NMR supplement monomers to structured trimers( Fig. 2) POGPOGPOGITGARGLAGPOGPdGPOGPOG As well as enabling the mechanism of this process to be defined, these experi ments are providing key information bout the molecular basis of diseases associated with mutations in the 0.8 encoding the collagen sequence (J Baum, pers. comm. An attractive alternative presents itself for proteins whose folding can be initiat- ed photochemically. A nice example is N0.4 the very recent study by Kaptein and coworkers of photoactive yellow protein (PYP), the motile bacterium Ectothiorhodospil 60 Light excitation induces the trans-cis iso merization of the p-coumaric acid cofac Time(min tor, which triggers a cycle of structural hanges in PYP yielding an intermediate, pB, that reverts to the native state pG in I s. Characterization of pB by revealed that it exhibits extensive struc. al and d ontrast 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(H, 5N)HsQC spectra recorded at different times after a laser pulse. Considerable variation in the build-up rates was found, with more 2 NMR folding profiles of a peptide(top) labeled with N at Gly 24(circles)and Ala 13(squares) id recovery for the more disorganized hows the time dependence regions of the protein. The major exce he tik. Thesisapsiar ance of the monomer peak sohie ties and the ap peafanme of the trimer peaks tion to this was in the neighborhood d onfor matia tf rv z4 titeat w ith mechanism (bottom) ivolving intermediates in which the correlates with high degrees of disorder. the unfolded state. (Taken with permission from ref. the chromophore controls the refolding of that part of the molecule. Although less generally applicable than stopped actions(such as those limited has provided novel insights into both flow methods, rapid photochemical trig ed to isomerize peptide bonds unfolding and folding reactions-lo. The gering of refolding (for example, usi very slow. In order to monitor rapid NMR enables data collection to begin tial to allow monitoring of very rapid reactions, stopped flow procedures within 100 ms of mixing, and has processes. 14 involving rapid mixing within the NMR allowed, for example, distinct steps in In our laboratories we have focused on ample tube are being developed?. the folding of dihydrofolate reductase to the development of a variety of comple xperiments of this type have recently be resolved and characterized through mentary NMR methods aimed at begun to transform NMR into a general repetitive collection of spectra during describing at the atomic level the struc and powerful technique for studying a the folding process(Fig. 1)0. A similar tural and dynamic changes taking place ide range of fundamental events in strategy is of course possible using two- during the folding of a protein from its folding dimensional(2D) approaches if the denatured state. The ultimate objective is One of the obvious requirements in reactions under investigation are suffi- to map out by experiment the energy these studies is obtaining sufficient reso- ciently slow. Baum and colleagues have surface of the folding reaction!. This lution to be able to monitor events at the exploited this in an extremely elegant requires the ability to monitor the envi- werful approach has been to use F bly of peptide fragments of collagen. 2. folding(for example, whether they are IR to study proteins in which specific By labeling the peptides with isN it has buried or exp o the inter-residue interac residues (particularly aromatic ones) been possible to record 2D HSQC spec- ularly to define have been replaced by fluorinated tra at intervals of as little as four min- tions or contacts that develop at analogs. This strategy has been pio- utes, and to observe the transition different stages of folding. The latter can peered by Frieden and coworkers, and of these peptides from disordered in principle be studied directly if nuclear nature structural biology. NMR supplement. july 1998NMR supplement nature structural biology • NMR supplement • july 1998 505 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 reso￾lution 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 pio￾neered 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 two￾dimemnsional (2D) approaches if the reactions under investigation are suffi￾ciently slow. Baum and colleagues have exploited this in an extremely elegant manner to study the folding and assem￾bly of peptide fragments of collagen11,12. By labeling the peptides with 15N it has been possible to record 2D HSQC spec￾tra at intervals of as little as four min￾utes, and to observe the transition of these peptides from disordered monomers to structured trimers (Fig. 2). As well as enabling the mechanism of this process to be defined, these experi￾ments 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 initiat￾ed 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 iso￾merization of the p-coumaric acid cofac￾tor, 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 struc￾tural 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 excep￾tion 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 trig￾gering of refolding (for example, using nanosecond laser pulses) has the poten￾tial to allow monitoring of very rapid processes13,14. In our laboratories we have focused on the development of a variety of comple￾mentary NMR methods aimed at describing at the atomic level the struc￾tural 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 envi￾ronments of individual residues during folding (for example, whether they are buried or exposed to solvent) and partic￾ularly to define the inter-residue interac￾tions or ‘contacts’ that develop at different stages of folding. The latter can in principle be studied directly if nuclear 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 2nd 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)