title: Experimental and Computational Analysis of Protein Stabilization by Gly-to-d-Ala Substitution: A Convolution of Native State and Unfolded State Effects.
abstract: The rational and predictable enhancement of protein stability is an important goal in protein design. Most efforts target the folded state, however stability is the free energy difference between the folded and unfolded states thus both are suitable targets. Strategies directed at the unfolded state usually seek to decrease chain entropy by introducing cross-links or by replacing glycines. Cross-linking has led to mixed results. Replacement of glycine with an l-amino acid, while reducing the entropy of the unfolded state, can introduce unfavorable steric interactions in the folded state, since glycine is often found in conformations that require a positive φ angle such as helical C-capping motifs or type I' and II″ β-turns. l-Amino acids are strongly disfavored in these conformations, but d-amino acids are not. However, there are few reported examples and conflicting results have been obtained when glycines are replaced with d-Ala. We critically examine the effect of Gly-to-d-Ala substitutions on protein stability using experimental approaches together with molecular dynamics simulations and free energy calculations. The data, together with a survey of high resolution structures, show that the vast majority of proteins can be stabilized by substitution of C-capping glycines with d-Ala. Sites suitable for substitutions can be identified via sequence alignment with a high degree of success. Steric clashes in the native state due to the new side chain are rarely observed, but are likely responsible for the destabilizing or null effect observed for the small subset of Gly-to-d-Ala substitutions which are not stabilizing. Changes in backbone solvation play less of a role. Favorable candidates for d-Ala substitution can be identified using a rapid algorithm based on molecular mechanics.
authors.affiliation: Department of Chemistry and ‡Laufer Center for Physical and Quantitative Biology, Stony Brook University , Stony Brook, New York 11794-3400, United States.
authors.forename: Junjie
authors.initials: J
authors.lastname: Zou
authors.affiliation: Department of Chemistry and ‡Laufer Center for Physical and Quantitative Biology, Stony Brook University , Stony Brook, New York 11794-3400, United States.
authors.forename: Benben
authors.initials: B
authors.lastname: Song
authors.affiliation: Department of Chemistry and ‡Laufer Center for Physical and Quantitative Biology, Stony Brook University , Stony Brook, New York 11794-3400, United States.
authors.forename: Carlos
authors.initials: C
authors.lastname: Simmerling
authors.affiliation: Department of Chemistry and ‡Laufer Center for Physical and Quantitative Biology, Stony Brook University , Stony Brook, New York 11794-3400, United States.
authors.forename: Daniel
authors.initials: D
authors.lastname: Raleigh
mesh_terms: D000409:Alanine / Q000737:chemistry; D000465:Algorithms; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry*; D002645:Chickens; D017127:Glycation End Products, Advanced; D005998:Glycine / Q000737:chemistry; D018398:Homeodomain Proteins / Q000737:chemistry*; D008840:Microfilament Proteins / Q000737:chemistry*; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D055550:Protein Stability; D058767:Protein Unfolding; D012709:Serum Albumin / Q000737:chemistry*; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry*; D000093423:Glycated Serum Albumin
chemical_list: D001426:Bacterial Proteins; D017127:Glycation End Products, Advanced; D018398:Homeodomain Proteins; D008840:Microfilament Proteins; D012709:Serum Albumin; D014157:Transcription Factors; C106185:engrailed homeobox proteins; C026615:villin; D000409:Alanine; D005998:Glycine; D000093423:Glycated Serum Albumin