Aggregation of partially folded intermediates populated during protein folding processes has been described for many proteins. Likewise, partially unfolded chains, generated by perturbation of numerous proteins by heat or chemical denaturants, have also been shown to aggregate readily. However, the process of protein aggregation from native-state conditions is less well understood. Granulocyte-colony stimulating factor (G-CSF), a member of the four-helix bundle class of cytokines, is a therapeutically relevant protein involved in stimulating the growth and maturation of phagocytotic white blood cells. Under native-like conditions (37 C [pH 7.0]), G-CSF shows a significant propensity to aggregate. Our data suggest that under these conditions, native G-CSF exists in equilibrium with an altered conformation, which is highly aggregation prone. This species is enriched in 1-2 M GdmCl, as determined by tryptophan fluorescence and increased aggregation kinetics. In particular, specific changes in Trp58 fluorescence report a local rearrangement in the large loop region between helices A and B. However, circular dichroism, reactivity toward cyanylation, and ANS binding demonstrate that this conformational change is subtle, having no substantial disruption of secondary and tertiary structure, reactivity of the free sulfhydryl at Cys17 or exposure of buried hydrophobic regions. There is no indication that this altered conformation is important to biological activity, making it an attractive target for rational protein stabilization.

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ABSTRACT.Trumenba (bivalent rLP2086) is a vaccine licensed for the prevention of meningococcal meningitis disease caused by Neisseria meningitidis serogroup B (NmB) in individuals 10-25 years of age in the USA. The vaccine is composed of two factor H binding protein (fHbp) variants that were recombinantly expressed in Escherichia coli as native lipoproteins: rLP2086-A05 and rLP2086-B01. The vaccine was shown to induce potent bactericidal antibodies against a broad range of NmB isolates expressing fHbp that were different in sequence from the fHbp vaccine antigens. Here, we describe the characterization of the vaccine antigens including the elucidation of their structure which is characterized by two distinct motifs, the polypeptide domain and the N-terminal lipid moiety. In the vaccine formulation, the lipoproteins self-associate to form micelles driven by the hydrophobicity of the lipids and limited by the size of the folded polypeptides. The micelles help to increase the structural stability of the lipoproteins in the absence of bacterial cell walls. Analysis of the lipoproteins in Toll-like receptor (TLR) activation assays revealed their TLR2 agonist activity. This activity was lost with removal of the O-linked fatty acids, similar to removal of all lipids, demonstrating that this moiety plays an adjuvant role in immune activation. The thorough understanding of the structure and function of each moiety of the lipoproteins, as well as their relationship, lays the foundation for identifying critical parameters to guide vaccine development and manufacture.

The predominantly ␤-sheet phage P22 tailspike adhesin contains eight reduced cysteines per 666 residue chain, which are buried and unreactive in the native trimer. In the pathway to the native trimer, both in vivo and in vitro transient interchain disulfide bonds are formed and reduced. This occurs in the protrimer, an intermediate in the formation of the interdigitated ␤-sheets of the trimeric tailspike. Each of the eight cysteines was replaced with serine by site-specific mutagenesis of the cloned P22 tailspike gene and the mutant genes expressed in Escherichia coli. Although the yields of native-like Cys>Ser proteins varied, sufficient soluble trimeric forms of each of the eight mutants accumulated to permit purification. All eight single Cys>Ser mature proteins maintained the high thermostability of the wild type, as well as the wild-type biological activity in forming infectious virions. Thus, these cysteine thiols are not required for the stability or activity of the native state. When their in vivo folding and assembly kinetics were examined, six of the mutant substitutions-C267S, C287S, C458S, C613S, and C635S-were significantly impaired at higher temperatures. Four-C290S, C496, C613S, and C635-showed significantly impaired kinetics even at lower temperatures. The in vivo folding of the C613S/C635S double mutant was severely defective independent of temperature. Since the trimeric states of the single Cys>Ser substituted chains were as stable and active as wild type, the impairment of tailspike maturation presumably reflects problems in the in vivo folding or assembly pathways. The formation or reduction of the transient interchain disulfide bonds in the protrimer may be the locus of these kinetic functions.Keywords: Protein folding; assembly; oligomers; cysteines; tailspike The ability of cysteine thiols to donate electrons and to switch between oxidized -S-S-and reduced -SH states contributes to many aspects of protein form and function, including catalysis (Lemere et al. 1995;Chapman et al. 1997;Rietsch and Beckwith 1998;Schick et al. 1998), metal binding (Christianson 1991;McCall et al. 2000), protein stability (Price-Carter et al. 1998), and protein folding.Cysteine thiols have been particularly valuable as reporters in protein folding pathways (Creighton 1992). The presence of disulfide-bonded intermediates in the folding of the extracellular proteins bovine pancreatic RNase A and bovine pancreatic trypsin inhibitor (BPTI) provided the first means for trapping transient folding intermediates and for ordering steps in protein folding pathways. A striking finding for BPTI was the demonstration of nonnative disulfide bonds in the productive pathway (Goldenberg 1992;Darby et al. 1995). Although these intermediates are short-lived, they participate in efficient formation of the correctly folded native state. Bovine pancreatic RNase A intermediates also contain nonnative disulfide bonds. These disulfide bonds can re-arrange even in the absence of exogenous oxidants (Song and Scheraga 2000), but i...