LT1 Determination & Prediction of 2 Structures

Question 1

Why is knowing protein structure important?

Answer 1

Know the active site and which amino acid residues are involved in ligand binding

Allows design of drugs to manipulate their activities

Question 2

What are the dihedral angles?

Answer 2

Phi and Psi angles are considered dihedral angles

Only the single covalent bonds can be rotated in the backbone

Partial double bond CANNOT rotate

Question 3

What defines positive and negative degree rotations?

Answer 3

Positive is assigned to right-hand rotation
Keep the atom in front at a 12 o’clock position and if the back atom is to the right then it is positive

Vice versa for negative values

Question 4

What are the range of dihedral angles?

Answer 4

-180 to +180

Question 5

How are alpha-helices defined by dihedral angles?

Answer 5

Whereas, both angles range from -180 to +180

In alpha helices (right-handed)
360 degrees / 3.6 amino acids in 1 turn = 100 degree turns, so 50 per dihedral angle

Question 6

How are beta-sheets defined by dihedral angles?

Answer 6

Beta-strand normally have
-140 degree = Phi
+140 degree = Psi

Question 7

What is circular dichroism, and how does it work?

Answer 7

Form of light absorption spectroscopy that allows determination of secondary structures = alpha and beta

Left- and right-handed circularly polarized light passes through a chiral molecule and after their magnitudes are different

Question 8

What are the two regions of CD bands for proteins?

Answer 8

Far UV region (amide region) = 170-250nm
Gives info about secondary structure = alpha displays 2 prominent bands at 222nm and 208nm beta only displays one at 251nm

Near UV region = 250-300nm
Originates from aromatic amino acids when their side chains immobilized in a folded protein and then transferred to asymmetric env (tertiary structure)

CD of aromatic residue is very small in absence of ordered structure = when unfolded protein or peptide

Question 9

What is circular dichoism useful for monitoring?

Answer 9

Structural transition of proteins = thermal unfolding or ligand binding

Question 10

What does mdeg tell us?

Answer 10

The observed raw ellipticity = needs to be converted to molar ellipticity

Question 11

How is the percentage of secondary structure in an unknown protein predicted?

Answer 11

If we assume, CD spectrum is a linear combination of CD spectra of each contributing secondary structure type = weighted by its abundance in polypeptide conformation

Use CD spectra of protein with known percentage of secondary structure by fitting the spectrum

Question 12

What does NMR allow us to determine?

Answer 12

Individual residue

Question 13

What is required for NMR?

Answer 13

Sequential assignment of backbone/side-chain resonances

Question 14

What does chemical shift index from NMR tell us?

Answer 14

The CSI provides information about the local secondary structure of proteins. By comparing the observed chemical shifts of specific backbone atoms (usually Cα or Cβ) to reference values for known secondary structures (like alpha helices, beta sheets, and random coils), researchers can infer the likely conformation of the protein segment.

Question 15

What do the chemical shift values tell us?

Answer 15

Positive CSI: Suggests the presence of an alpha helix.

Negative CSI: Suggests the presence of a beta sheet.

Values close to zero: Indicate regions that are likely disordered or in random coil conformations.

Question 16

What does nuclear overhause effect (NOE) tell us from NMR?

Answer 16

Measure the distance between two atoms can tell us if it is alpha-helix or beta-sheet

Question 17

What does J coupling constant from NMR tell us?

Answer 17

Determine Phi dihedral angle directly by measuring amount of splitting of a resonance peak (NH) by another coupled proton (H-alpha)

Question 18

Why is secondary structure prediction important?

Answer 18

Step towards knowing 3D structure
Understanding more about function of a protein without knowing its 3D structure

Question 19

How is accuracy of prediction calculated?

Answer 19

Addition of number of residues predicted correctly in each types of secondary structure
P alpha, P beta and P coil

Divided by total number of residues = gives a %

This value is known as Q3

Question 20

What is the improved Chous-Fasman method?

Answer 20

A computational approach used for predicting the secondary structure of proteins based on their amino acid sequences

Probability of finding a certain amino acid in a type of secondary structure

Search for stretches of sequence that favour certain types of secondary structure
The probability of an amino acid is not affected by neighbouring residues

So Q3 is around 50-60%
Need to consider neighbouring amino acids

Question 21

Why is GOR III method better than Chou-Fasman method?

Answer 21

GOR evaluates each residue PLUS adjacent 8 N-terminal and 8 C-terminal residue

If a particular amino acid is surrounded with residues that prefer to be in a helix = then it is likely in a helix even if its helical preference is quite low

Q3 ~ 65%

Question 22

What is the most commonly used way of predicting secondary structure?

Answer 22

Using neural network ~ 70-75% Q3

Align sequence with other related proteins of the same family

Find member w known structure and if significant matches between structure and sequence then assign secondary structure to corresponding residue

Question 23

Why is it easier to predict trans-membrane alpha-helices than in cytoplasmic proteins?

Answer 23

Transmembranes alpha helices are 20-30 residues long

High overall hydrophobicity

Short loops between helices that contain many positive charged residues