High-intensity, "pink" beam from an undulator was used in conjunction with microfabricated rapid-fluid mixing devices to monitor the early events in protein folding with time resolved small angle x-ray scattering. This Letter describes recent work on the protein bovine beta-lactoglobulin where collapse from an expanded to a compact set of states was directly observed on the millisecond time scale. The role of chain collapse, one of the initial stages of protein folding, is not currently understood. The characterization of transient, compact states is vital in assessing the validity of theories and models of the folding process.

Bovine ␤-lactoglobulin (␤-lg) has been used extensively as a model for studying protein folding. One of the problems preventing clarification of the folding mechanism is the incomplete reversibility from the unfolded state, probably caused by the thiol-disulfide exchange between a free thiol at Cys-121 and two disulfide bonds. We constructed and expressed three ␤-lg subtype A mutants in which Cys-121 was replaced by Ala, Ser, or Val (i.e. C121A, C121S, and C121V). We studied the reversibilities of these mutants from urea denaturation using circular dichroism, tryptophan fluorescence, reversedphase and gel-filtration high performance liquid chromatographies, and SDS-PAGE. The folded structure of each mutant was similar to that of wild-type ␤-lg. Ureainduced unfolding at pH 7.0 and 3.0 showed that although the C121S mutation notably decreases the stability, the destabilizing effects of the C121A and C121V mutations are less severe. For all of the mutants, complete refolding from the unfolded state in 8 M urea at both pH 7.0 and 3.0 was observed. Kinetics of the formation of the irreversibly unfolded species of wild-type ␤-lg in 8 M urea at pH 7.0 indicated that, first, an intramolecular thiol-disulfide exchange occurs to produce a mixture of species with non-native disulfide bonds followed by the intermolecular thiol-disulfide exchange producing the oligomers. These results indicate that intramolecular and intermolecular thiol-disulfide exchange reactions cause the low reversibility of wild-type ␤-lg especially at neutral pH and that the mutation of Cys-121 improves the reversibility, enabling us to study the folding of ␤-lg more exactly under various conditions.

Glycosylated variants of beta-lactoglobulin (BLG) were produced in the methylotrophic yeast Pichia pastoris to mimic the glycosylation pattern of glycodelin, a homologue of BLG found in humans. Glycodelin has three sites for glycosylation, corresponding to amino acids 63-65 (S1), 85-87 (S2) and 28-30 (S3) of BLG. These three sites were engineered into BLG to produce the variants S2, S12 and S123, which carried one, two and three glycosylation sites, respectively. The oligosaccharides on these BLG variants ranged from (mannose)(9)(N-acetylglucosamine)(2) (Man(9)GN(2)) to Man(15)GN(2) and were of the alpha-linked high mannose type. The variant S123 exhibited highest levels of glycosylation, with the range of glycans being Man(9-14)GN(2). Digestion of S123 with alpha-1,2 linkage specific mannosidase resulted in a single product corresponding to Man(6)GN(2). These results indicated a glycosylation pattern consisting of a Man(5)GN(2) structure extended by 4-9 mannose residues attached mainly by alpha-1,2 linkages. The results also indicated extension of the Man(5)GN(2) structure by a single alpha-1,6-linked mannose. The N-linked glycosylation pathway in P.pastoris is significantly different from that in Saccharomyces cerevisiae, with the addition of shorter outer chains to the core and no alpha-1,3 outer extensions.

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