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 folding is the nascent chain as it forms on the ribosome. The process of protein folding continues in a crowded molecular environment, in the presence of a variety of helper molecules, the most famous of which are the molecular chaperones whose major functions include the control of protein aggregation. Many small proteins, however, will refold efficiently in dilute aqueous solutions following 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, however, make this task challenging. The first is that folding is usually fast; many small proteins fold in milliseconds or less, although others may take considerably longer. The second, and perhaps most significant, is that the initial state from which the folding reaction is initiated is extremely heterogeneous1. The ultimate starting point of folding is a random coil, and proteins in strong denaturants approach this rather closely2. In the random coil state there are more accessible conformations for a
Fig. 1 Stopped-flow 19F NMR spectra of the refolding of 6-19F-tryptophan labeled Escherichia coli dihydrofolate 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 wellresolved 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).
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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 spectroscopy 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 hydrogens4. Much has been learned through these approaches, but recently, increasing 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 reactions frequently take place over minutes or hours, and some specific types of
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NMR supplement
Fig. 2 NMR folding profiles of a peptide (top) labeled with 15N at Gly 24 (circles) and Ala 13 (squares). O is the one-letter amino acid code for hydroxyproline. The central panel shows the time dependence of the cross peaks in an (1H-15N) HSQC spectrum of the peptide as it folds to form a collagen-like triple helix. The disappearance of the monomer peaks (solid lines) and the appearance of the trimer peaks (dashed lines) are faster for Gly 24 than for Ala 13. Gly 24 and Ala 13 follow 2 nd and 1st order kinetics respectively. The data are consistent with a mechanism (bottom) involving intermediates in which the local conformation of Gly 24 towards the chain end is largely helical while the more central Ala 13 is still in the unfolded state. (Taken with permission from ref. 12).
folding reactions (such as those limited by the need to isomerize peptide bonds involving proline residues) can also be very slow. In order to monitor rapid reactions, stopped flow procedures involving rapid mixing within the NMR sample tube are being developed7,8. Experiments of this type have recently begun to transform NMR into a general and powerful technique for studying a wide range of fundamental events in folding. One of the obvious requirements in these studies is obtaining sufficient resolution to be able to monitor events at the level of single residues. One extremely powerful approach has been to use 19F NMR to study proteins in which specific residues (particularly aromatic ones) have been replaced by fluorinated analogs. This strategy has been pioneered by Frieden and coworkers, and
has provided novel insights into both unfolding and folding reactions8–10. The ability to use one-dimemnsional (1D) NMR enables data collection to begin within 100 ms of mixing, and has allowed, for example, distinct steps in the folding of dihydrofolate reductase to be resolved and characterized through repetitive collection of spectra during the folding process (Fig. 1)10. A similar strategy is of course possible using twodimemnsional (2D) approaches if the reactions under investigation are sufficiently slow. Baum and colleagues have exploited this in an extremely elegant manner to study the folding and assembly of peptide fragments of collagen11,12. By labeling the peptides with 15N it has been possible to record 2D HSQC spectra at intervals of as little as four minutes, and to observe the transition of these peptides from disordered
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monomers to structured trimers (Fig. 2). As well as enabling the mechanism of this process to be defined, these experiments are providing key information about the molecular basis of diseases associated with mutations in the gene encoding the collagen sequence (J. Baum, pers. comm.). An attractive alternative presents itself for proteins whose folding can be initiated photochemically. A nice example is the very recent study by Kaptein and coworkers of photoactive yellow protein (PYP), the proposed photosensor of the motile bacterium Ectothiorhodospira halophilia (R. Kaptein, pers. comm.). Light excitation induces the trans-cis isomerization of the p-coumaric acid cofactor, which triggers a cycle of structural changes in PYP yielding an intermediate, pB, that reverts to the native state pG in ~1 s. Characterization of pB by NMR revealed that it exhibits extensive structural and dynamic disorder, in strong contrast to pG. The conversion of pB to pG can therefore be considered to be a folding reaction. It was monitored in detail by observing the recovery of pG cross peaks in a series of (1H,15N) HSQC spectra recorded at different times after a laser pulse. Considerable variation in the build-up rates was found, with more rapid recovery for the more disorganized regions of the protein. The major exception to this was in the neighborhood of the chromophore, where slow refolding correlates with high degrees of disorder, suggesting that retro-isomerization of the chromophore controls the refolding of that part of the molecule. Although less generally applicable than stopped flow methods, rapid photochemical triggering of refolding (for example, using nanosecond laser pulses) has the potential to allow monitoring of very rapid processes13,14. In our laboratories we have focused on the development of a variety of complementary NMR methods aimed at describing at the atomic level the structural and dynamic changes taking place during the folding of a protein from its denatured state. The ultimate objective is to map out by experiment the ‘energy surface’ of the folding reaction1. This requires the ability to monitor the environments of individual residues during folding (for example, whether they are buried or exposed to solvent) and particularly to define the inter-residue interactions or ‘contacts’ that develop at different stages of folding. The latter can in principle be studied directly if nuclear 505
NMR supplement Overhauser effects (NOEs) can be detected between specific nuclei. It will be necessary of course to interpret these in terms of structural ensembles, as we have discussed above, and to obtain information about the dynamic events associated with the polypeptide chain as folding takes place. We have used the family of c-type lysozymes and their structural homologs, the α-lactalbumins, as test systems for many of these experiments because the folding of these proteins has been studied in detail using a wide variety of other biophysical methods1,15,16. In addition, it is possible to alter the folding kinetics of some members of this family by factors of ~100 simply by changing the Ca2+ concentration in the refolding buffer. This turns out to be an extremely valuable factor in devising NMR experiments to probe different aspects of the folding process. One approach we have adopted to extract structural information from 1D experiments is to exploit photo-CIDNP17 (photochemically induced dynamic nuclear polarization). This technique, in which photo-excitation of a dye molecule can result in enhanced nuclear polarization of tryptophan, tyrosine and histidine residues to which it has access18 has been used to probe the accessibility of these residues in both native and denatured states19. We have found that it can be particularly powerful in time-resolved experiments (Fig. 3). Because polarization is induced in only a small number of residues, the resulting spectra are relatively well resolved. The approach also has a shorter experimantal dead time than conventional NMR, firstly because the polarization is produced during a ~50 ms light flash, a somewhat faster process than the spin-lattice relaxation required to polarize spins transferred into the NMR probe from a lower field region of the magnet. Secondly, efficient mixing is only needed in the small portion of the sample exposed to the laser flash, from which the signal is detected17. This rapid mixing approach, coupled with more conventional 1D experiments20 has enabled probing of the disordered collapsed state, formed rapidly after the initiation of refolding of these proteins, and monitoring of the rearrangement processes that occur subsequently. We are presently engaged in attempts to increase significantly the sensitivity of this experiment, and to develop 2D variants. This task has been substantially aided by the recognition that it is not necessary to record sequential spectra to monitor 506
Fig. 3 1H photo-CIDNP spectra (B) of the refolding of hen lysozyme after a simultaneous pH jump from 1.1 to 5.2, and dilution from 10 to 1.4 M urea17. The delay times are the intervals between the end of the 30 ms injection of protein solution into the refolding buffer and the beginnning of the 50 ms light flash that generates photo-CIDNP. Each spectrum is the result of a single injection. The first spectrum, at 30 ms, differs markedly from that of the denatured state in 10 M urea (A) and suggests a rapidly formed disordered collapsed state which reorganizes within a second to generate the native state whose spectrum is shown as (C).
kinetic events21. It turns out that this information can be extracted from a single 2D spectrum recorded while the time-dependent process takes place. If a reaction occurs during the accumulation of data in the experiment, it perturbs the line shapes and intensities of the cross-peaks in the resulting 2D spectrum. Computer simulation and kinetic model-fitting of these spectral features gives residue-specific rate constants for the folding reaction. This approach has been used already to probe the cooperativity of the formation of native-like structure in bovine α-lactalbumin during folding using a (1H-15N) HSQC experiment21 (Fig. 4), and to probe the structure of a folding intermediate with a non-native proline isomer formed in the refolding of ribonuclease T1 (J. Ballach, pers. comm.). Although such experiments are transforming the possibilities for NMR in studying folding, the detection of NOEs in collapsed and partially folded states
remains a major challenge. In many cases the intrinsic problems of dealing with heterogeneous systems are compounded by the existence of extensive exchange broadening of resonances resulting from slow interconversion of conformers within compact but disordered systems. Nevertheless, we have been able to show that extensive NOEs do exist at the earliest detectable stages of the folding of α-lactalbumin, and their characteristics indicate that the molecules have native-like compactness22. In order to begin to identify the specific NOEs within such species we have made use of the fact mentioned above that Ca2+ can profoundly change the folding kinetics of the protein. The idea is to generate NOEs in the partially folded state, and then to refold the protein rapidly to its native state22. Provided this refolding can be done rapidly compared with the nuclear relaxation rates, it is possible to transfer the NOEs to the well resolved spectrum of the native state for detection. Initial attempts to implement
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NMR supplement a
c
b
Fig. 4 (1H-15N) HSQC spectra of bovine α-lactalbumin at 3 oC during different stages of the folding process. a, Poorly resolved spectrum of the denatured state (A-state) at pH 2.0 recorded before the initiation of refolding. b, Kinetic spectrum accumulated during folding (30 min). c, Well resolved spectrum of the native (N) state at pH 7.0 recorded after the refolding reaction. The insets show enlargements of the region containing the Val 92 resonance of the N-state. The lower intensity of this resonance in spectrum (b) compared to (c), and the negative features above and below the central peak contain information on the local rate of formation of native structure21.
this radio frequency pulse-labeling method indicate the viability of this approach, suggesting that further development of this strategy could result in the information required to characterize the protein folding process in detail22. We are also developing a closely related experiment in which accessible aromatic side chains in the partially folded state are labeled by photo-CIDNP and then identified in the native state after rapid refolding. Concluding remarks We have outlined briefly in this article some of the approaches that are being developed to exploit the potential of NMR in kinetic studies of protein folding. These experiments are in the early stages of development and there are many opportunities for major advances in the future, including novel methods of initiating the folding process, for example by using electrochemical or photochemical techniques13,14, or temperature jumps23, as well as innovations in NMR methodology. The information emerging from these experiments is highly complementary to that emerging from NMR studies of equilibrium folding intermediates24, including those that probe the stabilities of interactions by subjecting these species to progressive denaturation25. In addition to the real-time kinetic experiments described here, there are exciting
NMR approaches based on magnetization transfer and line shape analysis that are providing information about folding kinetics, including events taking place on microsecond time scales26. In combination with other experimental approaches and theoretical advances, NMR is likely to play an increasingly important role in the quest to understand the mechanisms by which proteins fold. Acknowledgments We thank J. Balbach, V. Forge, J. A. Jones, N. A. J. van Nuland and S. L. Winder, for many of the ideas that have gone into the work from our own laboratories and that is discussed here. We are grateful to J. Baum, H. B. Gray, R. Kaptein and J. Balbach for kindly providing reprints of unpublished work, and to C. Freiden and J. Baum for copies of Figs 1 and 2. The Oxford Centre for Molecular Sciences is supported by BBSRC, EPSRC and MRC. The research of C.M.D. is also supported by the Howard Hughes Medical Institute and the Wellcome Trust.
Christopher M. Dobson is at the Oxford Centre for Molecular Sciences, New Chemistry Laboratory and Peter J. Hore is at the Physical and Theoretical Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QR, UK. Correspondence should be addressed to C.M.D. email:
[email protected] or P.J.H. email:
[email protected]
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