Thermodynamics of the helix -coil transition: Calorimetric and spectroscopic studies

Understanding of the rules governing protein folding has been the primary focus of structural biologists for the past 50 plus years. If one understands how a protein folds, then this knowledge can be used in the rational design of novel proteins with applications in therapeutics, pharmaceuticals, and industrial purposes. However, before we can imagine the grand applications for our work, we must first understand the basic principles underlying how the twenty amino acids fold into three basic structural motifs (alpha-helix, beta-strand, and coils) and become the assortment of proteins and enzymes that carry out the functions of life. The major goal of this thesis is to investigate the thermodynamic basis of helix formation.;The first part of this thesis is focused on the helical propensity of amino acid residues in different positions of the same a-helix. It has been previously established that the helical propensities of different amino acid residues in the middle of an alpha-helix in peptides and in proteins are very similar. The statistical analysis of the protein helices from the known three-dimensional structures shows no difference in the frequency of non-charged residues in the middle and at the C-terminus. Yet, experimental studies show distinctive differences for the helical propensities of non-charged residues in the middle and in the C-terminus in model peptides. Is this a general effect and is it applicable to protein helices or is it specific to the model alanine based peptides? To answer this question, the effects of substitutions at positions 28 (middle residue) and 32 (C2 position at the C-terminus) of the alpha-helix of ubiquitin, on the stability of this protein are measured using differential scanning calorimetry. The two data sets produce similar values for intrinsic helix propensity leading to a conclusion that non-charged amino acid residues at the solvent exposed positions in the middle and at the C-terminus of the alpha-helix have the same helical propensity. This conclusion is further supported with an excellent correlation between the helix propensity scale obtained for the two positions in ubiquitin with the experimental helix propensity scale established previously and with the statistical distribution of the residues in protein helices.;In the second part of this thesis, the alpha-helix is looked at as an isolated folding event. In isolation, the helix is free from the constraints of the rest of the protein and can be used to experimentally determine the energetics of helix formation. The temperature induced helix-coil transition in a series of host peptides was monitored using circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC). Combination of these two techniques allowed direct determination of the enthalpy of helix-coil transition for the studied peptides. (Abstract shortened by UMI.).