LT1 Determination & Prediction of 2 Structures Flashcards
Why is knowing protein structure important?
Know the active site and which amino acid residues are involved in ligand binding
Allows design of drugs to manipulate their activities
What are the dihedral angles?
Phi and Psi angles are considered dihedral angles
Only the single covalent bonds can be rotated in the backbone
Partial double bond CANNOT rotate
What defines positive and negative degree rotations?
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
What are the range of dihedral angles?
-180 to +180
How are alpha-helices defined by dihedral angles?
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
How are beta-sheets defined by dihedral angles?
Beta-strand normally have
-140 degree = Phi
+140 degree = Psi
What is circular dichroism, and how does it work?
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
What are the two regions of CD bands for proteins?
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
What is circular dichoism useful for monitoring?
Structural transition of proteins = thermal unfolding or ligand binding
What does mdeg tell us?
The observed raw ellipticity = needs to be converted to molar ellipticity
How is the percentage of secondary structure in an unknown protein predicted?
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
What does NMR allow us to determine?
Individual residue
What is required for NMR?
Sequential assignment of backbone/side-chain resonances
What does chemical shift index from NMR tell us?
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.
What do the chemical shift values tell us?
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.
What does nuclear overhause effect (NOE) tell us from NMR?
Measure the distance between two atoms can tell us if it is alpha-helix or beta-sheet
What does J coupling constant from NMR tell us?
Determine Phi dihedral angle directly by measuring amount of splitting of a resonance peak (NH) by another coupled proton (H-alpha)
Why is secondary structure prediction important?
Step towards knowing 3D structure
Understanding more about function of a protein without knowing its 3D structure
How is accuracy of prediction calculated?
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
What is the improved Chous-Fasman method?
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
Why is GOR III method better than Chou-Fasman method?
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%
What is the most commonly used way of predicting secondary structure?
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
Why is it easier to predict trans-membrane alpha-helices than in cytoplasmic proteins?
Transmembranes alpha helices are 20-30 residues long
High overall hydrophobicity
Short loops between helices that contain many positive charged residues
