Theory of NMR- J-coupling, structure, whatever.NMR measures the distance between protein atoms. They absorb radio waves at a resonating frequency until saturation is reached. J-coupling happens through bonds and means when nuclei influence each other, which is visible in NMR peaks (singlet, triplet, whatever).Nuclear Overhauser Effect: As excited nuclei relax, energy can be transferred to nearby nuclei by spin diffusion or saturation transfer. NOE makes contact maps to determine 3d structure NOEs between atoms on different side chains provide spatial information on which side chains are in contact with each other in the form of contact maps.NOEs provide geometric constraints on the structure, as they specify which atoms are in contact with each other. The polypeptide chain is folded in a computer into a 3D structure that agrees with the distance constraints / contact map. Remember that main chain bond angles limited, so there are constraints based on theory, as well.Conformational flexibility means many different structures that match the protein’s geometrical constraints, which results in few NOEs. NMR solution is an ensemble of many different possible structures that equally agree with the experimental data.HSQC NMR- overlay of nitrogen and hydrogen atoms. When a ligand binds a protein, its amino acids’ environment changes, therefore the acids’ chemical shift values also change. We can analyze these changes to determine the ligand binding site. We may use this, for example, to determine the different sites on a protein to which a drug binds, so that we can engineer a drug that binds to the protein with the greatest affinity for the protein.Protein folding is a chemical and thermodynamically describable process.Native or folded state is favorable simply because it is at a lower energy state.Gibbs free energy change = change in enthalpy minus (entropy times temperature).Enthalpy is the result of noncovalent interactions formed as the polypeptide chain folds up.Unfolded polypeptides can go through a huge number of conformations; this is called conformational entropy. This entropy decreases as the protein folds up.Entropy on its own is a measure of the number of accessible microstates in which a system can exist. There is a formula in the lecture notes.Conformational entropy increases upon denaturation: the universe likes to be random. Check the example equation in the lecture notes.So, why do proteins fold in the first place, if the universe likes being random?Simple- decrease in conformational entropy is paid for by the increase in entropy of the surrounding water molecules. Net entropy of the entire system tends to increase. Upon protein folding, hydrophobic residues move to the interior of the protein andordered water molecules are released, and entropy of the system increases: the hydrophobic effect is extremely favorable!Anfinsen experiment with ribonuclease A: a fellow called Anfinsen showed that denatured ribonuclease A could renature and refold to regain its structure; the disulfide cross-links, oncedisrupted, could reform. This demonstrated that amino acid sequence and by extension DNA sequence encoded a protein’s structure and function.Levinthal’s paradox- even a small polypeptide has millions of folding conformations, and randomly searching through them would take billions of years. How does protein folding takesuch little time, then, both in vivo and in vitro? The answer is that there must be a kinetically favorable pathway.The two models we have to address this problem are the condensation and nucleation models.Condensation models involve a hydrophobic collapse of non-polar residues to form a molten globule of protein, which restricts the conformational search space for the rest of the polypeptide.Nucleation models involve small regions of local structure that rapidly form first, around which the rest of the polypeptide folds.Both these models correctly describe reality, depending on the protein.Kinetic traps- if a protein incorrectly folds, but the resultant globule has a lower free energy and conformational entropy than the polypeptide, it can be thermodynamically unfavorable toget out of it. Some intermediates can be kinetic traps as well. Proteins need extra energy fromanother source to beat the trap, but sometimes, they need more energy than the quantity that can be given.Potential problems during folding:• Formation of incorrectly folded intermediates (including misplaced disulfides)• Aggregation of exposed hydrophobic residues• Proteins that are “trapped” in intermediate states, and unable to proceed down the energy funnel and reach the native state. Cells have proteins/machines that assist folding and untangle trapped intermediates:• Molecular Chaperones and Chaperonins• Protein Disulfide Isomerase• Peptidyl Prolyl IsomeraseChaperones are enzymes that assist folding by binding and releasing exposed hydrophobic regions within proteins as they fold, to prevent those hydrophobic residues from aggregating. The binding and release of substrate is controlled by ATP hydrolysis and nucleotide exchange.Chaperonins provide an ideal folding environment- it provides a cage of sorts for the chaperones and misfolded proteins to interact. We don’t know exactly how this works, but weknow certain areas and instances of it