphous replacement) - soak crystals in heavy atoms (HA) while keeping the protein (P) isomorphic (same form), then we take the difference in structure factors, F(HA+P) - F(HA) = F(P), distance shown in Patterson map
- MR (molecular replacement) - homology, need at least 35% sequence identity, performs rotational and translational operations to match model with unknown
Methods for phase improvement:
- solvent flattening - ignore solvent envelope from e- density and mask it out
- histogram matching
- non-crystallographic symmetry (NCS) averaging
Sunday, March 15, 2009
Mechanistic Basis of Enzyme-Targeted Drugs
Most often, subtle changes occur in the side
chain and main chain positions of the enzyme in order to
bind the substrates transition state optimally.
(Pauling).
http://pubs.acs.org/doi/full/10.1021/bi050247e
James G. Robertson*
Softzymics, Inc., Princeton, New Jersey 08540
Biochemistry, 2005, 44 (15), pp 5561–5571
DOI: 10.1021/bi050247e
Publication Date (Web): March 22, 2005
Some interesting paragraphs from the paper ...
Thus, it should be stated again. Enzymes are catalysts,
and the catalytic event is at least as important if not more
important than the binding event. Enzyme catalysis progresses
through binding events, conformational changes, one or more
transition states, or reaction intermediates, and product
release, and all of these steps occur with defined rate
constants. The rate constants define a thermodynamic profile
that can be used for drug design, and this differentiates
enzymes from all other target classes.
the larger the increase in replication time, the more toxic the drug
In
addition, purely kinetic information can be used to predict
potential drug toxicity. As an example, a detailed pre-steady-
state kinetic analysis of human mitochondrial DNA poly-
merase has been used to predict the potential toxicity of
marketed antiviral drugs.
One
of the major lessons to be learned from marketed drugs is
that nature has designed enzymes to perform a selective
chemical reaction, and therefore, it is likely that the most
potent drugs to be discovered or designed will be related to
the substrate structure, reactivity, or electrostatic potential
surface of intermediate(s) or transition state(s).
chain and main chain positions of the enzyme in order to
bind the substrates transition state optimally.
(Pauling).
http://pubs.acs.org/doi/full/10.1021/bi050247e
James G. Robertson*
Softzymics, Inc., Princeton, New Jersey 08540
Biochemistry, 2005, 44 (15), pp 5561–5571
DOI: 10.1021/bi050247e
Publication Date (Web): March 22, 2005
Some interesting paragraphs from the paper ...
Thus, it should be stated again. Enzymes are catalysts,
and the catalytic event is at least as important if not more
important than the binding event. Enzyme catalysis progresses
through binding events, conformational changes, one or more
transition states, or reaction intermediates, and product
release, and all of these steps occur with defined rate
constants. The rate constants define a thermodynamic profile
that can be used for drug design, and this differentiates
enzymes from all other target classes.
the larger the increase in replication time, the more toxic the drug
In
addition, purely kinetic information can be used to predict
potential drug toxicity. As an example, a detailed pre-steady-
state kinetic analysis of human mitochondrial DNA poly-
merase has been used to predict the potential toxicity of
marketed antiviral drugs.
One
of the major lessons to be learned from marketed drugs is
that nature has designed enzymes to perform a selective
chemical reaction, and therefore, it is likely that the most
potent drugs to be discovered or designed will be related to
the substrate structure, reactivity, or electrostatic potential
surface of intermediate(s) or transition state(s).
Friday, February 27, 2009
huntington's disease, alzheimer and apoptosis
feb '09 Nature:
Good and bad cell death by Donald W. Nicholson:
...
Whereas Alzheimer's disease affects neurons and synaptic junctions of the cerebral cortex, Huntington's disease is characterized by progressive and inexorable deterioration of neurons that project to the striatum region of the brain. Caspase-6-mediated breakdown of huntingtin, the protein that is mutated in Huntington's disease, is necessary for neuronal dysfunction and degeneration in this disorder. Whether the circuitry involved in APP cleavage, DR6 triggering and caspase activation have broad, overlapping mechanistic commonalities in the development of the nervous system, response to injury and disease-associated neurodegeneration is not known. But the links are intriguing and warrant further attention.
--proposed: loss of NGF (nerve growth factor) resulting in APP (amyloid-beta precursor protein) cleavage to generate N-APP (amino terminal portion of APP) that binds to DR6 (death receptor) and activate caspases
on a side note,
- domain swapping between amyloid (misfolded proteins) and prions have been linked to Alzheimer's as well
- not all amyloids are bad, some are functional, maybe nanomaterials?
http://repositorio-aberto.up.pt/bitstream/10216/7161/10/Text.pdf
Huntington’s disease is an autosomal-dominant, progressive neurodegenerative
disorder which affects 4-7 in every 100 000 individuals, worldwide.
Huntington’s disease designation comes from the name of an English doctor, who
vividly characterized this disorder, George Huntington. Several doctors in the 19th century
noticed the hereditary nature of the disease but it was George Huntington who introduced
the term “chorea”, from the Greek word for dance, to describe the involuntary “dance-like”
movements shown by his patients (Elliotson, 1832; Hayden, 1981; Harper P in Bates,
2002; Folstein, 1989).
Good and bad cell death by Donald W. Nicholson:
...
Whereas Alzheimer's disease affects neurons and synaptic junctions of the cerebral cortex, Huntington's disease is characterized by progressive and inexorable deterioration of neurons that project to the striatum region of the brain. Caspase-6-mediated breakdown of huntingtin, the protein that is mutated in Huntington's disease, is necessary for neuronal dysfunction and degeneration in this disorder. Whether the circuitry involved in APP cleavage, DR6 triggering and caspase activation have broad, overlapping mechanistic commonalities in the development of the nervous system, response to injury and disease-associated neurodegeneration is not known. But the links are intriguing and warrant further attention.
--proposed: loss of NGF (nerve growth factor) resulting in APP (amyloid-beta precursor protein) cleavage to generate N-APP (amino terminal portion of APP) that binds to DR6 (death receptor) and activate caspases
on a side note,
- domain swapping between amyloid (misfolded proteins) and prions have been linked to Alzheimer's as well
- not all amyloids are bad, some are functional, maybe nanomaterials?
http://repositorio-aberto.up.pt/bitstream/10216/7161/10/Text.pdf
Huntington’s disease is an autosomal-dominant, progressive neurodegenerative
disorder which affects 4-7 in every 100 000 individuals, worldwide.
Huntington’s disease designation comes from the name of an English doctor, who
vividly characterized this disorder, George Huntington. Several doctors in the 19th century
noticed the hereditary nature of the disease but it was George Huntington who introduced
the term “chorea”, from the Greek word for dance, to describe the involuntary “dance-like”
movements shown by his patients (Elliotson, 1832; Hayden, 1981; Harper P in Bates,
2002; Folstein, 1989).
Thursday, February 26, 2009
protein structure -- exam2 -- domains, tertiary, quaternary structure, folding, membrane proteins
super secondary structures - motifs (lec 8)
what is a motif:
- simplest combination of sec. structures
- break down domains, you get a motif
alpha-helix
- 4 helix bundle - anti-parallel helix form hydrophobic core
- coil-coil - insoluble, helices are parallel, pattern repeats every two turns (heptad repeat, 7 residues, 3.5 residues per turn) a and d are nonpolar, eg keratin
- apha-loop-alpha - binds calcium, eg troponin-C
beta-motif
- beta-hairpin - anti-parallel strands connected by a hairpin (2-5 residues long), eg bovine trypsin inhibitor, erabutoxin
- beta-meander - series of anti-parallel strands connected by hairpins, often by large loops (4123)
- beta-sandwich - 90 degree packing of two sheets eg T-cell surface glycoprotein CD8
- beta-alpha-beta - parallel beta strands, loop 1 forms the active site (loop out of the C-term of beta strand), also called strand-loop-helix-loop-strand, usually right handed, helix shields hydrophobic residues, eg. triosephosphate isomerase (4-beta-alpha-beta-alpha)
helix-domains (lec 9) - all helices
**Q2 - draw coiled coil motif using helical wheels
- indicate which residues form the main contact
- indicate which residues form the electrostatic interactions
a. 4 helix bundle - coiled-coil interaction (heptad repeat, a-d = hydrophobic, g-e = electrostatic interaction), eg lysozyme, cytochrome, human growth, hormone, Rop (c2) dimer, hemagglutinin body
b. globin - 8 helices where alternating helix interact, each helix 7-28 residues long, no motif, pack to bind heme, eg hemoglobin, myoglobin
- helix packing (due to geometry and electrostatics):
a. knobs in holes - coiled coil - 20 degrees, eg GCN4, each side chain in the hydrophobic region of one of the alpha helices can contact 4 side chains from the second alpha helix, the side chain of a residue in position "d" in one helix is directed into a hole at the surface of the second helix surface surrounded by one d-residue, two a-residues, and one e-residue. (n, n-3, n+4, n+1) (trick: 4-3=+1, there's a - and a +)
b. ridges in grooves - globins - 50 degrees, fitting ridges of side chains of one helix onto the grooves between side chains of the other helix
i. i+4 (common) (25) and i+3 (45) = 45-25 = 20 in coiled-coil (rare)
ii. i+4 and i+4 = 25+25 = 50 in globin
iii. i+4 and i+4 pack at 90 degrees (notches) because of Gly
two ways helices pack together
* knob and knotch - glycine forms notch - no side chain
* ridges and grooves
beta-domains (lec 10) - all antiparallel sheets, except for beta-helix (no alpha helices)
- up and down beta-sheets (same as beta meander)
a. barrels, eg retinol-binding beta-barrel (2 sheets packed against each other), porin,
b. propeller-like, eg neuraminidase (influenza virus), c4, 6 sheets (each 4 strands) connected to form a barrel, loops create a funnel like active site
- greek-key in beta barrel - right handed (fold to right), 4123, eg. gamma-crystalin
- greek-key in jelly roll barrel - 81274563, all anti-parallel eg. hemagglutinin binding site
- beta-helix - (not alpha-helix) - beta strands that look like a helix, two parallel beta sheets (strands in the sheet are parallel), strands connected by hairpins - Gly rich, bind Ca2+ eg alkaline protease (2 sheets)
- draw greek key motif and jelly roll barrel
- what is the only beta domain that uses parallel sheets rather than anti-parallel sheets?
- beta-helix domain can use parallel sheets
-
hydrophobic forces (bottom part of alpha helix) stabilise beta strands in alpha/beta domains
question 7 (lec 11)
draw alpha/beta topology for 4 3 1 2, all parallel, find active site (crevice) (it's at the top, between 3 and 1), there's a long loop
-draw solid line, going to right (thumb points to right), so it points out of paper (fingers curl towards you)
-2-3 below the plane, use dashed line point in paper, go to left,
- right-hand connection of beta-alpha-beta
****protein-class*
muramidase - N-term all alpha, huge 27 alpha helices (looks like a big horseshoe)
bacterial alkaline protease - all beta, beta helix
t-cell surface glycoprotein cd8 - beta sandwhich motif, all beta
triophosphate isomerase (tmc)** - alpha-beta class, alpha-beta barrel
retinol-bind - all beta, beta barrel
tnc** - ef hand, all alpha, binds calcium
neuraminidase - all beta, beta propeller
lec 11 - alpha/beta domains
alpha/beta domain types:
a. alpha/beta tim barrel (alpha/beta barrel) - 8 parallel strands in the centre surrounded by helices, forms a closed cylinder, helices on one side, eg triose phosphate isomerase, snorkling effect - res 1,5 polar point in (alternate strand), res 3 hydrophobic point in (alternate strand), res 2,4 hydrophobic point out, facing helices
b. alpha/beta open twisted beta sheet (mixed beta sheet) - helices on both sides of the sheet, forms a long crevice at the switch point - forms the active site, eg Rossman Fold, Lactate Dehydrogenase, bovine carboxypeptidase A, around 6 strands, each with only 5-6 residues in length, doubly-wound topology, if you go to the right, helix wil
l be pointing towards you.
c. alpha/beta horse shoe - leucine rich motif (20-30) - forms stabilizing hydrophobic core between beta-strand, loop and alpha-helix, a large curve parallel sheet with helices on the outside, eg placental ribonuclease inhibitor
- right-handed beta-alpha-beta motif (works via hydrophobic association), both beta-strands and helices are parallel but strands are anti-parallel to helices
protein structure hierarchy:
primary-amino acid sequence
secondary-turns, loops, alpha-helices, beta-sheets
super secondary structure(motifs) -coiled-coil, 4 helix bundle, alpha-loop-helix, beta-meander, beta hairpin, greek-key, beta-alpha-beta(parallel beta strand)
domains: stable unit, all alpha - helix bundle, leucine zipper, globin, all beta-jelly roll, beta barrel, beta propeller, beta helix, greek key barrel, alpha/beta (parallel strands)-barrel, open twisted sheet, horseshoe fold, alpha+beta (anti-parallel beta strands)
quaternary structure: 2+ protein complex, virus
loops - low sequence conservation allows for higher divergence and specificity (compared to more stable sec. struct)
Take home message: the secondary structure provides a stable scaffold where the loops provide active site / specific binding site of the protein. you can predict the location of the active site with alpha/beta domains (topology diagram), not so with alpha and beta domains
q9. protein folding (lec 12)
- disordered proteins are big, non-dense, charged and hydrophillic, low complexity, no sec. structure
- molten globular state (formed quickly by hydrophobic collapse)
- types of intramolecular and function
****-two types of intramolecular (chaperone as part of the protein, eg pre-pro insulin) function (typeI - for tertiary, typeII - for quaternary)
BPTI (bovine trypsin inhibitor) has 3 S-S bonds to guide folding pathway
- cis-trans formation - rate limiting step (not as much for proline)
- chaperones - groel-groes, ClpB (shuriken)
http://www.pnas.org/content/90/15/6924.abstract
(Type I - tertiary structure) The N-terminal propeptide of subtilisin, a serine protease, functions as an intramolecular chaperone (help in protein folding) which is crucial for proper folding of the active enzyme.
Type II - C-term helps in assembly of quaternary structure
q10. conformation change (lec 13)
serpin protease inhibitor complex - loop become beta strand to all anti-parallel beta sheet
serpin alone - mix sheet
serpin-trypsin complex promote degredation, trypsin is disrupted - protease prone region exposed, digested by protease, structure fall aparts
q11. too much info (lec 13)
-homotetramer more common than heterotetramer
give example of heteromultimer-photosynthetic reaction centre, hemoglobin, cdk2-cyclin complex, ovalbumin-trypsin complex, tryptophan synthase, F1-atpase, pea lectin,
homo-multimer-hiv protease (homodimer->symmetry CN -cyclical single fold symmetry 360/N, DN - dihedral 2-fold (180 degrees) rotational, helical -microtubules, viral coats) (need to be a dimer to form functional active site), c4 (homotetramer: K+ channel), hcc dimer domain swapped - C2
D7-GroEL chaperone, D2-lactate dehydrogenase, C2-equine alcohol dehydrogenase
C2-symmetry => symmetric contact (two identical contacts)
assymetric contact => forms tubes
protein-protein interface: center of interface, like protein-interior, hydrophobic
q12. hemoglobin (lec 13) (heterotetramer: pea lectin, photosynthetic reaction centre, tryptophan synthase, f1-atpase)
in hemoglobin: pseudo horizontal symmetry, c2-hemoglobin (vertical)
c4-k+ channel
c4-PFK-phosphofructokinase, homotetrameric protein with negative feedback inhibition, allostery effects, binds substrate F6P (cooperative binding), allosteric effector ADP, and inhibitor PEP (later products), no allosteric effects for ATP (2nd atp noncooperative)
pg 114, a/b structure, changed from R (relaxed) to T (tensed) state (substrate is bound)
hexokinase-induced fit
morpheein-eg porphobilinogen synthase, homo oligorimization of subunits to quaternary structure which depends on the environment (pH, [salt], water, lipid, chaperones)
quaternary structure = eg enzyme complex
lec14: domain swaps
- RNAse A
- prions
- amyloids (misfolded proteins)
- Human cystatin C (HCC) L68Q mutation => HCCA (brain hemorrhage)
- HCC dimer - alpha/beta, C2 symmetry, connecting hinge region forms a loop (open beta-sheet interface)
- induce S-S bonds to HCC to stabilize protein and prevent dimer swaps
q13. properties essential at interface (quaternary, conformational dynamics lec 13 )
-hydrophobic at interior
-peripheral-like exterior, charged and polar
- cdk2 (conformational change) binds cyclin complex, in cell cycle - PSTAIRE and T-loop and Glue51 (E51), when active (cyclin binds to cdk2), the active site is open and is ready to phosphorylate the substrate
- calmodulin / TnC helix melting (EF hand motifs, binds Ca2+)
- the serpin fold is a SERine Protease INhibitor
- trypsin is a serine protease (digestive enzymes)
- alpha1-antitrypsin has a serpin fold
- serpin-serine protein inhibitor, binds trypsin and degrades it
2 stats, active (metastable, mixed beta sheet) and latent/very stable, all anti-parallel
- serine superfamily (eg ovalbumin protein in blood) protease inhibitors, serpin binds bpti, becomes very stable and results in 40% disruption of native trypsin structure, degrading it****
http://en.wikipedia.org/wiki/Serpin
All typically have three β-sheets (termed A, B and C) and eight or nine α-helices (hA-hI) (see figure 4). Serpins also possess an exposed region termed the reactive centre loop (RCL) that in inhibitory molecules includes the specificity determining region and forms the initial interaction with the target protease, has Arg residue serving as bait
Structural studies on serpins also revealed that inhibitory members of the family undergo an unusual conformational change, termed the Stressed to Relaxed (S to R) transition.
The RCL of a serpin acts as a substrate for its cognate protease. However, after the RCL is cleaved, but prior to hydrolysis of the acyl-enzyme intermediate, the serpin rapidly undergoes the S to R transition.
..... so inshort, serpins are like mouse traps for serine proteases (eg trypsin) (mouse), where the bait is the P1 residue in the loop, when it's cleaved by the protease, the loop snaps back swinging along the trypsin and the loop becomes a new beta strand that is anti-parallel between beta strand 5 and 15 of sheet A, forming a stable conformation (latent form, hyperstable), while disrupting the structure of the serine protease (mouse) is 40% disrupted
- phosphofructokinase (homotetramer, alpha+beta, 2 domains - ATP binding site, and fructose-6-phosphate) and hemoglobin - heterotetramer R relaxed to T tensed state, allostery / cooperative binding
q14. membrane protein intro (lec 15)
-detergent needed to purify membrane protein, to solubilize protein=extract protein from membrane, detergent like lipids
- 1. overexpress 2. separation of membrane 3. extraction from membrane 4. chromatograpy / affinity 5. crystallize
- pdc - protein detergent complex
- aim is to reduce micelle-micelle forces (hydrophobic interaction between detergents) and maximize directional electrostatic force between protein-protein interactions
q15. membrane protein intro (lec 15)
-interface, aromatic residues Trp, Tyr, Phe, interior-aliphatic residues-Ala, Val, Leu, Ile
-types, single tm anchor, polytopic, monotopic, beta-strand porin type
- topology: region of embedded in membrane and n or c are in / out
q16. alpha helical membrane protein (K+ channel - lec 16)
- label key features (dehydration of K+ ion when passing through selectivity filter, composed of 8 oxygen from backbone carbonyls, Na+ too small), selectivity and rate of diffusion (energy balance between solvated and naked K+ ions in the cavity and in the pores)
- *dipole, point to same direction
q17, alpha helical membrane protein (K+ channel - lec 16)
monoclonal antibody useful in crystallisation, detergent - hydrophobic - too random, no direction,
electrostatics - more directional, so better packing, better resolution
-increase hydrophilic surface, Fab antibody fragment becomes part of k+ channel,
kinemage: ch12, kin4
q18. beta membrane protein (lec 17)
- beta-porin - eg OprP (mediates phosphate), OpcA, TolC - very long protein that spans the periplasmic surface of gram-negative bateria (140 A), OprM
- Arg ladder for phosphate
- short loops in inside (periplasmic of gram-negative bacteria) of cell
- long loops in outside of cell (extra cellular), sometimes act as a lid (loop 5)
- eyelet, the core, if you rotate 90 (label calcium ions - sphere), has Ca2+ ions
kinemage: porin, hydrophobic outside, inside pore: one side +, other side -, trypto aromatics outside
q19, beta-membrane protein (lec 17)
- draw an autotransporter (in gram neg - bacteria only), 3 parts, n-term signal peptide, passenger (virulence factor), transporter (becomes a beta barrel, embed itself on the membrane to allow passenger to pass through)
- describe function
- hydrophobic belt (around 30A)
- Wza protein has alpha-helices in outer membrane
what is a motif:
- simplest combination of sec. structures
- break down domains, you get a motif
alpha-helix
- 4 helix bundle - anti-parallel helix form hydrophobic core
- coil-coil - insoluble, helices are parallel, pattern repeats every two turns (heptad repeat, 7 residues, 3.5 residues per turn) a and d are nonpolar, eg keratin
- apha-loop-alpha - binds calcium, eg troponin-C
beta-motif
- beta-hairpin - anti-parallel strands connected by a hairpin (2-5 residues long), eg bovine trypsin inhibitor, erabutoxin
- beta-meander - series of anti-parallel strands connected by hairpins, often by large loops (4123)
- beta-sandwich - 90 degree packing of two sheets eg T-cell surface glycoprotein CD8
- beta-alpha-beta - parallel beta strands, loop 1 forms the active site (loop out of the C-term of beta strand), also called strand-loop-helix-loop-strand, usually right handed, helix shields hydrophobic residues, eg. triosephosphate isomerase (4-beta-alpha-beta-alpha)
helix-domains (lec 9) - all helices
**Q2 - draw coiled coil motif using helical wheels
- indicate which residues form the main contact
- indicate which residues form the electrostatic interactions
a. 4 helix bundle - coiled-coil interaction (heptad repeat, a-d = hydrophobic, g-e = electrostatic interaction), eg lysozyme, cytochrome, human growth, hormone, Rop (c2) dimer, hemagglutinin body
b. globin - 8 helices where alternating helix interact, each helix 7-28 residues long, no motif, pack to bind heme, eg hemoglobin, myoglobin
- helix packing (due to geometry and electrostatics):
a. knobs in holes - coiled coil - 20 degrees, eg GCN4, each side chain in the hydrophobic region of one of the alpha helices can contact 4 side chains from the second alpha helix, the side chain of a residue in position "d" in one helix is directed into a hole at the surface of the second helix surface surrounded by one d-residue, two a-residues, and one e-residue. (n, n-3, n+4, n+1) (trick: 4-3=+1, there's a - and a +)
b. ridges in grooves - globins - 50 degrees, fitting ridges of side chains of one helix onto the grooves between side chains of the other helix
i. i+4 (common) (25) and i+3 (45) = 45-25 = 20 in coiled-coil (rare)
ii. i+4 and i+4 = 25+25 = 50 in globin
iii. i+4 and i+4 pack at 90 degrees (notches) because of Gly
two ways helices pack together
* knob and knotch - glycine forms notch - no side chain
* ridges and grooves
beta-domains (lec 10) - all antiparallel sheets, except for beta-helix (no alpha helices)
- up and down beta-sheets (same as beta meander)
a. barrels, eg retinol-binding beta-barrel (2 sheets packed against each other), porin,
b. propeller-like, eg neuraminidase (influenza virus), c4, 6 sheets (each 4 strands) connected to form a barrel, loops create a funnel like active site
- greek-key in beta barrel - right handed (fold to right), 4123, eg. gamma-crystalin
- greek-key in jelly roll barrel - 81274563, all anti-parallel eg. hemagglutinin binding site
- beta-helix - (not alpha-helix) - beta strands that look like a helix, two parallel beta sheets (strands in the sheet are parallel), strands connected by hairpins - Gly rich, bind Ca2+ eg alkaline protease (2 sheets)
- draw greek key motif and jelly roll barrel
- what is the only beta domain that uses parallel sheets rather than anti-parallel sheets?
- beta-helix domain can use parallel sheets
-
hydrophobic forces (bottom part of alpha helix) stabilise beta strands in alpha/beta domains
question 7 (lec 11)
draw alpha/beta topology for 4 3 1 2, all parallel, find active site (crevice) (it's at the top, between 3 and 1), there's a long loop
-draw solid line, going to right (thumb points to right), so it points out of paper (fingers curl towards you)
-2-3 below the plane, use dashed line point in paper, go to left,
- right-hand connection of beta-alpha-beta
****protein-class*
muramidase - N-term all alpha, huge 27 alpha helices (looks like a big horseshoe)
bacterial alkaline protease - all beta, beta helix
t-cell surface glycoprotein cd8 - beta sandwhich motif, all beta
triophosphate isomerase (tmc)** - alpha-beta class, alpha-beta barrel
retinol-bind - all beta, beta barrel
tnc** - ef hand, all alpha, binds calcium
neuraminidase - all beta, beta propeller
lec 11 - alpha/beta domains
alpha/beta domain types:
a. alpha/beta tim barrel (alpha/beta barrel) - 8 parallel strands in the centre surrounded by helices, forms a closed cylinder, helices on one side, eg triose phosphate isomerase, snorkling effect - res 1,5 polar point in (alternate strand), res 3 hydrophobic point in (alternate strand), res 2,4 hydrophobic point out, facing helices
b. alpha/beta open twisted beta sheet (mixed beta sheet) - helices on both sides of the sheet, forms a long crevice at the switch point - forms the active site, eg Rossman Fold, Lactate Dehydrogenase, bovine carboxypeptidase A, around 6 strands, each with only 5-6 residues in length, doubly-wound topology, if you go to the right, helix wil
l be pointing towards you.c. alpha/beta horse shoe - leucine rich motif (20-30) - forms stabilizing hydrophobic core between beta-strand, loop and alpha-helix, a large curve parallel sheet with helices on the outside, eg placental ribonuclease inhibitor
- right-handed beta-alpha-beta motif (works via hydrophobic association), both beta-strands and helices are parallel but strands are anti-parallel to helices
protein structure hierarchy:
primary-amino acid sequence
secondary-turns, loops, alpha-helices, beta-sheets
super secondary structure(motifs) -coiled-coil, 4 helix bundle, alpha-loop-helix, beta-meander, beta hairpin, greek-key, beta-alpha-beta(parallel beta strand)
domains: stable unit, all alpha - helix bundle, leucine zipper, globin, all beta-jelly roll, beta barrel, beta propeller, beta helix, greek key barrel, alpha/beta (parallel strands)-barrel, open twisted sheet, horseshoe fold, alpha+beta (anti-parallel beta strands)
quaternary structure: 2+ protein complex, virus
loops - low sequence conservation allows for higher divergence and specificity (compared to more stable sec. struct)
Take home message: the secondary structure provides a stable scaffold where the loops provide active site / specific binding site of the protein. you can predict the location of the active site with alpha/beta domains (topology diagram), not so with alpha and beta domains
q9. protein folding (lec 12)
- disordered proteins are big, non-dense, charged and hydrophillic, low complexity, no sec. structure
- molten globular state (formed quickly by hydrophobic collapse)
- types of intramolecular and function
****-two types of intramolecular (chaperone as part of the protein, eg pre-pro insulin) function (typeI - for tertiary, typeII - for quaternary)
BPTI (bovine trypsin inhibitor) has 3 S-S bonds to guide folding pathway
- cis-trans formation - rate limiting step (not as much for proline)
- chaperones - groel-groes, ClpB (shuriken)
http://www.pnas.org/content/90/15/6924.abstract
(Type I - tertiary structure) The N-terminal propeptide of subtilisin, a serine protease, functions as an intramolecular chaperone (help in protein folding) which is crucial for proper folding of the active enzyme.
Type II - C-term helps in assembly of quaternary structure
q10. conformation change (lec 13)
serpin protease inhibitor complex - loop become beta strand to all anti-parallel beta sheet
serpin alone - mix sheet
serpin-trypsin complex promote degredation, trypsin is disrupted - protease prone region exposed, digested by protease, structure fall aparts
q11. too much info (lec 13)
-homotetramer more common than heterotetramer
give example of heteromultimer-photosynthetic reaction centre, hemoglobin, cdk2-cyclin complex, ovalbumin-trypsin complex, tryptophan synthase, F1-atpase, pea lectin,
homo-multimer-hiv protease (homodimer->symmetry CN -cyclical single fold symmetry 360/N, DN - dihedral 2-fold (180 degrees) rotational, helical -microtubules, viral coats) (need to be a dimer to form functional active site), c4 (homotetramer: K+ channel), hcc dimer domain swapped - C2
D7-GroEL chaperone, D2-lactate dehydrogenase, C2-equine alcohol dehydrogenase
C2-symmetry => symmetric contact (two identical contacts)
assymetric contact => forms tubes
protein-protein interface: center of interface, like protein-interior, hydrophobic
q12. hemoglobin (lec 13) (heterotetramer: pea lectin, photosynthetic reaction centre, tryptophan synthase, f1-atpase)
in hemoglobin: pseudo horizontal symmetry, c2-hemoglobin (vertical)
c4-k+ channel
c4-PFK-phosphofructokinase, homotetrameric protein with negative feedback inhibition, allostery effects, binds substrate F6P (cooperative binding), allosteric effector ADP, and inhibitor PEP (later products), no allosteric effects for ATP (2nd atp noncooperative)
pg 114, a/b structure, changed from R (relaxed) to T (tensed) state (substrate is bound)
hexokinase-induced fit
morpheein-eg porphobilinogen synthase, homo oligorimization of subunits to quaternary structure which depends on the environment (pH, [salt], water, lipid, chaperones)
quaternary structure = eg enzyme complex
lec14: domain swaps
- RNAse A
- prions
- amyloids (misfolded proteins)
- Human cystatin C (HCC) L68Q mutation => HCCA (brain hemorrhage)
- HCC dimer - alpha/beta, C2 symmetry, connecting hinge region forms a loop (open beta-sheet interface)
- induce S-S bonds to HCC to stabilize protein and prevent dimer swaps
q13. properties essential at interface (quaternary, conformational dynamics lec 13 )
-hydrophobic at interior
-peripheral-like exterior, charged and polar
- cdk2 (conformational change) binds cyclin complex, in cell cycle - PSTAIRE and T-loop and Glue51 (E51), when active (cyclin binds to cdk2), the active site is open and is ready to phosphorylate the substrate
- calmodulin / TnC helix melting (EF hand motifs, binds Ca2+)
- the serpin fold is a SERine Protease INhibitor
- trypsin is a serine protease (digestive enzymes)
- alpha1-antitrypsin has a serpin fold
- serpin-serine protein inhibitor, binds trypsin and degrades it
2 stats, active (metastable, mixed beta sheet) and latent/very stable, all anti-parallel
- serine superfamily (eg ovalbumin protein in blood) protease inhibitors, serpin binds bpti, becomes very stable and results in 40% disruption of native trypsin structure, degrading it****
http://en.wikipedia.org/wiki/Serpin
All typically have three β-sheets (termed A, B and C) and eight or nine α-helices (hA-hI) (see figure 4). Serpins also possess an exposed region termed the reactive centre loop (RCL) that in inhibitory molecules includes the specificity determining region and forms the initial interaction with the target protease, has Arg residue serving as bait
Structural studies on serpins also revealed that inhibitory members of the family undergo an unusual conformational change, termed the Stressed to Relaxed (S to R) transition.
The RCL of a serpin acts as a substrate for its cognate protease. However, after the RCL is cleaved, but prior to hydrolysis of the acyl-enzyme intermediate, the serpin rapidly undergoes the S to R transition.
..... so inshort, serpins are like mouse traps for serine proteases (eg trypsin) (mouse), where the bait is the P1 residue in the loop, when it's cleaved by the protease, the loop snaps back swinging along the trypsin and the loop becomes a new beta strand that is anti-parallel between beta strand 5 and 15 of sheet A, forming a stable conformation (latent form, hyperstable), while disrupting the structure of the serine protease (mouse) is 40% disrupted
- phosphofructokinase (homotetramer, alpha+beta, 2 domains - ATP binding site, and fructose-6-phosphate) and hemoglobin - heterotetramer R relaxed to T tensed state, allostery / cooperative binding
q14. membrane protein intro (lec 15)
-detergent needed to purify membrane protein, to solubilize protein=extract protein from membrane, detergent like lipids
- 1. overexpress 2. separation of membrane 3. extraction from membrane 4. chromatograpy / affinity 5. crystallize
- pdc - protein detergent complex
- aim is to reduce micelle-micelle forces (hydrophobic interaction between detergents) and maximize directional electrostatic force between protein-protein interactions
q15. membrane protein intro (lec 15)
-interface, aromatic residues Trp, Tyr, Phe, interior-aliphatic residues-Ala, Val, Leu, Ile
-types, single tm anchor, polytopic, monotopic, beta-strand porin type
- topology: region of embedded in membrane and n or c are in / out
q16. alpha helical membrane protein (K+ channel - lec 16)
- label key features (dehydration of K+ ion when passing through selectivity filter, composed of 8 oxygen from backbone carbonyls, Na+ too small), selectivity and rate of diffusion (energy balance between solvated and naked K+ ions in the cavity and in the pores)
- *dipole, point to same direction
q17, alpha helical membrane protein (K+ channel - lec 16)
monoclonal antibody useful in crystallisation, detergent - hydrophobic - too random, no direction,
electrostatics - more directional, so better packing, better resolution
-increase hydrophilic surface, Fab antibody fragment becomes part of k+ channel,
kinemage: ch12, kin4
q18. beta membrane protein (lec 17)
- beta-porin - eg OprP (mediates phosphate), OpcA, TolC - very long protein that spans the periplasmic surface of gram-negative bateria (140 A), OprM
- Arg ladder for phosphate
- short loops in inside (periplasmic of gram-negative bacteria) of cell
- long loops in outside of cell (extra cellular), sometimes act as a lid (loop 5)
- eyelet, the core, if you rotate 90 (label calcium ions - sphere), has Ca2+ ions
kinemage: porin, hydrophobic outside, inside pore: one side +, other side -, trypto aromatics outside
q19, beta-membrane protein (lec 17)
- draw an autotransporter (in gram neg - bacteria only), 3 parts, n-term signal peptide, passenger (virulence factor), transporter (becomes a beta barrel, embed itself on the membrane to allow passenger to pass through)
- describe function
- hydrophobic belt (around 30A)
- Wza protein has alpha-helices in outer membrane
Saturday, February 7, 2009
lec8 - super secondary structure - motifs
super secondary structure (motif): Associations of secondary structural elements through sidechain interactions. (almost like domains).
eg alpha-alpha, beta-alpha-beta, beta-beta
a turn states that the α-carbons of residues i and i+3 must be within 7.0 Å.
90 degree corners, beta-corner and alpha-alpha corner, because of Gly
Simplest Motif with a Specific function:
- helix-turn-helix: DNA binding
- helix-loop-helix (EF domain): Ca2+ (binds to loop) binding eg t
roponinC in muscles - coiled-coil - very strong, insoluble, 2 amphipathic parallel helices interacts with hydrophobic edge in the middle, eg. alpha-keratin in hair
- helix bundle - 4 anti-parallel helix bundle, hydrophobic in the middle
- beta-hairpin turn (2-5 res), simples motif involving strands eg b
ovine trypsin inhibitor
- beta-meander (up-and-down) - 4 anti-parallel strands, order of sequence is the same order as strands/connection
- greek-key - 4 strand anti-parallel, looks like a loop that was bended
beta-alpha-beta - parallel beta-strands,
right-handed, helix forms a shield
Friday, January 30, 2009
Senataxin -- SETX
- Large 2677 amino acids
- Coiled-coil domain composed of helices
- Involved in DNA ds-break response
- Found in cytoplasm and nucleoplasm
- Binds nucleotides and ATP
- UvrD Helicase domain at C-term http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=127317
- Phosphoprotein (PTM -- post translational modification)
- No PDB structures yet
- Paralogs of UPF1 and IGHMBP2 proteins
- Homologs of yeast SEN1P, chimps, mouse SETX
- OMIM ( disease, Online Mendelian Inheritance in Men) -- ALS4 and AOA2 -- neurodegenerative disease that causes poor coordination with hands, eyes, limbs, speech.
- http://www.uniprot.org/uniprot/Q7Z333#section_comments
NP_055861 , Q7Z333
- http://www.genecards.org/cgi-bin/carddisp.pl?gene=Setx
- 1-Litre of Tears
- 3 isoforms
Wednesday, January 28, 2009
Turns and loops
Turns
- serve to change the direction of the polypeptide chain, so if chain is going north, then a U-turn will change direction to south
- involves 4 residues, h-bond between carbonyl of residue i and amide of residue i+3
- type I is the most common
- turns are mostly found in the surface, so residues are usually charged and polar
- type I' and II' are mirror image conformations and are rare because of steric hindrance
Loops
- longer than turns, between 6 and 16 residues long
- term used to describe 2 stranded beta-sheet
- beta-hairpin (loop) like turns, generally polar, flexible and are often found in active sites, bin
Secondary structure - Beta sheets
- carbonyls alternate in direction
- side-chains alternate in direction (pleated, doubling the layers because CA alternates up and down the sheet)
- zig-zag
- phi: -135, psi: +135 (top-left or Rama plot)
- pitch: 7A tall, 2 residues per turn, 3.5A rise between residues
- beta strands together form beta sheets via H-bonding between main-chain backbone carbonyls and amides
- vs. alpha helices, beta-sheets have H-bonds with different segments of peptide, 0.1A H-bond shorter, both are amphipathic
- left-hand twisted in terms of angle between strand crossings
- uninvolved strands on the edges can wrap around and be involved in H-bond with the
other strand to form barrels
- Greek Key Motif (3214 or 4123 topology) (number in the order of translation, ie N-term is 1 and the C-term is the last number)
- anti-parallel - strongest, H-bond perfectly 180 degrees horizontal, dipoles cancel with other strand, more solvent accessible, some bifuricate forming beta-bulge
- parallel - weaker, H-bond in an angle, dipole aligns with other strand, producing a net dipole on one side = unstable, usually burried, less twisted
- mixed - mixture of parallel and anti-parallel strands
Connection Types
- hairpin - When the backbone enters the same end of the sheet that it left.
- right-handed crossover - When the backbone enters the opposite end.
Monday, January 26, 2009
Non-covalent interactions
* C-C covalent bond requires 83 kcal/mol (~360 kJ/mol) to break
* 1 kcal/mol ~ 4 kJ/mol
* H-bond (O-H....N) is about 7 kcal/mol
* bond length is from H-donor to H-acceptor
* London forces, a special type of van der Waals attraction, is when two aromatics are on top of each other, induced dipoles
* charge-charge (electrostatic) strongest IN VACUUM F=kq1q2/r^2*e (Coulomb's Law) e = dielectric, in H2O, e ~ 80, interior of protein e ~ 2 (almost vacuum)
* dipole(mu) (permanent and induced), diff. in electronegativity, Molecules with no netchargebut with asymmetric distribution of charge (e.g. CO or H2O) –Polar (permanent dipole),
* peptide bond has dipole, eg H2O, vector toward q+, mu = qx q=charge, x=distance
* van der Waals (short range than charge-charge), very weak but plays important role in stability
* van der Waals (vdw) radii rv=R1+R2 H(1.2A), **C(1.7), N(1.5), O(1.4) (radii decrease because # protons increase but # orbitals stay the same)
* Lennard-Jones potential, e=(1/r)^12-(1/r)^6 balance between vdw attractions (1/r)^6 and repulsions (1/r)^12 http://en.wikipedia.org/wiki/Lennard-Jones_potential
* hydrogen bonds, between H-bond donor (O or N) and H-bond acceptor (O or N), strong and specific non-convalent interaction, 2.6-3.5A H-bond length, 180 degrees, straight H-bond is strongest, found in secondary structure, alpha-helices, at 2.55A, the h-bond is very strong and there's a low barrier hydrogen bond (LBHB)
* water, unique because of H-bonds, sphere of hydration enables salt to dissolve, water can steal hydrogen bonds from alpha-helices, breaking it
* hydrophobic interaction - lipids aggregate because it takes less energy to form a single cage than 2 separate water cages
Summary
* hydrophobic effect and van der Waal's forces
** weakest
** for stability of folded protein
** non-specific (doesn't matter what orientation is the water cage, as long as it helps minimize energy)
vs
* hydrogen bonding and electrostatic (charge-charge)
** strongest
** for single folded state formation, NOT stability of folded state
** highly specific
* 1 kcal/mol ~ 4 kJ/mol
* H-bond (O-H....N) is about 7 kcal/mol
* bond length is from H-donor to H-acceptor
* London forces, a special type of van der Waals attraction, is when two aromatics are on top of each other, induced dipoles
* charge-charge (electrostatic) strongest IN VACUUM F=kq1q2/r^2*e (Coulomb's Law) e = dielectric, in H2O, e ~ 80, interior of protein e ~ 2 (almost vacuum)
* dipole(mu) (permanent and induced), diff. in electronegativity, Molecules with no netchargebut with asymmetric distribution of charge (e.g. CO or H2O) –Polar (permanent dipole),
* peptide bond has dipole, eg H2O, vector toward q+, mu = qx q=charge, x=distance
* van der Waals (short range than charge-charge), very weak but plays important role in stability
* van der Waals (vdw) radii rv=R1+R2 H(1.2A), **C(1.7), N(1.5), O(1.4) (radii decrease because # protons increase but # orbitals stay the same)
* Lennard-Jones potential, e=(1/r)^12-(1/r)^6 balance between vdw attractions (1/r)^6 and repulsions (1/r)^12 http://en.wikipedia.org/wiki/Lennard-Jones_potential* hydrogen bonds, between H-bond donor (O or N) and H-bond acceptor (O or N), strong and specific non-convalent interaction, 2.6-3.5A H-bond length, 180 degrees, straight H-bond is strongest, found in secondary structure, alpha-helices, at 2.55A, the h-bond is very strong and there's a low barrier hydrogen bond (LBHB)
* water, unique because of H-bonds, sphere of hydration enables salt to dissolve, water can steal hydrogen bonds from alpha-helices, breaking it* hydrophobic interaction - lipids aggregate because it takes less energy to form a single cage than 2 separate water cages
Summary
* hydrophobic effect and van der Waal's forces
** weakest
** for stability of folded protein
** non-specific (doesn't matter what orientation is the water cage, as long as it helps minimize energy)
vs
* hydrogen bonding and electrostatic (charge-charge)
** strongest
** for single folded state formation, NOT stability of folded state
** highly specific
Sunday, January 25, 2009
Post translational modifications (PTM)
Main chain modification
* Proteolytic cleavage
** Signal peptidase (preprotein): "molecular postal code", N(basic, +ve charged region), H(hydrophobic region), C(specificity region, has Ala-X-Ala consensus sequence in positions -3,-2,-1), eg secretion systems, in eukarytotes (co-translational translocation in ER) vs Post-Translational translocation in prokaryotes http://en.wikipedia.org/wiki/Secretory_pathway
** Zymogen (proprotein) eg trypsin digestive enzyme, from trypsinogen (inactive) to trypsin (active form)
** pre-pro peptide hormone: eg insulin, forms hexamer with Zn, signal is cleaved by signal peptidase and delivered to ER, then an intermediate (proprotein) form where it's needed to position the two chains in the correct place to form disulfide bonds, the intermediate peptide is cleaved by PC1 and PC2 endoprotease then digested by exoprotease to produce active form
** viral RNA - is used to make polyprotein (for viral coats) - protein that, after synthesis, is cleaved to produce several functionally distinct polypeptides.
** Inteins - intron-exon like at a protein level (has exteins too)
* N-terminal modifications
** Acetylation - added by Acetyl-Coa at N-term, blocks sequencing (for side-chains, it's Lys and Arg, because methyls CH3 are electron donating groups, so want to donate electrons to + charge groups)
** Myristylation - 14 carbon myristol(fatty acid) group, associate protein with membranes
Side chain modification
* glycosylation - proteins covalently linked with carbohydrate (for proteins bound to membrane)
** n-linked - N-linked (ND2 from Asn, N is single letter code for Asn) to sugar GlcNAc, occurs cotranslationally in ER, consensus Ans-X-Ser/Thr
** o-linked - O-linked (OG from Ser/Thr) to sugar GalNAc (both vowels, o, a, OH pointing same direction as CH2OH) and occurs post-translationally in Golgi (another vowel o)
* methylation - add methyl Lys or Arg
* phosphorylation - regulate activity, kinase phosphorylate proteins, groups with hydroxyl are phosphorylated: ser (happens 1000times)/thr(100times)/tyr(1time)
* sulfation - happens in Tyr, happens permanently, for stabilizing, NOT for regulatory modification, eg fibrinogen, donor is PAPS,
* prenylation/lipidation - adds 15 carbon farnesyl or 20 carbon geranylgeranyl group to Cys at carboxy terminus, consensus is CAAX (A is any aliphatic residue except Ala)
* hydroxylation - vitamin c-dependent modification - Pro and Lys hydroxylation (add OH), eg collagen
* carboxylation - vitamin k-dependent modification - Carboxylation of Glu (so two carbonyls), for blood clotting, chelating Ca2+ ions
* disulfide bond formation - oxidized 2 Cys residues in ER (oxidizing environment), in vivo, glutathione (glutamine+cysteine+glycine) (GSH) -> GSSG (glutatione disulfide) (oxidized form) -> these are the oxidizers
* citrullination (deimination) - neutralize arginine+ charge
* deamidation - replace amide of asparagine with carbonyl, changing it to aspartic acid
Summary:
http://en.wikipedia.org/wiki/Prenylation
Cysteine - Disulfide bond formation, Prenylation
N-termini - Acetylation, Myristorylation, Methylation
Aginine - Methylation, Citrullination (deamination) (NH to O)
C-term - Amidation
Asparagine - N-Glycosylation, deamiDation (Asparagine to Aspartic Acid and IsoAspartic Acid)
Ubiquitination - E1 activating, E2 conjugating, E3 ubiquitin protean ligase
Glutamate - Carboxylation (Vit K dependent)
Hydroxyl Groups (S/T) - Phosphorylation, O-linked Glycosylation
Tyrosine - Sulfation
Proline - Hydroxylation (Vit C dependent)
Lysine - Hydroxylation (Vit C dependent), Methylation
* Proteolytic cleavage
** Signal peptidase (preprotein): "molecular postal code", N(basic, +ve charged region), H(hydrophobic region), C(specificity region, has Ala-X-Ala consensus sequence in positions -3,-2,-1), eg secretion systems, in eukarytotes (co-translational translocation in ER) vs Post-Translational translocation in prokaryotes http://en.wikipedia.org/wiki/Secretory_pathway
** Zymogen (proprotein) eg trypsin digestive enzyme, from trypsinogen (inactive) to trypsin (active form)
** pre-pro peptide hormone: eg insulin, forms hexamer with Zn, signal is cleaved by signal peptidase and delivered to ER, then an intermediate (proprotein) form where it's needed to position the two chains in the correct place to form disulfide bonds, the intermediate peptide is cleaved by PC1 and PC2 endoprotease then digested by exoprotease to produce active form
** viral RNA - is used to make polyprotein (for viral coats) - protein that, after synthesis, is cleaved to produce several functionally distinct polypeptides.
** Inteins - intron-exon like at a protein level (has exteins too)
* N-terminal modifications
** Acetylation - added by Acetyl-Coa at N-term, blocks sequencing (for side-chains, it's Lys and Arg, because methyls CH3 are electron donating groups, so want to donate electrons to + charge groups)
** Myristylation - 14 carbon myristol(fatty acid) group, associate protein with membranes
Side chain modification
* glycosylation - proteins covalently linked with carbohydrate (for proteins bound to membrane)
** n-linked - N-linked (ND2 from Asn, N is single letter code for Asn) to sugar GlcNAc, occurs cotranslationally in ER, consensus Ans-X-Ser/Thr
** o-linked - O-linked (OG from Ser/Thr) to sugar GalNAc (both vowels, o, a, OH pointing same direction as CH2OH) and occurs post-translationally in Golgi (another vowel o)
* methylation - add methyl Lys or Arg
* phosphorylation - regulate activity, kinase phosphorylate proteins, groups with hydroxyl are phosphorylated: ser (happens 1000times)/thr(100times)/tyr(1time)
* sulfation - happens in Tyr, happens permanently, for stabilizing, NOT for regulatory modification, eg fibrinogen, donor is PAPS,
* prenylation/lipidation - adds 15 carbon farnesyl or 20 carbon geranylgeranyl group to Cys at carboxy terminus, consensus is CAAX (A is any aliphatic residue except Ala)
* hydroxylation - vitamin c-dependent modification - Pro and Lys hydroxylation (add OH), eg collagen
* carboxylation - vitamin k-dependent modification - Carboxylation of Glu (so two carbonyls), for blood clotting, chelating Ca2+ ions
* disulfide bond formation - oxidized 2 Cys residues in ER (oxidizing environment), in vivo, glutathione (glutamine+cysteine+glycine) (GSH) -> GSSG (glutatione disulfide) (oxidized form) -> these are the oxidizers
* citrullination (deimination) - neutralize arginine+ charge
* deamidation - replace amide of asparagine with carbonyl, changing it to aspartic acid
Summary:
http://en.wikipedia.org/wiki/Prenylation
Cysteine - Disulfide bond formation, Prenylation
N-termini - Acetylation, Myristorylation, Methylation
Aginine - Methylation, Citrullination (deamination) (NH to O)
C-term - Amidation
Asparagine - N-Glycosylation, deamiDation (Asparagine to Aspartic Acid and IsoAspartic Acid)
Ubiquitination - E1 activating, E2 conjugating, E3 ubiquitin protean ligase
Glutamate - Carboxylation (Vit K dependent)
Hydroxyl Groups (S/T) - Phosphorylation, O-linked Glycosylation
Tyrosine - Sulfation
Proline - Hydroxylation (Vit C dependent)
Lysine - Hydroxylation (Vit C dependent), Methylation
Friday, January 23, 2009
Secondary structure -- Alpha Helices
* Secondary structure -- protein folding brought by linking carbonyl and amide groups of the backbone together by means of hydrogen bonds
Types: alpha-helices, beta-sheets, turns and loops
Linus Pauling -- predicted right-handed alpha-helix and planar peptide groups
Why do alpha helices form?
- Solved by Linus Pauling, to neutralize the main-chain atom charges (NH and C=O) making hydrophobic cores possible through hydrogen bonding
- Formed by phi (X) and psi (Y) angles of -60 and -50 degrees (bottom left quadrant of Ramachandran plot)
Alpha-helix measurements
- 3.6 residues per turn (H-bond froms between n and residue n+4)
- 5.4A patch, so one full turn is 5.4A tall
- Therefore, each additional residue gives a rise of 1.5A per residue (5.4A/3.6residues)
- Mostly right-handed, thumb points in the direction of translation (N-termini to C-termini), finger curls in the helix curling direction
end of helices (N+) usually found on the surface of the proteins
side-chains in alpha-helices are outside and points downward looking like a christmas tree
**bad Proline, produces steric, no H in NH bond to donate, so interferes with H-bond pattern
solvents (h2O) also causes bends
preference for (aliphatic): ala, leu, glu, met
against (hydroxyl): pro, gly, ser, tyr
Locations
* surface helices: amphipathic (high hydrophobic moment, 50% hydrophobic 50% hydrophilic
* membrane helices: hydrophobic (low hydrophobic moment, all hydrophobic)
* soluble helices: hydrophilic (low hydrophobic moment, all hydrophilic)
helical wheel to help determine hydrophobic moment (50-50 hydrophobic, hydrophilic)
Non-common helices / variations:
- pi helix (n+5)(4.3-16) (short and stuby, more residues per turn)
- 3-10 helix (n+3) (3 residues = 10 atoms per turn)
groove - the through, bottom, A long narrow furrow or channel.
ridge - the one pointing up, A long narrow elevation on the ocean floor.
http://www.cryst.bbk.ac.uk/PPS2/course/section9/9_helhel.html
- i+4n ridge (more common) than i+3n (slightly more slanted) ridge
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