Genetic Analysis of Protein Stability and Function

Abstract

There is tremendous variability in the importance of individual amino acids in protein sequences. On the one hand, nonconservative residue substitutions can be tolerated with no loss of activity at many residue positions, especially those exposed on the protein surface. On the other hand, destabilizing mutations can occur at a large number of different sites in a protein, and for many proteins such mutations account for more than half of the randomly isolated missense mutations that confer a defective phenotype. At sites that are key determinants of stability or activity, even residue substitutions that are generally considered to be conservative (e.g., Glu in equilibrium Asp, Asn in equilibrium Asp, Ile in equilibrium Leu, Lys in equilibrium Arg and Ala in equilibrium Gly) can have severe phenotypic effects. Unfortunately, this means that there is no simple way to infer the likely effect of an amino acid substitution on the basis of sequence information alone. A nonconservative Gly----Arg substitution could be phenotypically silent at one position while a conservative Asn----Asp change could lead to complete loss of activity at another position. For proteins whose structures are known, it is often possible to predict whether particular residue substitutions will be destabilizing, as long as detailed estimates of the destabilization energy are not required. Substitutions that introduce polar groups, large cavities, or overly large side chains into the hydrophobic core are potentially the most destabilizing. Substitutions that disrupt hydrogen bonding or electrostatic interactions can also have significant effects, although the destabilization caused by these substitutions is smaller than that caused by severe core mutations. Destabilizing substitutions that involve replacing glycines in turns, or introducing prolines into alpha-helices and other disallowed positions are also reasonably common. Finally, most solvent exposed residues can apparently be freely substituted without serious effects on protein stability. Although exceptions may occur, these generalizations serve to summarize a large body of information and can be rationalized in physical and chemical terms. It is an especially encouraging result that proteins appear to tolerate most substitutions, even those that are destabilizing, without significant changes in the native structure. For proteins whose structures are known, this means that it is reasonable to interpret mutant phenotypes in terms of the wild-type structure. For proteins whose structures are not known, it is reasonable to infer that mutations that reduce activity without affecting stability are directly involved in function.(ABSTRACT TRUNCATED AT 400 WORDS)

Mutational analysis of the BPTI folding pathway: I. Effects of aromatic → leucine substitutions on the distribution of folding intermediates

“…This sensitivity of the folding intermediates to the aromatic 4 Leu substitutions contrasts with the generally observed tolerance of proteins to amino acid replacements. Many studies of mutant protein structures indicate that the overall fold of a protein is remarkably tolerant to amino acid replacements (Alber, 1989;Pakula & Sauer, 1989;Goldenberg, 1992a;Matthews, 1993a). Even when a replacement leads to a large destabilization, the result is generally a protein that fails to fold at all, rather than one that folds to a radically different conformation (Lattman & Rose, 1993).…”

Detecting Site-Specific Physicochemical Selective Pressures: Applications to the Class I HLA of the Human Major Histocompatibility Complex and the SRK of the Plant Sporophytic Self-Incompatibility System

“…In most proteins, only a few residues at regions of low variability are functionally crucial (for example, those residues with catalytic function at or close to the active site), whereas, the majority of other residues play a role in maintaining the appropriate three-dimensional structure of the protein to ensure its functionality (PAKULA and SAUER 1989). Therefore, most AA sites are either unlikely to change or only preferentially change over time, depending on the strength of site-specific purifying selection.…”

Misfolded proteins impose a dosage-dependent fitness cost and trigger a cytosolic unfolded protein response in yeast

“…proteomics | stability | heat shock | evolutionary rate M ost new genetic mutations arising in protein-coding sequences decrease the likelihood that the encoding protein will fold properly (1,2). Misfolding reduces the concentration of functional proteins, squanders cellular time and energy on production of useless proteins (3), and generates misfolded proteins that may harm cells (4).…”