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A nuclear magnetic resonance (NMR) technique has been devised to probe the structures of disordered, partially folded states of proteins at the level of individual amino acid residues. Chemically induced dynamic nuclear polarization (CIDNP) is first generated in exposed aromatic side-chains of the denatured state and then transferred to the high-resolution NMR spectrum of the native state by stimulating rapid refolding of the protein. Crucial improvements in sensitivity were achieved by carrying out the polarization-producing photochemistry in a deoxygenated sample of the disordered state of the protein in a magnetic field of 4.0 T and recording the (1)H NMR spectrum of the refolded native state at 9.4 T (400 MHz). Application of this method to the low pH molten-globule state of alpha-lactalbumin reveals remarkably nativelike environments for the aromatic residues in the primary hydrophobic core of the protein. This result provides compelling evidence that the detailed fold of a protein can be established prior to the formation of the cooperative close-packed native structure.

Two new techniques offering considerable improvements in the quality of 1H photo-CIDNP spectra of proteins are demonstrated. Both focus on the problem of progressive photo-degradation of the flavin dye used to generate polarization in exposed tryptophan, tyrosine and histidine side-chains. One approach uses rapid addition and removal of protein/flavin solution between light flashes to mix the NMR sample and introduce fresh dye into the laser-irradiated region. The other involves chemical oxidation of photo-reduced flavin by the addition of hydrogen peroxide. In both cases a larger number of scans can be accumulated before the flavin is exhausted than would otherwise be possible. The techniques are demonstrated by 600 MHz CIDNP-NOESY spectroscopy of bovine holo-alpha-lactalbumin, and by real-time CIDNP observation of the refolding of bovine apo-alpha-lactalbumin following rapid dilution from a high concentration of chemical denaturant.

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We report the combined use of real-time photo-CIDNP NMR and stopped-flow fluorescence techniques to study the kinetic refolding of a set of mutants of a small globular protein, HPr, in which each of the four phenylalanine residues has in turn been replaced by a tryptophan residue. The results indicate that after refolding is initiated, the protein collapses around at least three, and possibly all four, of the side-chains of these residues, as (i) the observation of transient histidine photo-CIDNP signals during refolding of three of the mutants (F2W, F29W, and F48W) indicates a strong decrease in tryptophan accessibility to the flavin dye; (ii) iodide quenching experiments show that the quenching of the fluorescence of F48W is less efficient for the species formed during the dead-time of the stopped-flow experiment than for the fully native state; and (iii) kinetic fluorescence anisotropy measurements show that the tryptophan side-chain of F48W has lower mobility in the dead-time intermediate state than in both the fully denatured and fully native states. The hydrophobic collapse observed for HPr during the early stages of its folding appears to act primarily to bury hydrophobic residues. This process may be important in preventing the protein from aggregating prior to the acquisition of native-like structure in which hydrophobic residues are exposed in order to play their role in the function of the protein. The phenylalanine residue at position 48 is likely to be of particular interest in this regard as it is involved in the binding to enzymes I and II that mediates the transfer of a phosphoryl group between the two enzymes.

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