Determination of alpha-helix propensities within the context of a folded protein Blaber et al. J. Mol. Biol 1994

Ser44 and Val131 are two of a very limited number of possible sites in T4 Lysozyme that fulfill the criteria of an unperturbed site. 1)Site located in canonical alpha helix phi = -60 psi = -45 2)Site in interior of helix not in N or C cap 3)No strong sidechain interaction 4)Site exposed to solvent 5)No crystal contacts

Virtually all side chains stabilize helix correlation between subs at 44 and 131

Proline severely disrupts helix

Substitutions can have unintended consequences The Val131Asp mutation leads to a large conformational change in the distant residue Arg 154 leading to the formation of a salt-bridge. But careful crystal structure is at pH 6.7 while stability measurement was taken at pH 3.0

Virtually all side chains stabilize helix correlation between subs at 44 and 131

by tweaking the  1 angle Thr 44 can hydrogen bond to the carbonyl carbon of Asn 40. Some interactions are very sensitive to geometry so subtle conformational change can have large energetic effects Subtle changes lead to new interactions

Side-chain conformations cluster into well-defined rotamers

The statistical approach to analyzing the rules that govern protein structure. -Instead of making mutants yourself, make use of the diversity of protein structures found in nature. -Using known protein structure avoids surprises from un- intended structural changes introduced by mutations. -Using a very large number of structures ensures that effects specific to a particular protein will cancel out.

Most residues in helices prefer g + conformation Phe, Trp and Tyr are the exceptions. They like to adopt trans. Ser in position 44 actually adopts an unusual rotatmer. Should this site have been selected. Ser sometimes also adopts g - conformer, because It can form hydrogen bonds to i-3 carbonyl carbon.

The observed changes in free energy associated with different replacements within an alpha-helix are small (0 to 1.0 kcal/mol). At the same time relatively small changes in the conformation of the folded structure, or different assumptions as to the nature of the unfolded state, can easily alter estimates…. by several 10th of a kcal/mol Blaber et al. J. Mol. Biol 1994

Summary Protein melting curves in conjunction with site-directed mutagenesis allow us to measure energetic contributions to protein stability. Statistical analysis of known protein structures provide an independent way to assess conformational energetics. BUT -Protein melting has to be a two state process -We have to make assumptions about thermodynamic properties (i.e. ∆C p folded = ∆C p unfolded ) -We are comparing folded and unfolded state, but we have no idea what unfolded state looks like. -Mutations can have unintended consequences. -> Better check structures of mutants.