The formation of hydrogen-bonded structure in the folding reaction of ubiquitin, a small cytoplasmic protein with an extended (3-sheet and an a-helix surrounding a pronounced hydrophobic core, has been investigated by hydrogendeuterium exchange labeling in conjunction with rapid mixing methods and two-dimensional NMR analysis. The time course of protection from exchange has been measured for 26 backbone amide protons that form stable hydrogen bonds upon refolding and exchange slowly under native conditions. Amide protons in the (3-sheet and the a-helix, as well as protons involved in hydrogen bonds at the helix/sheet interface, become 80% protected in an initial 8-ms folding phase, indicating that the two elements of secondary structure form and associate in a common cooperative folding event. Somewhat slower protection rates for residues 59, 61, and 69 provide evidence for the subsequent stabilization of a surface loop. Most probes also exhibit two minor phases with time constants of about 100 ms and 10 s. Only two of the observed residues, Gln-41 and Arg-42, display significant slow folding phases, with amplitudes of 37% and 22%, respectively, which can be attributed to native-like folding intermediates containing cis peptide bonds for Pro-37 and/or Pro-38. Compared with other proteins studied by pulse labeling, including cytochrome c, ribonuclease, and barnase, the initial formation of hydrogen-bonded structure in ubiquitin occurs at a more rapid rate and slowfolding species are less prominent.A central goal of experimental protein folding studies is to obtain structural and kinetic information on intermediate states between the disordered polypeptide chain of a denatured protein and the folded structure of a native protein. The existence of protein folding pathways with well-defined intermediates is now widely accepted (1, 2), but we know little about the nature of folding intermediates in precise structural terms. Because of the cooperative nature of foldingunfolding transitions, partially folded forms are rarely observed at equilibrium, and kinetic intermediates tend to be too short-lived for the direct application of high-resolution structural tools such as two-dimensional NMR.Our approach to this problem makes use of pulsed hydrogen-deuterium (H-D) exchange in conjunction with rapid mixing methods and two-dimensional NMR analysis (3, 4). In a typical pulse-labeling experiment, the protein is initially unfolded in a D20 denaturant solution so that the labile backbone amide protons (NH) are replaced by deuterons (ND). Refolding, initiated by rapid dilution of the denaturant, is allowed to proceed in D20 (or in H20 at mildly acidic pH, where NH exchange is slow) for various times before the partially refolded protein is briefly exposed to H20 at basic pH. Amide hydrogens that are still exposed at this time become protonated during the labeling pulse, while those that are already protected from exchange (e.g., by forming hydrogen bonds) remain deuterated. After the labeling pulse, the protein is allo...

Ubiquitin adopts a non-native folded structure in 60% methanol solution at low pH. Two-dimensional nuclear magnetic resonance (2D NMR) was used to measure the hydrogen-exchange rates of backbone amide protons of ubiquitin in both native and methanol forms, and to characterize the structure of ubiquitin in the methanol state. Protection factors (the ratios of experimentally determined exchange rates to the rates calculated for an unfolded polypeptide) for protons in the native form of ubiquitin range from less than 10 to greater than 10(5). Most of the protons that are protected from exchange are located in regions of hydrogen-bonded secondary structure. The most strongly protected backbone amide protons are those of residues comprising the hydrophobic core. Hydrogen exchange from ubiquitin in methanol solution was too rapid to measure directly by 2D NMR, so a labeling scheme was employed, in which exchange with solvent occurred while the protein was in methanol solution. Exchange was quenched by dilution with aqueous buffer after the desired labeling time, and proton occupancies were measured by 1H NMR of the native form of the protein. Protection factors for protons in the methanol form of ubiquitin range from 2.6 to 42, with all protected protons located in hydrogen-bonded structure in the native form. Again, the most strongly protected protons are those of residues in the hydrophobic core. Comparison of the patterns of the hydrogen-exchange rates in the native and methanol forms indicates that almost all of the native secondary structure persists in the methanol form, but that it is almost uniformly destabilized by 4-6 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)

Despite the requirement for a functional signal sequence in protein export, little is known of the conformational properties and membrane interactions of these highly hydrophobic amino terminal extensions on nearly all exported proteins. The Escherichia coli lambda phage receptor signal sequence was studied in phospholipid monolayers by circular dichroism and Fourier transform infrared spectroscopy; the signal peptide was shown to prefer an alpha-helical conformation when inserted into the lipid phase. However, interaction with the lipid surface without insertion induced the signal sequence, which is unstructured in bulk aqueous solution, to adopt a beta structure. These observations are combined in a model for the initial steps in signal sequence-membrane interaction in vivo.

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Wild-type and pseudorevertant signal peptides of the lamB gene product of Escherichia coli interact with lipid systems whereas a nonfunctional deletion mutant signal peptide does not. This conclusion is based on interaction of synthetic signal peptides with a lipid monolayer-water surface, conformational changes induced by presence of lipid vesicles in an aqueous solution of signal peptide, and capacities of the peptides to promote vesicle aggregation. Analysis of the signal sequences and previous conformational studies suggest that these lipid interaction properties may be attributable to the tendency of the functional signal peptides to adopt alpha-helical conformations. Although the possibility of direct interaction between the signal peptide and membrane lipids during protein secretion is controversial, the results suggest that conformationally related amphiphilicity and consequent membrane affinity of signal sequences are important for function in vivo.