NMR supplement Kinetic studies of protein folding using NMR spectroscopy Christopher M. Dobson and Peter J Hore Recent progress has advanced our abilities to use NMR spectroscopy to follow- in real time -the structural and dynamic changes taking place during protein folding In a cell, the starting point of protein rant). This allows the use of biophysical polypeptide than there are molecules in on the ribosome. The process of protein in real time. Two aspects of folding, how- of folding can involve extremely diverse folding continues in a crowded molecu- ever, make this task challenging. The structural ensembles until the very last lar environment, in the presence of a first is that folding is usually fast; many stages of the reaction. This complicates ariety of helper molecules, the most small proteins fold in milliseconds or substantially the analysis of the results of famous of which are the molecular chap- less, although others may take consider- structural studies erones whose major functions include ably longer. The second, and perhaps In order to combat these problems, the control of protein aggregation. Many most significant, is that the initial state one approach has been to utilize a wide small proteins, however, will refold effi- from which the folding reaction is initi- range of spectroscopic techniques, each ciently in dilute aqueous solutions fol- ated is extremely heterogeneous!. The able to monitor the formation of specific lowing transfer from a denaturing ultimate starting point of folding is a aspects of protein structure, in stopped environment(such as 6 M guanidinium random coil, and proteins in strong and quenched flow mode 3. NMR spe chloride) into one where the native state denaturants approach this rather close- troscopy has played a significant role is thermodynamically stable (such as ly?. In the random coil state there are here through its ability to analyze the hat produced by dilution of the denatu- more accessible conformations for a distributions of hydrogen and deuterium in labile sites in proteins, and through pulse labeling to follow in a site-specific manner the formation of structure that Native resonances example as a result of the formation of hydrogen bonds involving amide hydro gens. Much has been learned through to use nmr directly to follow folding In principle such experiments could allow he detailed analysis of the structural ensembles populated at different stages ng reaction, a fundamentally the level of detail in which we are able to define the folding The use of NMR to study reactions of time(sec)1.5 A proteins in real time' started nearly 30 years ago with the objective of studying enzymatic mechanisms. 6. Not long afterwards efforts were made to stud 30& Unfolded resonances protein folding and unfolding, and a variety of experimental strategies were developed for this purpose(reviewed in Fig. 1 Stopped-flow F NMR spectra of the refolding of 6.19F-tryptophan labeled Escherichia coli dihy. ref. 7). Particular emphasis was placed on slow reactions to overcome the kinetics and chemical shifts suggest the formation of an intermediate that is unable to bind short periods of time. Unfolding reac- and 133, and little if any native side chain environment around tryptophans 22 and 74. The resonance tions frequently take place over minutes beled 47i is that of Trp 47 in the intermediate. (Taken from ref. 10 with permission) or hours, and some specific types of nature structural biology . NMR supplement. july 1998NMR supplement 504 nature structural biology • NMR supplement • july 1998 In a cell, the starting point of protein folding is the nascent chain as it forms on the ribosome. The process of protein folding continues in a crowded molecu￾lar environment, in the presence of a variety of helper molecules, the most famous of which are the molecular chap￾erones whose major functions include the control of protein aggregation. Many small proteins, however, will refold effi￾ciently in dilute aqueous solutions fol￾lowing transfer from a denaturing environment (such as 6 M guanidinium chloride) into one where the native state is thermodynamically stable (such as that produced by dilution of the denatu￾rant). This allows the use of biophysical techniques to follow the folding process in real time. Two aspects of folding, how￾ever, make this task challenging. The first is that folding is usually fast; many small proteins fold in milliseconds or less, although others may take consider￾ably longer. The second, and perhaps most significant, is that the initial state from which the folding reaction is initi￾ated is extremely heterogeneous1. The ultimate starting point of folding is a random coil, and proteins in strong denaturants approach this rather close￾ly2. In the random coil state there are more accessible conformations for a polypeptide than there are molecules in the test tube. This means that the process of folding can involve extremely diverse structural ensembles until the very last stages of the reaction. This complicates substantially the analysis of the results of structural studies. In order to combat these problems, one approach has been to utilize a wide range of spectroscopic techniques, each able to monitor the formation of specific aspects of protein structure, in stopped and quenched flow mode3. NMR spec￾troscopy has played a significant role here through its ability to analyze the distributions of hydrogen and deuterium in labile sites in proteins, and through ‘pulse labeling’ to follow in a site-specific manner the formation of structure that protects against solvent exchange, for example as a result of the formation of hydrogen bonds involving amide hydro￾gens4. Much has been learned through these approaches, but recently, increas￾ing progress has been made on strategies to use NMR directly to follow folding. In principle such experiments could allow the detailed analysis of the structural ensembles populated at different stages of the folding reaction, and transform fundamentally the level of detail in which we are able to define the folding process. Kinetic NMR approaches The use of NMR to study reactions of proteins in ‘real time’ started nearly 30 years ago with the objective of studying enzymatic mechanisms5,6. Not long afterwards efforts were made to study protein folding and unfolding, and a variety of experimental strategies were developed for this purpose (reviewed in ref. 7). Particular emphasis was placed on slow reactions to overcome the intrinsic difficulties in accumulating NMR spectra of adequate quality in short periods of time. Unfolding reac￾tions frequently take place over minutes or hours, and some specific types of Kinetic studies of protein folding using NMR spectroscopy Christopher M. Dobson and Peter J. Hore Recent progress has advanced our abilities to use NMR spectroscopy to follow — in real time — the structural and dynamic changes taking place during protein folding. Fig. 1 Stopped-flow 19F NMR spectra of the refolding of 6-19F-tryptophan labeled Escherichia coli dihy￾drofolate reductase following dilution from 5.5 to 2.75 M urea at 5 °C in the presence of 3.8 mM NADP+. The disappearance of the five resonances of the unfolded state, clustered between -46.0 and -46.6 p.p.m., and the growth of the more widely dispersed native peaks are clearly seen in this well￾resolved set of spectra. Each spectrum represents the sum of 41 separate rapid dilution experiments. The kinetics and chemical shifts suggest the formation of an intermediate that is unable to bind NADP+ strongly, having a native-like side chain environment in the regions around tryptophans 30, 47 and 133, and little if any native side chain environment around tryptophans 22 and 74. The resonance labeled 47i is that of Trp 47 in the intermediate. (Taken from ref. 10 with permission)