6152
Biochemistry 1993, 32, 6 152-6 156
Investigation of Ribonuclease T 1 Folding Intermediates by Hydrogen-Deuterium Amide Exchange-Two-Dimensional NMR Spectroscopy+ Leisha S. Mullins, C. Nick Pace, and Frank M. Raushel' Departments of Chemistry and Medical Biochemistry and Genetics and Center for Macromolecular Design, Texas A&M University, College Station, Texas 77843 Received January 1 1 , 1993; Revised Manuscript Received April 6, 1993
ABSTRACT: The rate of hydrogen bond formation at individual amino acid residues in ribonuclease TI
(RNase T I ) has been investigated by the hydrogen-deuterium exchange2D N M R (HDEx-2D N M R ) technique (Udgaonkar & Baldwin, 1988; Rder et al., 1988) to gain insight into the mechanism and pathways of protein folding. The HDEx-2D N M R technique combines rapid mixing and 2D N M R 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-hydrogenbonded 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 6 0 4 0 % of the protein molecules showing rapid hydrogen bond formation (12-119 s-l) in the a-helix and the 8-sheet. The remaining 2 0 4 0 % of the molecules are protected in a slow phase with a rate constant that has a lower limit of 0.01 s-*. If the rate constants in this first phase are arbitrarily subdivided into two classes, fast ( 2 2 5 s-l) and intermediate (C25 s-l), 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. The HDEx-2D N M R results indicate that, in the early stages of folding, RNase TI folds on at least two parallel pathways, each having at least one intermediate. We propose that these two intermediates resemble a native-like intermediate and a "nonstructured" molten globule.
There is intense interest in the mechanism of folding of proteins both in vivo and in vitro. On the basis of recent studies, the mechanism of protein folding in vivo has become more complicated as it has become clearer that a number of other proteins are involved, but the mechanism of protein folding in vitro has become less complicated as some of the more complex models have been eliminated. Experimental results supportingat least three descriptionsof protein folding pathways in vitro have been presented (Baldwin, 1989,1990). These depictions include molten globules (Kuwajima, 1989), the framework model (Kim & Baldwin, 1982), and the subdomain model (Oas & Kim, 1988). The molten globule model postulates a compact intermediate with considerable formation of secondary structure. In the framework model, the initial formation of the secondary structural elements precedes subsequent folding events. The subdomain model proposes the initial formation of small domains of secondary and tertiary structure that later dock to form the nativeprotein structure. The three models are complementaryin that each predicts a small number of sequential pathways with a finite number of transiently formed intermediates. The integration of one or more of these models into a unified description of protein folding requires a more detailed structural characterization of protein folding intermediates. This has, however, been difficult to achieve experimentally due to the transient nature of these intermediates. This limitation has been overcome, in part, by the development of the hydrogendeuteriumexchange-2D NMR (HDEx-2D NMR) technique This work was supported in part by the Robert A. Welch Foundation (A-840 and A1060), the NIH (GM 37034), and the Texas Advanced Research Program. * Address correspondence to this author at the Department of Chemistry.
0006-2960/93/0432-6152$04.00/0
by Udgaonkar and Baldwin (1988) and Roder et al. (1988). The HDEx-2D NMR technique enables the structural elucidation of transiently formed folding intermediates and thus permits a more detailed structural characterization of protein folding pathways. This information can therefore provide valuable insight into the parameters governing secondary and tertiary structure formation during protein folding. The HDEx-2D NMR technique exploits the difference in the chemical exchange rates of hydrogen-bonded and nonhydrogen-bondedamide residues. The protein is unfolded in DzO to allow all amide protons to exchange with deuterium. At time to the denaturant is rapidly diluted below the critical denaturant concentration and the protein allowed to refold. After various times, t l , the refolding protein is exposed to an elevated pH HzO pulse for a time t 2 during which all nonhydrogen-bonded amide deuterons exchange for protons (Udgaonkar & Baldwin, 1988; Roder et al., 1988). The pH is then reduced to enable the protein to complete the folding process under conditions where all of the amide exchange reactions are quenched. The protein at this point has a mixture of hydrogen and deuterium labels whose occupancy can be determined by 2D J-correlated (COSY) NMR spectroscopy (Bax & Freeman, 1981) for the slowly exchanging amide sites of the fully folded protein. The folded and unfolded portions of the protein can therefore be differentiated as a function of the refolding time, tl, because only the portions of the protein which have folded during the period tl will contain the deuterium label.' The time course for deuterium protection thus provides a measure of the rate of hydrogen bond formation for each of these residues during the folding process. We have now investigated the rate of hydrogen bond formation at individual amino acid positions in ribonuclease 0 1993 American Chemical Society
Biochemistry, Vol. 32, No. 24, I993 6153
Folding Pathway for RNase T1 T1 (RNase T1) by the HDEx-2D NMR technique. RNase T1 is an excellent model for studies of the mechanism of protein folding. RNase T1 is a small compact globular protein of 104 amino acids with a single hydrophobic core formed between a 4.5-turn a-helix and an antiparallel @-sheet. RNase TI has four prolines, two of which are in the cis conformation (Pro-39 and Pro-55) and two of which are in the trans conformation (Pro-60 and Pro-71). The native conformation of RNase T I has two disulfide bonds forming a large loop (Cys-6 to Cys-103) and a small loop (Cys-2 to Cys- 10). The three-dimensional structure has been determined to 1.5-A resolution by X-ray crystallography (MartinezOyanedel et al., 1991) and the complete NMR spectrum assigned for the resonances observed at 500 MHz (Hoffmann & Ruterjans, 1988). The energetics (Pace, 1990) and the kinetics (Kiefhaber et al., 1990-a
