A naturally occurring protective system in urea-rich cells: mechanism of osmolyte protection of proteins against urea denaturation.
Trimethylamine N-oxide (TMAO) is a solute concentrated in the urea-rich cells of elasmobranchs and coelacanth to offset the damaging effects of urea on intracellular protein structure and function. On the basis of transfer free energy measurements, favorable interaction of TMAO with amino acid side chains promote protein denaturation. This effect is more than offset by highly unfavorable TMAO-peptide backbone interactions that not only oppose denaturation but also provide stabilization against denaturation by urea. By combining transfer free energies of side chains and backbone with surface area exposure in the native and unfolded states of ribonuclease T1, the transfer free energies of native and unfolded protein from water to 1 M TMAO are estimated as 1.7 and 5.9 kcal/mol, respectively. These estimates agree favorably with the respective values of 1.2 and 5.4 kcal/mol determined experimentally by Lin and Timasheff [(1994) Biochemistry 33, 12695-12701]. The unfavorable transfer free energies of native and unfolded protein from water to TMAO provides a molecular level rationale for preferential hydration of proteins by osmolytes. Promotion of denaturation by urea is found to be offset by TMAO in a manner that is roughly additive of the combined effects of both solutes. The favorable interaction of urea with the backbone provides the dominant driving force for protein unfolding by this denaturant, and the unfavorable interaction of TMAO with backbone is the dominant force opposing urea denaturation. In solutions that contain significant organic solute concentration, the ascendance of the role of the peptide backbone over that of side chains can explain many observed effects in protein denaturation and stability induced by a variety of stabilizing and destabilizing organic solutes.
Wang
A
A
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77555-1052, USA.
Bolen
D W
DW
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D054659:Diketopiperazines; D018940:Elasmobranchii / Q000378:metabolism*; D008744:Methylamines / Q000378:metabolism; D016877:Oxidants / Q000378:metabolism; D010879:Piperazines / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D012995:Solubility; D013696:Temperature; D013816:Thermodynamics; D014508:Urea / Q000378:metabolism* / Q000494:pharmacology; D014882:Water-Electrolyte Balance
D000596:Amino Acids; D054659:Diketopiperazines; D008744:Methylamines; D016877:Oxidants; D010879:Piperazines; D014508:Urea; C005855:trimethyloxamine