Investigation of ribonuclease T1 folding intermediates by hydrogen-deuterium amide exchange-two-dimensional NMR spectroscopy.
The rate of hydrogen bond formation at individual amino acid residues in ribonuclease T1 (RNase T1) has been investigated by the hydrogen-deuterium exchange-2D NMR (HDEx-2D NMR) technique (Udgaonkar & Baldwin, 1988; Rder et al., 1988) to gain insight into the mechanism and pathways of protein folding. The HDEx-2D NMR technique combines rapid mixing and 2D NMR methods to follow the protection of backbone amide deuterons from exchange with solvent protons as a function of folding time. The technique depends on the difference in the exchange rates of hydrogen-bonded and non-hydrogen-bonded amide residues so that as the protein folds, the amide residues involved in hydrogen bonding are protected from exchange with solvent to give structural information about early folding events. The observed time course for deuterium protection was followed for 24 backbone amide residues that form stable hydrogen bonds in RNase T1. The time courses are biphasic with 60-80% of the protein molecules showing rapid hydrogen bond formation (12-119 s-1) in the alpha-helix and the beta-sheet. The remaining 20-40% of the molecules are protected in a slow phase with a rate constant that has a lower limit of 0.01 s-1. If the rate constants in this first phase are arbitrarily subdivided into two classes, fast (> or = 25 s-1) and intermediate (< 25 s-1), then the amide residues that are found in the hydrophobic core are in the fast class while those located on the periphery of the three-dimensional structure are in the intermediate class.(ABSTRACT TRUNCATED AT 250 WORDS)
Mullins
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Department of Chemistry, Texas A&M University, College Station 77843.
Pace
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Raushel
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D000577:Amides; D003903:Deuterium; D006859:Hydrogen; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D017510:Protein Folding; D006163:Ribonuclease T1 / Q000737:chemistry*
D000577:Amides; D006859:Hydrogen; D003903:Deuterium; D006163:Ribonuclease T1