Williamson Protein Structure, CD, Fl

Question 1

Types of alpha proteins- ridges in grooves

Answer 1

Side chains on the surface form ridges of sidechains separated by grooves
Can be used to tell if it is a I+3 or I+4 helix by how far apart the numbers are
Helices pack ridge to groove
When you lay the helices on top of one another you get interaxial angles- 50 degrees for two I+4 and 20 for a 4 and 3

Question 2

Coiled coils

Answer 2

Can be parallel or anti parallel
Globular and fibrous
Individual right handed, but form left handed super coil
The left handed twist means there is 3.5 residues per turn
There is a heptad repeat where the pattern repeats every 7 residues
Salt bridges between e and g residues

Question 3

Coiled coil with leucine zipper

Answer 3

Residues a and d are leucine or isoleucine
E and g are charged and help alignment
Helices interact by knobs in holes where the side chains of one helix fit into gaps in the other
The 18 degree offset produces the coiled coil

Question 4

B propeller

Answer 4

Neuraminidase
Beta propeller
6 overlapping blades

Question 5

B helix

Answer 5

Pectate lyase C
Parallel B helix
Interior is filled by side chains
Made from 3 parallel sheets
Some polar and charged residues pair
Extreme stability

Question 6

Packing in beta barrels

Answer 6

Shear number- the residue offset
Number of strands and shear number define geometry
Top layers of the barrel may be polar to protect the hydrophobic core

Question 7

A/B proteins

Answer 7

Have a b-a-b motif
Parallel beta strands
Helices pack against the strands forming a hydrophobic core

2 main types

• closed 8 strand barrel. TIM barrel (triose phosphate isomerase). Funnel shaped active site is formed by loop connecting c terminal regions of beta strands to helix
• open sheet with helices both sides. Helices occur on the same face of the sheet. For helices to be on both sides, a crossover is needed. Active sites are formed where the strand order is reversed

Question 8

a + b

Answer 8

Have helices and sheets but is not ordered
The sheet here is mainly antiparallel
Hydrophobic cores are formed by barrels, sandwiches or ridge and groove

Question 9

Small proteins

Answer 9

BPTI- bovine pancreatic trypsin inhibitor
58 residues
Folding dependent on disulphides
Some stabilised by metal binding

Question 10

Post translational modifications

6 types

Answer 10

Acetylation, cytoplasmic proteins n terminus, degradation?
Glycosylation- n linked (Asn) or o linked (Ser/thr) added in ER before and after folding. Solubility and interaction
Y-carboxylation- Glu in ER, adds Ca binding seen in blood clotting
Hydroxylation- pro and Lys. ER, needs glycine after, collagen folding
Phosphorylation- Ser, thr, Tyr, ER post folded regulatory
Disulphide- Cys, ER secreted and membrane for stability

Question 11

Catalytic triad

Answer 11

Ser- his - asp
Found in chymotrypsin, trypsin and elastase
Convergent evolution with subtilisin of bacteria

Question 12

Physical principle of fluorescence

Answer 12

Always lower than absorbed as lost as heat
Q= no. Of excited molecules that fluoresce/total excited molecules
In proteins, only Tyr and Trp
Trp dominates as more intense and energy transfer Tyr -> Trp

Question 13

Trp dependent on microenvironment

Answer 13

Fluorescent intensity- quenching groups COO-, His, disulphides and aromatics
External quenchers- acrylic idea and iodide
Polar 350
Hydrophobic 310

Question 14

FRET

Answer 14

Emission of donor overlaps acceptor
Aligned parallel electron dipoles interact
Excited and ground state overlap = lost as heat
Fluorescence at a longer wavelength as lost as heat- stokes shift
Fluorescence lifetime
Polarisation dependent on transition moment of electron returning to ground state
Polarisation difference = fluorescence anisotropy

Question 15

Fluorimeter

Answer 15

Single wavelength

Measure at 90 degrees

Question 16

2 types of fluorophores

Answer 16

Intrinsic- chemical groups Phe, Tyr Trp cofactors

Extrinsic- fusion GFP. Cys residue attachment.

Question 17

Biological applications of fluorescence

4

Answer 17

Ligand binding- mastoparin binding to calmodulin. Trp sidechain moves from polar to hydrophobic so wavelength change for binding
Denaturantion- will move away from quenching groups. Measure of conformational stability

Folding transitions in adenylate kinase- open has acceptor and donor far apart. FRET will occur in substrate bound form

Sensitivity assay for MMP activity- cleavage of peptide will stop quenching of Mca by FRET. Cleavage of peptide increases the fluorescence of Mca fusion

Question 18

Energy transfer efficiency

Answer 18

Ro^6 / (Ro^6 + R6)
R is distance, Ro is Forster distance (distance where transfer is 50% efficient)
Efficiency of transfer is proportional to 1/R^6

Must be between 0.5Ro and 1.5Ro

Question 19

What is CD?

Answer 19

Differential absorbance of L and R polarised light
Sensitivity to chiral its and molecular symmetry
Two waves of same amplitude wavelength and phase in perpendicular planes
Plane polarised light at 45 to competent waves
The waves are then 90 degrees out of phase giving circularly polarised light
90= right polarised -90= left polarised
Superposition gives plane polarised

Question 20

Absorption in a CD medium

Answer 20

Materials absorbing left and right polarised light exhibit CD
Absorbs more right than left -> elliptically polarised
This will twist the same as the least absorbed

Question 21

Components of a CD machine

Answer 21

Monochromatic and linear polariser
Photo elastic modulator (linear to left and right)
Detector- intensity is difference between peak and trough
Spectra

Question 22

Units for CD measurements

Answer 22

A = Al -Ar
E = El - ER (or just A) /c*l
A= theta/32982
For mean molar, divide again by N

Question 23

Far UV and near UV

Answer 23

Far 190-250 peptide bonds. Secondary structure.
Near 250-300 aromatics. Aromatic sidechains, will be asymmetric. The more mobile, there will be less asymmetry and lower signal. Up to 2mg protein. Tertiary structure.

Question 24

CD applications

Answer 24

Folded state
Secondary structure contents
Comparing native and recombinant proteins
Stability

Intercept of delta G against urea gives conformational stability