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

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.

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

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

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

  3. Effect of AA substitutions at position 44 relative to Gly Virtually all side chains stabilize helix relative to glycine. Why?

  4. Proline severely disrupts helix

  5. Correlation between energetic effects of substitutions at pos 44 and 131

  6. Why introducing acidic residues at pos. 131 leads to greater helix stabilization than introducing it at pos 44 WT V131D

  7. Subtle changes lead to new interactions 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

  8. 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

  9. 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.

  10. Side-chain conformations cluster into well-defined rotamers

  11. 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. ∆Cp folded = ∆Cp 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.

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