Gram-negative:
- pathogenic
- has an extra empy space later, periplasmic surface
Many species of Gram-negative bacteria are pathogenic, meaning that they can cause disease in a host organism. This pathogenic capability is usually associated with certain components of Gram-negative cell walls, in particular the lipopolysaccharide (also known as LPS or endotoxin) layer.[1] In humans, LPS triggers an innate immune response characterized by cytokine production and immune system activation. Inflammation is a common result of cytokine(from the greek cyto=cell,kinesis=movement) production, which can also produce host toxicity.
Lipid A is a lipid component of an endotoxin held responsible for toxicity of Gram-negative bacteria. It is the innermost of the three regions of the lipopolysaccharide (LPS, also called endotoxin) molecule, and its hydrophobic nature allows it to anchor the LPS to the outer membrane. While its toxic effects can be damaging, the sensing of lipid A by the human immune system may also be critical for the onset of immune responses to Gram-negative infection, and for the subsequent successful fight against the infection.
Gram-positive cell walls typically lack the outer membrane found in Gram-negative bacteria.
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
quotes on honesty
http://www.quotegarden.com/honesty.html
If you tell the truth you don't have to remember anything. ~Mark Twain
If you tell the truth you don't have to remember anything. ~Mark Twain
Wednesday, February 25, 2009
Exam writing tips
http://www.gillmacmillan.ie/ecom/Library3.nsf/5dc7eb74f29342f580256ead004e0898/f3162d25f998d70a80256cdf0043d58d?OpenDocument
resolvconf: Error: /etc/resolv.conf must be a symlink
fix:
/etc$ sudo ln -s /etc/resolvconf/run/resolv.conf
/etc$ sudo ln -s /etc/resolvconf/run/resolv.conf
Wednesday, February 18, 2009
Saturday, February 14, 2009
ところ - tokoro - by the way
Wednesday, February 11, 2009
Sunday, February 8, 2009
Animes bring back memories
Slayers Revolution
http://www.veoh.com/browse/videos/category/comedy/watch/v160847724B4gk8mt#watch%3Dv1733031747W6R34e
Kimi Ga Nozomu Eien: Next Season (4-part OAV)
http://www.baka-updates.com/search/search?searchitem=Kimi+Ga+Nozomu+Eien++&submit.x=6&submit.y=7&submit=submit&searchradio=releases
.... hmmm ... so I just finished watching the 4 eps, overall, it went ok, still felt a bit uneasy though *sigh*
Hajime No Ippo New Challenger
http://www.veoh.com/collection/sliphive/watch/v174420702FZtQEdE
http://www.veoh.com/browse/videos/category/comedy/watch/v160847724B4gk8mt#watch%3Dv1733031747W6R34e
Kimi Ga Nozomu Eien: Next Season (4-part OAV)
http://www.baka-updates.com/search/search?searchitem=Kimi+Ga+Nozomu+Eien++&submit.x=6&submit.y=7&submit=submit&searchradio=releases
.... hmmm ... so I just finished watching the 4 eps, overall, it went ok, still felt a bit uneasy though *sigh*
Hajime No Ippo New Challenger
http://www.veoh.com/collection/sliphive/watch/v174420702FZtQEdE
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
getting lost ...
went horribly lost going back from the dentist @ fraser *sigh*, nice experience but mom didn't appreciate it as much :(, went to 3 different cities, over 2 diff bridge :D
〜に (ni) Meaning: (location marker, time marker, direction marker)
Friday, February 6, 2009
Learning Japanese Books
A dictionary of basic Japanese sentence patterns / Naoko Chino.
Japanese step by step : an innovative approach to speaking and reading Japanese / Gene Nishi.
PL 539.5 E5 N56 2000
| PL 619 C69 2000 Title | A dictionary of basic Japanese sentence patterns / Naoko Chino. |
| Published | Tōkyō : Kōdansha Intānashonaru, 2000. |
Japanese step by step : an innovative approach to speaking and reading Japanese / Gene Nishi.
PL 539.5 E5 N56 2000
| Title | Japanese step by step : an innovative approach to speaking and reading Japanese / Gene Nishi. |
| Published | New York : McGraw-Hill, c2000. |
How to write and publish a scientific paper
Day, R.A. 1983. How to write and publish a scientific paper. Second edition. ISI Press,
Philadelphia, Pennsylvania, 181 pp. wise and witty.
T11 D33 1998
The keyword is reproducibility -- provide a written document showing what he or she did, why it was done, how it was done, and what was learned from it.
"a naturalist's life would be a happy one if he had only to observe and never to write" -- Darwin 1985
Philadelphia, Pennsylvania, 181 pp. wise and witty.
T11 D33 1998
The keyword is reproducibility -- provide a written document showing what he or she did, why it was done, how it was done, and what was learned from it.
"a naturalist's life would be a happy one if he had only to observe and never to write" -- Darwin 1985
Thursday, February 5, 2009
japanese resources
Japanese Online Learning
http://smart.fm/lists (aka http://www.iknow.co.jp)
http://www.genkienglish.net/genkijapan/learnjapanesenumbers.htm
http://lang-8.com/
http://www.thejapanesepage.com/beginners/hiragana/wa
Romaji to hiragana/katakana http://www.whiteagle.net/jap/
Quizes and such, fun phrase testing too
http://www.easyjapanese.org/lesson01.html
LernJ Japanese learning adventure
http://lrnj.com/
http://japanese.about.com/blpod.htm Japanese Phrase of the Day
http://en.wikipedia.org/wiki/Japanese_verb_conjugations
Anime Lyrics
http://www.animelyrics.com/anime/x/exdream.htm
Newsletters
http://www.yookoso.com/
Kanji List
http://www.ajalt.org/kanmana/index_e.html
Japanese School - $190 for 200hrs.
http://www.vjls-jh.com
http://learnjapanese.elanguageschool.net/
http://www.escapeartist.com/japan/japan3.htm
Sight-seeing in Kyoto
http://www.kyoto-np.co.jp/kp/topics/eng/eng.html
http://www.japan-guide.com/e/e2158.html
http://www.geocities.com/japanfaq/FAQ-Manners.html
http://www.japan-guide.com/
Important Phrases
http://www.tripadvisor.com/Travel-g294232-s604/Japan:Important.Phrases.html
http://www.theforeigner-japan.com/
Nice Pics about Kyoto
http://www.insidekyoto.com/2008_05_01_archive.html
Trip planner
http://www.hyperdia.com/
http://wikitravel.org/en/Kyoto
http://www.kyoto.travel/
http://guidetojapanese.org/
http://wikitravel.org/en/Japanese_phrasebook
http://nihongo-dekimasu.blogspot.com
http://rs643.rapidshare.com/files/208184313/conversationaljapanese.pdf
http://www.megaupload.com/?d=A4TFJEQQ japanese phrasebook
http://www.nihongoperapera.com/nintendo-ds.html
http://naruhodojapan.blogspot.com/2007/05/nazotte-oboeru-otona-no-kanji-renshu.html
Japanese Events
http://www.japan-guide.com/event/?aMONTH=7&aYEAR=2009
http://smart.fm/lists (aka http://www.iknow.co.jp)
http://www.genkienglish.net/genkijapan/learnjapanesenumbers.htm
http://lang-8.com/
http://www.thejapanesepage.com/beginners/hiragana/wa
Romaji to hiragana/katakana http://www.whiteagle.net/jap/
Quizes and such, fun phrase testing too
http://www.easyjapanese.org/lesson01.html
LernJ Japanese learning adventure
http://lrnj.com/
http://japanese.about.com/blpod.htm Japanese Phrase of the Day
http://en.wikipedia.org/wiki/Japanese_verb_conjugations
Anime Lyrics
http://www.animelyrics.com/anime/x/exdream.htm
Newsletters
http://www.yookoso.com/
Kanji List
http://www.ajalt.org/kanmana/index_e.html
Japanese School - $190 for 200hrs.
http://www.vjls-jh.com
http://learnjapanese.elanguageschool.net/
http://www.escapeartist.com/japan/japan3.htm
Sight-seeing in Kyoto
http://www.kyoto-np.co.jp/kp/topics/eng/eng.html
http://www.japan-guide.com/e/e2158.html
http://www.geocities.com/japanfaq/FAQ-Manners.html
http://www.japan-guide.com/
Important Phrases
http://www.tripadvisor.com/Travel-g294232-s604/Japan:Important.Phrases.html
http://www.theforeigner-japan.com/
Nice Pics about Kyoto
http://www.insidekyoto.com/2008_05_01_archive.html
Trip planner
http://www.hyperdia.com/
http://wikitravel.org/en/Kyoto
http://www.kyoto.travel/
http://guidetojapanese.org/
http://wikitravel.org/en/Japanese_phrasebook
http://nihongo-dekimasu.blogspot.com
http://rs643.rapidshare.com/files/208184313/conversationaljapanese.pdf
http://www.megaupload.com/?d=A4TFJEQQ japanese phrasebook
http://www.nihongoperapera.com/nintendo-ds.html
http://naruhodojapan.blogspot.com/2007/05/nazotte-oboeru-otona-no-kanji-renshu.html
Japanese Events
http://www.japan-guide.com/event/?aMONTH=7&aYEAR=2009
Anime OP/Closing musics
X-TV
Ex-Dream
http://www.youtube.com/watch?v=ritJ1NUitv8
http://www.animelyrics.com/anime/x/exdream.htm
Chrno Crusade
Sayonara Solitia
Farewell, Solitaire
www.youtube.com/watch?v=WEueSBnZr-E
http://www.animelyrics.com/anime/chrnocrusade/sayonarasolitia.htm
さよならャ潟eィア
[クロノクルセイド ED]
歌 ：千葉紗子
作詞：梶浦由記
作曲：梶浦由記
編曲：梶浦由記
大好きと思うからね
傷ついたり 躊躇ったり
冷たい頬を寄せ合って
心が生まれた
いつも今すぐに会いたい
無口になるほど好きよ
優しさどうしたら見えるの？
抱きしめてもっと強く
暖かな胸を信じるよ
さよならャ潟eィア
明日へ……
小さな私だから
全部でも足りないよね
何にも隠さないで
貴方にあげたい
まだ白い夜明けを見送って
こんなに大事な人に
どうして巡り会えたのと
痛いほど繋ぐ指で
寂しさ消える夢を見るの
さよならャ潟eィア
もう一人じゃないから
明日目覚めるの
貴方と……
大好きな人だからね
側にいる 守ってる
貴方へ繋がる大地に
生まれて良かった
Ex-Dream
http://www.youtube.com/watch?v=ritJ1NUitv8
http://www.animelyrics.com/anime/x/exdream.htm
Chrno Crusade
Sayonara Solitia
Farewell, Solitaire
www.youtube.com/watch?v=WEueSBnZr-E
http://www.animelyrics.com/anime/chrnocrusade/sayonarasolitia.htm
さよならャ潟eィア
[クロノクルセイド ED]
歌 ：千葉紗子
作詞：梶浦由記
作曲：梶浦由記
編曲：梶浦由記
大好きと思うからね
傷ついたり 躊躇ったり
冷たい頬を寄せ合って
心が生まれた
いつも今すぐに会いたい
無口になるほど好きよ
優しさどうしたら見えるの？
抱きしめてもっと強く
暖かな胸を信じるよ
さよならャ潟eィア
明日へ……
小さな私だから
全部でも足りないよね
何にも隠さないで
貴方にあげたい
まだ白い夜明けを見送って
こんなに大事な人に
どうして巡り会えたのと
痛いほど繋ぐ指で
寂しさ消える夢を見るの
さよならャ潟eィア
もう一人じゃないから
明日目覚めるの
貴方と……
大好きな人だからね
側にいる 守ってる
貴方へ繋がる大地に
生まれて良かった
ので (no de) (because, and )
# 彼の話はあまりにも馬鹿げていたので誰も信じなかった。 [ex #481]
His story was too ridiculous for anyone to believe.
# このように平易な英語で書かれているので、この本は初心者に役立つ。 [ex #482]
Written as it is in plain English, this book is useful for beginners.
# 彼はお金に困っていたので、昨日スーパーマーケットで万引きを働いた。 [ex #483]
He was hard up for money and so lifted goods in a supermarket yesterday.
# 風邪をひいたので学校に行かない [ex #5695]
Iwon't go to school because I cought a cold
His story was too ridiculous for anyone to believe.
# このように平易な英語で書かれているので、この本は初心者に役立つ。 [ex #482]
Written as it is in plain English, this book is useful for beginners.
# 彼はお金に困っていたので、昨日スーパーマーケットで万引きを働いた。 [ex #483]
He was hard up for money and so lifted goods in a supermarket yesterday.
# 風邪をひいたので学校に行かない [ex #5695]
Iwon't go to school because I cought a cold
Quotes from a fwded email
Your mind is like a parachute...it functions only when open.
The best vitamin for making friends..... B1
Of all the things you wear, your expression is the most important.
If you want your dreams to come true, you mustn't oversleep.
'Be kinder than necessary because everyone you meet is fighting some kind of battle..'
A sharp tongue can cut your own throat
Life is too short to wake up with regrets. So love the people who
treat you right. Forget about the ones who don't. Believe
everything happens for a reason. If you get a second chance, grab it with both hands. If it changes your life, let it.
Nobody said life would be easy, they just promised it would be worth it.
Friends are like balloons; once you let them go, you might not get them back. Sometimes we get so busy with our own lives and problems that we may not even notice that we've let them fly away. Sometimes we are so caught up in who's right and who's wrong that we forget what's right and wrong.
Sometimes we just don't realize what real friendship means until it is too late. I don't want to let that happen so I'm gonna tie you to my heart so I never lose you.
Ideas won't work unless ' You' do.
You lie the loudest when you lie to yourself.
If you lack the courage to start, you have already finished.
One thing you can't recycle is wasted time.
The heaviest thing you can carry is a grudge.
One thing you can give and still keep...is your word.
The happiness of your life depends on the quality of your thoughts.
The best vitamin for making friends..... B1
Of all the things you wear, your expression is the most important.
If you want your dreams to come true, you mustn't oversleep.
'Be kinder than necessary because everyone you meet is fighting some kind of battle..'
A sharp tongue can cut your own throat
Life is too short to wake up with regrets. So love the people who
treat you right. Forget about the ones who don't. Believe
everything happens for a reason. If you get a second chance, grab it with both hands. If it changes your life, let it.
Nobody said life would be easy, they just promised it would be worth it.
Friends are like balloons; once you let them go, you might not get them back. Sometimes we get so busy with our own lives and problems that we may not even notice that we've let them fly away. Sometimes we are so caught up in who's right and who's wrong that we forget what's right and wrong.
Sometimes we just don't realize what real friendship means until it is too late. I don't want to let that happen so I'm gonna tie you to my heart so I never lose you.
Ideas won't work unless ' You' do.
You lie the loudest when you lie to yourself.
If you lack the courage to start, you have already finished.
One thing you can't recycle is wasted time.
The heaviest thing you can carry is a grudge.
One thing you can give and still keep...is your word.
The happiness of your life depends on the quality of your thoughts.
Tuesday, February 3, 2009
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