A key paradigm in the biology of adaptation holds that urea affects protein function by increasing the fluctuations of the native state, while trimethylamine N-oxide (TMAO) affects function in the opposite direction by decreasing the normal fluctuations of the native ensemble. Using urea and TMAO separately and together, hydrogen exchange (HX) studies on RNase A at pH* 6.35 were used to investigate the basic tenets of the urea:TMAO paradigm. TMAO (1 M) alone decreases HX rate constants of a select number of sites exchanging from the native ensemble, and low urea alone increases the rate constants of some of the same sites. Addition of TMAO to urea solutions containing RNase A also suppresses HX rate constants. The data show that urea and TMAO independently or in combination affect the dynamics of the native ensemble in opposing ways. The results provide evidence in support of the counteraction aspect of the urea:TMAO paradigm linking structural dynamics with protein function in urea-rich organs and organisms. RNase A is so resistant to urea denaturation at pH* 6.35 that even in the presence of 4.8 M urea, the native ensemble accounts for >99.5% of the protein. An essential test, devised to determine the HX mechanism of exchangeable protons, shows that over the 0-4.8 M urea concentration range nearly 80% of all observed sites convert from EX2 to EX1. The slow exchange sites are all EX1; they do not exhibit global exchange even at urea concentrations (5.8 M) well into the denaturation transition zone, and their energetically distinct activated complexes leading to exchange gives evidence of residual structure. Under these experimental conditions, the use of DeltaG(HX) as a basis for HX analysis of RNase A urea denaturation is invalid. Study holds ProTherm entries: 14306, 14307, 14308, 14309 Extra Details: 0.1M imidazole was added in the experiment fluctuations; rate constants; structural dynamics;,protein function
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:45 p.m.
|Number of data points||8|
|Proteins||Ribonuclease pancreatic ; Ribonuclease pancreatic|
|Assays/Quantities/Protocols||Experimental Assay: Cm pH:7.05 ; Experimental Assay: m pH:7.05 ; Experimental Assay: dG_H2O pH:7.05 ; Experimental Assay: Cm pH:6.35 ; Experimental Assay: m pH:6.35 ; Experimental Assay: dG_H2O pH:6.35|
|Libraries||Mutations for sequence KETAAAKFERQHMDSSTSAASSSNYCNQMMKSRNLTKDRCKPVNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCAYKTTQANKHIIVACEGNPYVPVHFDASV|