Integrating chemistry, biophysics and physiology in the evolution of mammalian Myoglobins

Abstract

This work describes an integration between chemistry, molecular biophysics, physiology, sequence evolution and bioinformatics to better understand the evolution of mammalian myoglobins (Mb) in terms of their primary biochemical function (i.e., O2 binding) and their thermodynamic stability (i.e., folding free energy). First, we merge a large set of previously reported thermochemical data for Mb mutants with a physiological model of O2 delivery in the skeletal muscle cells to quantify the functional proficiency of Mb mutants under various physiological conditions. We find that O2-storage and –transport are distinct functions which depend on O2 partial pressure and conclude that conserved residues in wild type (WT) Mb were fixated under a selection pressure of low ܲைమ . Second, we present an integrated model of convective O2-transport and O2-affinity of mutant Mb to quantify the impacts of mutations in Mb on the aerobic dive limits (ADL) of Weddell seals (Leptonychotes weddellii). We show that wild-type Mb traits are only superior under specific physiological conditions that critically prolong the ADL, action radius, and fitness of the seals. Third, we deal with the observation of higher folding stabilities (i.e., ΔGfolding) of cetacean Mbs compared to their terrestrial counterparts. Using ancestral sequence reconstruction, maximum likelihood and Bayesian tests to describe the evolution of cetacean Mbs, and experimentally calibrated computation of stability effects of mutations (i.e., ΔΔGfolding), we observe accelerated evolution in cetaceans and identify seven positively selected sites in Mb. We show that these sites contribute to Mb stabilization by favoring hydrophobic folding, structural integrity, and intra-helical hydrogen bonds. Finally, we ask a fundamental question that how a general protein phenotype such as folding stability, that was shown as an example to be positively selected in cetacean Mbs in the iv third part of this thesis, affects the rate of protein evolution. Using a model that combines explicit evolution of Mb sequences, folding stability, and application of maximum likelihood (ML) estimation of evolution rate (ER), we find that ER predicted by ML methods is highly correlated with ER from simulations using the explicit sequence information by counting the number of synonymous and nonsynonymous mutations fixed in the population. We show that this agreement is strongest in the regime of high stability where proteins are mostly evolving neutrally. In the unstable regime where protein evolution is dominated by selection for stabilizing mutations we detect a weak yet significant positive selection for specific residues in the sequences from simulations of the order of dN/dS ~ 1.5. Overall, this thesis provides one of the first examples in the study of evolution of function and thermodynamic stability in a mammalian protein with implications for fitness. We quantify and highlight the biological relevance for the selection of a higher concentration of Mb in the skeletal muscle of marine mammals and provide an explanation for the increase in folding stability of Mb. Moreover, this thesis provides number of theoretical findings directly testable with future experimental studies regarding the function, physiology and thermodynamic stability of mammalian Mbs.