Common and unique strategies of myoglobin evolution for deep-sea adaptation of diving mammals

Abstract

Summary Myoglobin (Mb) is highly concentrated in the myocytes of diving mammals such as whales and seals, in comparison with land animals, and its molecular evolution has played a crucial role in their deep-sea adaptation. We previously resurrected ancestral whale Mbs and demonstrated the evolutional strategies for higher solubility under macromolecular crowding conditions. Pinnipeds, such as seals and sea lions, are also expert diving mammals with Mb-rich muscles. In the present study, we resurrected ancestral pinniped Mbs and investigated their biochemical and structural properties. Comparisons between pinniped and whale Mbs revealed the common and distinctive strategies for the deep-sea adaptation. The overall evolution processes, gaining precipitant tolerance and improving thermodynamic stability, were commonly observed. However, the strategies for improving the folding stability differed, and the pinniped Mbs exploited the shielding of hydrophobic surfaces more effectively than the whale Mbs.

Improvement of Protein Solubility in Macromolecular Crowding during Myoglobin Evolution

“…The O 2 -storage protein myoglobin (Mb) is known to be expressed in myocytes of diving mammals such as seals and whales at much higher concentrations than those of land animals, which contributes to their diving ability in oceans. − Mirceta et al (2013) indicated that diving animal Mbs have developed increased positive charges during evolution from their ancient land animal predecessors to adapt to the deep-sea environment, based on the prediction of amino acid sequences of their ancestral Mbs, and concluded that the higher Mb concentration in their muscle tissues is enabled by the electrostatic repulsion between Mb molecules. Dasmeh et al (2013) suggested that thermodynamic stabilities of whale Mbs had increased during the molecular evolution for deep sea adaptation, based on their theoretical prediction of folding stability from the 3D models of the ancestral Mbs. , We have synthesized ancient whale and seal Mbs and experimentally confirmed that both the positive surface charges and folding stability had been increased during evolution. , Furthermore, solubility tests were performed in the presence of PEG and indicated that the diving mammal Mbs had evolved increased tolerance against precipitant or macromolecular crowding, rather than increased solubility in a dilute buffer solution. However, chemical mechanisms of the precipitant tolerance remain unclear.…”

“…16,17 We have synthesized ancient whale and seal Mbs and experimentally confirmed that both the positive surface charges and folding stability had been increased during evolution. 18,19 Furthermore, solubility tests were performed in the presence of PEG and indicated that the diving mammal Mbs had evolved increased tolerance against precipitant or macromolecular crowding, rather than increased solubility in a dilute buffer solution. However, chemical mechanisms of the precipitant tolerance remain unclear.…”

“…As we and other researchers previously described, the Mb concentrations in diving mammal myocytes could be higher because of increased folding stability, as well as the solubility increase by the electrostatic repulsion between Mb molecules with their larger Z Mb . 16,18,19,31 The increases of stability and solubility seem to occur independently since solubility increased by residue replacements in the early phases of evolution before the stability increased by further replacements in the late phases for both whales and seals. 18,19 The solubility values on the vertical axes of Figure 1 correspond to the range of Mb concentration from 0.01 mg/mL or 0.58 μM (log S = −2) to 1000 mg/mL or 58 mM (log S = 3).…”

“…16,18,19,31 The increases of stability and solubility seem to occur independently since solubility increased by residue replacements in the early phases of evolution before the stability increased by further replacements in the late phases for both whales and seals. 18,19 The solubility values on the vertical axes of Figure 1 correspond to the range of Mb concentration from 0.01 mg/mL or 0.58 μM (log S = −2) to 1000 mg/mL or 58 mM (log S = 3). On the other hand, the in vivo Mb concentrations of land mammals were reported to be less than ∼7 mg/mL or ∼0.4 mM, and those of deep-diving mammals were found to be ∼60 mg/mL or 3−4 mM.…”

“…These hsMb data show that β has the minimal value at solution pH that is close to the p I value or Z Mb = 0 and increases with | Z Mb | at the both sides of the positive and negative Z Mb values. The β values for each Z Mb of the whale and seal Mbs including their ancestral proteins at neutral pH, , as well as those of swMb at acidic and basic pH, were also plotted with the relationship between β and Z Mb for hsMb (Figure A). These data for ancient and extant Mbs are roughly consistent with those of hsMb at various pH.…”