Inferring stabilizing mutations from protein phylogenies : application to influenza hemagglutinin

One selection pressure shaping sequence evolution is the requirement that a protein fold with sufficient stability to perform its biological functions. We present a conceptual framework that explains how this requirement causes the probability that a particular amino acid mutation is fixed during evolution to depend on its effect on protein stability. We mathematically formalize this framework to develop a Bayesian approach for inferring the stability effects of individual mutations from homologous protein sequences of known phylogeny. This approach is able to predict published experimentally measured mutational stability effects (ΔΔG values) with an accuracy that exceeds both a state-of-the-art physicochemical modeling program and the sequence-based consensus approach. As a further test, we use our phylogenetic inference approach to predict stabilizing mutations to influenza hemagglutinin. We introduce these mutations into a temperature-sensitive influenza virus with a defect in its hemagglutinin gene and experimentally demonstrate that some of the mutations allow the virus to grow at higher temperatures. Our work therefore describes a powerful new approach for predicting stabilizing mutations that can be successfully applied even to large, complex proteins such as hemagglutinin. This approach also makes a mathematical link between phylogenetics and experimentally measurable protein properties, potentially paving the way for more accurate analyses of molecular evolution

A Genetic Linkage Map of the Mouse Using Restriction Landmark Genomic Scanning (Rlgs)

We have developed a multiplex method of genome analysis, restriction landmark genomic scanning (RLGS) that has been used to construct genetic maps in mice. Restriction landmarks are end-labeled restriction fragments of genomic DNA that are separated by using high resolution, two-dimensional gel electrophoresis identifying as many as two thousand landmark loci in a single gel. Variation for several hundred of these loci has been identified between laboratory strains and between these strains and Mus spretus. The segregation of more than 1100 RLGS loci has been analyxed in recombinant inbred (RI) strains and in two separate interspecific genetic crosses. Genetic maps have been derived that link 1045 RLGS loci to reference loci on all of the autosomes and the X chromosome of the mouse genome. The RLGS method can be applied to genome analysis in many different organisms to identify genomic loci because it used end-labeling of restriction landmarks rather than probe hybridization. Different combinations of restriction enzymes yield different sets of RLGS loci providing expanded power for genetic mapping

Energetics of aliphatic deletions in protein cores

Although core residues can sometimes be replaced by shorter ones without introducing significant changes in protein structure, the energetic consequences are typically large and destabilizing. Many efforts have been devoted to understand and predict changes in stability from analysis of the environment of mutated residues, but the relationships proposed for individual proteins have often failed to describe additional data. We report here 17 apoflavodoxin large-to-small mutations that cause overall protein destabilizations of 0.6–3.9 kcal.mol−1. By comparing two-state urea and three-state thermal unfolding data, the overall destabilizations observed are partitioned into effects on the N-to-I and on the I-to-U equilibria. In all cases, the equilibrium intermediate exerts a “buffering” effect that reduces the impact of the overall destabilization on the N-to-I equilibrium. The performance of several structure-energetics relationships, proposed to explain the energetics of hydrophobic shortening mutations, has been evaluated by using an apoflavodoxin data set consisting of 14 mutations involving branching-conservative aliphatic side-chain shortenings and a larger data set, including similar mutations implemented in seven model proteins. Our analysis shows that the stability changes observed for any of the different types of mutations (LA, IA, IV, and VA) in either data set are best explained by a combination of differential hydrophobicity and of the calculated volume of the modeled cavity (as previously observed for LA and IA mutations in lysozyme T4). In contrast, sequence conservation within the flavodoxin family, which is a good predictor for charge-reversal stabilizing mutations, does not perform so well for aliphatic shortening ones