Molecular evolution and structure-function relationships of myoglobin in diving mammals

• Main Author: Mirceta, Scott Jon
• Published: University of Liverpool 2011
• Subjects: 599.14
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.569549

Remarkable feats of breath-hold endurance are observed in diving mammals, with some species routinely diving for an hour. During most mammalian dives metabolism remains aerobic in nature, which is accomplished by restricting the blood flow to parts of the body through peripheral vaso-constriction and bradycardia. This mechanism preserves essential oxygen (02) for heart and brain function, but also means some parts of the body, including locomotory muscles, become isolated and have to rely on O2 stored within the tissues. Due to the isolation of skeletal muscles, mammalian divers must be able to buffer large quantities of H+ ions due to the production of CO2 during aerobic metabolism and acidic end products of anaerobic metabolism once muscle O2 stores have been consumed. The protein responsible for storing molecular 02 is myoglobin (Mb), a small 17 kDa monomeric globular haemoprotein with the primary function of reversible O2 binding and facilitated diffusion of O2 to the mitochondria. A hallmark of mammalian divers is increased Mb concentrations ([Mb]. with divers exhibiting concentrations up to thirty times those seen in non-diving species. Previous research has found that proteins at high concentrations are prone to form aggregations leading to non-functioning protein. This raises the question of Mb solubility at such high concentrations as observed in mammalian divers. The central hypothesis of this thesis is that mammalian myoglobin has undergone previously unrecognised, parallel and adaptive evolution in several lineages of mammalian divers that has profoundly increased their maximal physiological diving capacity. To test the hypothesis, Mb amino acid sequences of 124 mammals, including 24 newly determined sequences, are analysed for the content and individual buffering properties of their ionisable amino acids. This is used to calculate the specific Mb buffer value (~Mb) for each species, which is experimentally verified by acid-base titration of purified Mb. Together with known [Mb], the contribution of Mb to whole muscle buffer capacity WmuscleMb) is then quantified. Amino acid sequences are assessed for substitutions that increase modelled net Mb charge in mammalian divers compared to terrestrial species and predictions are confirmed by measuring electrophoretic mobility of purified Mb. Observed changes in Mb buffer properties and net surface charge are mapped on a composite mammalian phylogeny to test whether they are significantly linked to the evolution of diving behaviour. Observed molecular changes in Mb amino acid sequence are integrated with diving capacity, producing a model that allows prediction of maximal dive duration from Mb amino acid sequence and body mass. Using ancestral Mb sequence reconstructions and body mass estimates, the model is applied to infer the evolution of maximal diving capacity in the cetacean lineage. Results suggest a general trend towards increasing ~Mb due to increased Mb histidine content in mammalian divers.βmuscleMb is significantly higher in divers compared to terrestrial species, and can account for up to 45%, of the increase in whole muscle buffering observed in diving mammals. This study shows a remarkable trend in all diving species to significantly increase the net charge of the Mb protein, which would convey increased Mb solubility. This is supported by a significant correlation between Mb net charge and maximal [Mb]. Evolutionary analysis shows that high Mb net charge is significantly linked to the occurrence of diving. Contrary to previous findings, the model developed here finds that increases in [Mb] convey greater increases in dive duration than similar increases in body mass. This study provides novel in sights into how cumulative substitutions on the molecular surface of Mb can have profound adaptive effects on the physiological properties conveyed to the whole animal. It suggests that adaptation to diving in multiple lineages of mammals involved not only evolution of increased expression levels of Mb, but also substantial qualitative changes to the protein to avoid aggregation and increase solubility and buffering.