Pulsed hydrogen exchange/mass spectrometry, a new method for studying protein folding, has been used to investigate folding of cytochrome c on the 5 ms to 15 s time scale. Cytochrome c, unfolded in guanidine hydrochloride/D2O, was allowed to refold in a high-speed quenched-flow apparatus and pulse-labeled with protium to identify unfolded regions. Intact, labeled cytochrome c was digested into fragments which were analyzed by HPLC electrospray ionization mass spectrometry to determine the level of deuterium in each fragment. Bimodal distributions of deuterium were found for most segments, indicating that regions represented by these segments were either unfolded or completely folded in the intact polypeptide prior to labeling. This behavior is consistent with cooperative, localized folding which occurs in less than 10 ms in individual molecules. Deuterium levels found in the fragments were normalized to levels found in the same fragments derived from folded cytochrome c, pulse-labeled in the same manner, to indicate the percentage of cytochrome c that was folded. These results show that the N/C-terminal regions fold cooperatively on a time scale extending from less than the mixing time of the apparatus (5 ms) to as long as 15 s, and that the other regions also fold cooperatively. However, these regions do not begin to fold until 30 ms after mixing. In addition to providing new information on cytochrome c folding, these results demonstrate that pulse-hydrogen exchange/mass spectrometry is complementary to NMR in some respects and advantageous in others. Results of this study form the foundation required to extend the pulsed hydrogen exchange approach to folding studies of proteins too large to be analyzed by NMR.

The authors describe the discovery of a new class of inhibitors to an essential Streptococcus pneumoniae cell wall biosyn-thesis enzyme, MurF, by a novel affinity screening method. The strategy involved screening very large mixtures of diverse small organic molecules against the protein target on the basis of equilibrium binding, followed by iterative ultrafiltration steps and ligand identification by mass spectrometry. Hits from any affinity-based screening method often can be relatively nonselective ligands, sometimes referred to as "nuisance" or "promiscuous" compounds. Ligands selective in their binding affinity for the MurF target were readily identified through electronic subtraction of an empirically determined subset of promiscuous compounds in the library without subsequent selectivity panels. The complete strategy for discovery and identification of novel specific ligands can be applied to all soluble protein targets and a wide variety of ligand libraries.

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Inactivation of pancreatic RNAase A occurs in guanidinium chloride (GdmCl) at low concentrations before the unfolding of the molecule as a whole can be detected [Liu and Tsou (1987) Biochim. Biophys. Acta 916, 455-464]. We have now shown that the rate of digestion of the RNAase molecule by either trypsin or proteinase K increases significantly at low concentrations of GdmCl where the enzyme is largely inactivated, but fluorescence and absorption measurements reveal no conformational changes. N-Terminal sequence analysis of the peptide fragments generated shows that proteolysis occurs primarily at or near the active site. The decrease in activity of RNAase at low concentrations of GdmCl is therefore due to partial unfolding of the molecule, particularly at the active site and not to an inhibition by the denaturant.

It was found that in the chemical ionization (isobutane) mass spectra of some asymmetric secondary alcohols and a-amino acids, when a pair of enantiomers (such as R-and S-Zphenylbutyric anhydride, R-and S-mandelic acid, R-and S-Zmethylbutanoic acid or R-and Sa-phenyl ethyl amine) were used as reaction reagents, the relative abundances of characteristic ions formed by the stereoselective reaction between sample and reagent of the same configuration were much higher than those ions formed by the sample and a reagent of a different configuration. The absolute configuration of the sample molecule may be predicted by examination of mass spectra of the sample measured with R-and Sreagent respectively. This approach proved to be a convenient way for determination of the absolute configurations of organic molecules on a micromole level by mass spectrometry.