Protein Structure and Function Flashcards
What do atoms in proteins undergo that can be detected by infra-red or Raman spectroscopes?
Small-scale vibrational and rotational motions.
When do large multi-domain proteins undergo large movements?
Upon ligand binding
What can proteins do spontaneously but rarely?
Undergo transient but complete unfolding.
What evidence from using quenching reagents to observe protein dynamics are there?
That fluorescence of aromatic amino-acids is instantly quenched by close proximity of chain small molecules and that internal residue are also quenched. This suggests that the solvents can ‘dissolve’ into the protein or the protein undergo breathing motions to expose the buried regions.
What is used to monitor hydrogen exchange?
Mass spectrometry or NMR. Monitors the extent of exchange versus time.
What are protons in proteins?
Very sensitive to their environment.
What is the rate of hydrogen exchange dependent on?
pH, location of each amide within a protein, the degree of burial from the solvent, frequency of partial unfolding and hydrogen bonding in the native state.
Why is it not possible to use 1D hydrogen exchange for large proteins?
There are too many proton signals with similar chemical shift and the width of each peak increases resulting in a degenerate spectrum.
What is used for large proteins?
2D hydrogen exchange. Minimises overlapping or clustering of signals by using two parameters to sort out the data.
What two parameters are used in 2D hydrogen exchange?
Isoelectric focussing and SDS-PAGE.
A fingerprint of all -NH groups except for what is generated in 2D hydrogen exchange?
Proline.
What three pieces of evidence of protein dynamics were found using NMR?
Ring flipping, dynamic regions and a richness of dynamics from ps to seconds.
Which residues cannot rotate their rings?
Tryptophan and histidine as they are too large and require 360 degree rotation.
What two pieces of evidence of protein dynamics were found using crystallography?
No density and B-factors and that small proteins give different structures.
What is the temperature factor B?
The B-factor which is the extent of smearing due to the local flexibility of the polypeptide. Low B-factors indicate cold, static areas and high B-factors indicate hot, dynamic areas.
How can small proteins give different structures?
Crystallising at different temperatures and their relative positions of the backbone carbon on different crystal lattices.
How are molecular dynamics analysed?
Atoms of known structure are given a certain velocity and a random motion. The magnitude of the force acting on each of these atoms is then calculated. As the original position and velocity is known the effect of the force can be used to calculate the new positions of all the atoms. For molecular systems the force acting on each atom is defined as a sum of terms which include bonded and non-bonded interactions.
What information can protein dynamics reveal?
Highly detailed information at a molecular level, a rich pattern of atomic movements and rates of inter-conversion and relative stabilities of each conformation.
What does roughness in the native well of an energy landscape of a protein lead to?
Discrete but randomly inter converting states.
Why are protein dynamics important?
For multi-enzyme complexes, active site availability, ligand binding and catalysis.
What are the two types of empirical observations that can be used to observe protein dynamics?
Proteins undergoing small and large scale conformational changes and oxygen release from oxyhaemoglobin.
What are some methods of detecting macro molecular interactions?
Direct visualisation, genetic methods, biochemical binding assays and biophysical assays.
Why is quantitative characterisation of binding and intermolecular interactions required?
To find specific vs. non-specific association, stable vs. transient complexes, strong vs. weak interactions, the type of interactions involved, the reaction between ligand/protein structure and strength and understanding of regulation of affinity by post-translational modification and solution conditions.
What is a method of direct visualisation?
Using fluorescence microscopy.
