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
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