Hydrogen exchange in native and alcohol forms of ubiquitin.
Ubiquitin adopts a non-native folded structure in 60% methanol solution at low pH. Two-dimensional nuclear magnetic resonance (2D NMR) was used to measure the hydrogen-exchange rates of backbone amide protons of ubiquitin in both native and methanol forms, and to characterize the structure of ubiquitin in the methanol state. Protection factors (the ratios of experimentally determined exchange rates to the rates calculated for an unfolded polypeptide) for protons in the native form of ubiquitin range from less than 10 to greater than 10(5). Most of the protons that are protected from exchange are located in regions of hydrogen-bonded secondary structure. The most strongly protected backbone amide protons are those of residues comprising the hydrophobic core. Hydrogen exchange from ubiquitin in methanol solution was too rapid to measure directly by 2D NMR, so a labeling scheme was employed, in which exchange with solvent occurred while the protein was in methanol solution. Exchange was quenched by dilution with aqueous buffer after the desired labeling time, and proton occupancies were measured by 1H NMR of the native form of the protein. Protection factors for protons in the methanol form of ubiquitin range from 2.6 to 42, with all protected protons located in hydrogen-bonded structure in the native form. Again, the most strongly protected protons are those of residues in the hydrophobic core. Comparison of the patterns of the hydrogen-exchange rates in the native and methanol forms indicates that almost all of the native secondary structure persists in the methanol form, but that it is almost uniformly destabilized by 4-6 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)
Pan
Y
Y
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta 30332-0400.
Briggs
M S
MS
D006859:Hydrogen / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D000432:Methanol / Q000737:chemistry*; D017510:Protein Folding; D011522:Protons; D014452:Ubiquitins / Q000737:chemistry*
D011522:Protons; D014452:Ubiquitins; D006859:Hydrogen; D000432:Methanol