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 information about the dynamic ssociated with the polypeptide c出 B We have used the family of c-type mm1500 lysozymes and their structural homologs, the a-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 methods. 15, 16. In addi it is possible to alter the folding kinet ics 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 different aspects of the folding rocess. One approach we have adopted to extract structural information from ID experiments is to exploit photo-CIDNPI7 photochemically induced dynamic nuclear polarization). This technique, in which photo-excitation of a dye molecule rTTT can result in enhanced nuclear polariza- tion of tryptophan, tyrosine and histidine residues to which it has accessis has been δ/ppm used to probe the accessibility of these Fig 3"H photo CIDNP spectra(B)of the refolding of hen lysozyme after a simultaneous pH jump states. We have found that it can be par- end of the 30 ms injection of protein solution into the refolding buffer and the beginnning of t ticularly powerful in time-resolved exper- 50ms light flash that generates photo-CIDNP. Each spectrum is the result of a single injection. Th iments(Fig. 3). Because polarization is suggests a rapidly formed disordered collapsed state which reorganizes within a second to gener. induced in only a small number of ate the native state whose spectrum is shown as(c) residues, the resulting spectra are relative ly well resolved. The approach also has a shorter experimantal dead time than con- kinetic events. It turns out that this infor- remains a major challenge. In many cases ventional NMR, firstly because the polar- mation can be extracted from a single 2d the intrinsic problems of dealing with het ization is produced during a-50 ms light spectrum recorded while the time-depen- erogeneous systems are compounded by flash, a somewhat faster process than the dent process takes place. If a reaction the existence of extensive exchange broad spin-lattice relaxation required to polar- occurs during the accumulation of data in ening of resonances resulting from slow ize spins transferred into the NMR probe the experiment, it perturbs the line shapes interconversion of conformers within com- from a lower field region of the magnet. and intensities of the cross-peaks in the pact but disordered systems. Nevertheless, Secondly, efficient mixing is only needed resulting 2D spectrum. Computer simula- we have been able to show that extensive in the small portion of the sample tion and kinetic model-fitting of these NOEs do exist at the earliest detectable exposed to the laser flash, from which the spectral features gives residue-specific rate stages of the folding of a-lactalbumin, and nal is detected i? onstants for the folding reaction. This their characteristics indicate that the mole- This rapid mixing approach, coupled approach has been used already to probe cules have native-like compactness 22. In with more conventional ID experiments the cooperativity of the formation of order to begin to identify the specific NOEs has enabled probing of the disordered col- native-like structure in bovine a-lactalbu- within such species we have made use of psed state, formed rapidly after the initi- min during folding using a('H-I5N) the fact mentioned above that Ca2+can ation of refolding of these proteins, and HSQC experiment(Fig 4), and to probe profoundly change the folding kinetics of monitoring of the rearrangement process- the structure of a folding intermediate the protein. The idea is to generate NOEs in es that occur subsequently. We are present- with a non-native proline isomer formed the partially folded state, and then to refold ly engaged in attempts to increase in the refolding of ribonuclease Tl (. the protein rapidly to its native state2z significantly the sensitivity of this experi- Ballach, pers. comm) Provided this refolding can be done rapidly ment, and to develop 2D variants. This Although such experiments ar d with the nuclear relaxation rates task has been substantially aided by the forming the possibilities for NMR it is possible to transfer the NoEs to the recognition that it is not necessary to ing folding, the detection of ell resolved spectrum of the native state record sequential spectra to monitor collapsed and partially folded for detection. Initial attempts to implement nature structural biology . NMR supplement. july 1998NMR supplement 506 nature structural biology • NMR supplement • july 1998 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 addi￾tion, it is possible to alter the folding kinet￾ics 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 polariza￾tion 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 par￾ticularly powerful in time-resolved exper￾iments (Fig. 3). Because polarization is induced in only a small number of residues, the resulting spectra are relative￾ly well resolved. The approach also has a shorter experimantal dead time than con￾ventional NMR, firstly because the polar￾ization is produced during a ~50 ms light flash, a somewhat faster process than the spin-lattice relaxation required to polar￾ize 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 col￾lapsed state, formed rapidly after the initi￾ation of refolding of these proteins, and monitoring of the rearrangement process￾es that occur subsequently. We are present￾ly engaged in attempts to increase significantly the sensitivity of this experi￾ment, 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 kinetic events21. It turns out that this infor￾mation can be extracted from a single 2D spectrum recorded while the time-depen￾dent 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 simula￾tion 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-lactalbu￾min 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 trans￾forming the possibilities for NMR in study￾ing folding, the detection of NOEs in collapsed and partially folded states remains a major challenge. In many cases the intrinsic problems of dealing with het￾erogeneous systems are compounded by the existence of extensive exchange broad￾ening of resonances resulting from slow interconversion of conformers within com￾pact 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 mole￾cules 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 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 gener￾ate the native state whose spectrum is shown as (C)