Chapter 7 Protein Folding and Intracellular Transport: Evaluation of Conformational Changes in Nascent Exocytotic Proteins
Publisher Summary
This chapter describes various assays that have been developed to analyze the in vivo process of protein folding or oligomerization within the ER. These assays are divided into two groups—namely, (1) assays that probe conformational changes in the nascent polypeptide chain and (2) assays that follow the assembly of the polypeptides into oligomers. To determine the time course in vivo of folding and assembly of membrane or secretory proteins, it is necessary to label the nascent polypeptides with a radioactive amino acid, usually [35S]methionine or [35S]cysteine. Optimally, the period of incorporation of isotope should be short relative to the time course of folding so that only the completely unfolded polypeptide will be labeled at the start of the chase period. However, in practice it may be difficult to achieve, because initial folding of the polypeptide may commence even before translation and translocation of the chain is completed. The assays for analysis of protein folding using antibodies specific for different conformational states depend on the availability of antibodies that recognize different conformational forms of the protein of interest, and in particular antibodies that discriminate between unfolded and fully folded forms of the polypeptide chain. Polyclonal or monoclonal antibodies raised against reduced and alkylated polypeptides are likely to recognize the fully unfolded conformations and may not recognize the folded molecule.
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Cited by (29)
Functional Analysis of the Transmembrane Domain in Paramyxovirus F Protein-Mediated Membrane Fusion
2009, Journal of Molecular BiologyTo enter cells, enveloped viruses use fusion-mediating glycoproteins to facilitate the merger of the viral and host cell membranes. These glycoproteins undergo large-scale irreversible refolding during membrane fusion. The paramyxovirus parainfluenza virus 5 mediates membrane merger through its fusion protein (F). The transmembrane (TM) domains of viral fusion proteins are typically required for fusion. The TM domain of F is particularly interesting in that it is potentially unusually long; multiple calculations suggest a TM helix length between 25 and 48 residues. Oxidative cross-linking of single-cysteine substitutions indicates the F TM trimer forms a helical bundle within the membrane. To assess the functional role of the paramyxovirus parainfluenza virus 5 F protein TM domain, alanine scanning mutagenesis was performed. Two residues located in the outer leaflet of the bilayer are critical for fusion. Multiple amino acid substitutions at these positions indicate the physical properties of the side chain play a critical role in supporting or blocking fusion. Analysis of intermediate steps in F protein refolding indicated that the mutants were not trapped at the open stalk intermediate or the prehairpin intermediate. Incorporation of a known F protein destabilizing mutation that causes a hyperfusogenic phenotype restored fusion activity to the mutants. Further, altering the curvature of the lipid bilayer by addition of oleic acid promoted fusion of the F protein mutants. In aggregate, these data indicate that the TM domain plays a functional role in fusion beyond merely anchoring the protein in the viral envelope and that it can affect the structures and steady-state concentrations of the various conformational intermediates en route to the final postfusion state. We suggest that the unusual length of this TM helix might allow it to serve as a template for formation of or specifically stabilize the lipid stalk intermediate in fusion.
The paramyxovirus, simian virus 5, fusion (F) protein contains seven amino acids between heptad repeat B (a domain required for a biologically active fusion protein) and the presumptive boundary of the transmembrane (TM) domain. The role of the seven membrane proximal residues in stability and fusion promotion was examined by construction of a series of insertion, substitution, and deletion mutants, as manipulation of this region to enable proteolytic cleavage would facilitate production of a soluble F protein. The majority of the mutant F proteins both oligomerized and had kinetics of intracellular transport similar to those of wild-type (wt) F protein. All mutant F proteins were expressed at the cell surface at or near the same level as the wt F protein. However, by using both a qualitative lipid mixing assay and a quantitative content mixing assay for membrane fusion, it was found that mutant F proteins containing insertions in the region between heptad repeat B and the TM domain were unable to induce fusion, whereas the mutant F proteins containing substitutions in this region, together with three of the four mutants with deletions in this region, could induce fusion. Four of the F protein mutants contained a Factor Xa cleavage site, IEGR; however, Factor Xa treatment of cell surfaces released either none or only very small amounts (<1% of total protein) of the soluble heterodimer F1+ F2. As an alternative method of generating soluble F protein, a glycosyl phosphatidylinositol (GPI) anchor was added to the F protein at three membrane-proximal positions. The highest level of surface expression was observed when the final molecule did not contain a significant insertion of amino acids into the membrane proximal region. Two F-GPI mutants reached the surface at approximately 20% of the levels seen with the wt F protein, and approximately 25% of the cell surface population of these mutants could be cleaved with phosphatidylinositol phospholipase C (PI-PLC) to yield soluble F protein. However, all the F-GPI mutants oligomerized aberrantly and failed to promote fusion. Taken together, these data indicate that the spacing of the region immediately adjacent to the presumptive boundary of the TM domain is extremely important for the fusogenic activity of the SV5 F protein.
Expression of influenza B virus hemagglutinin containing multibasic residue cleavage sites
1997, VirologyThe hemagglutinin (HA) protein of influenza B virus contains a single arginine residue at its cleavage site and the HA0precursor is not cleaved to the HA1and HA2subunits by tissue culture cell-associated proteases. To investigate if an HA protein could be obtained that could be cleaved by an endogenous cellular protease, the cDNA for HA of influenza B/MD/59 virus was subjected to site-specific mutagenesis. Three HA mutant proteins were constructed, through substitution or insertion of arginine residues, that have 4, 5, or 6 basic residues at their cleavage sites. Chemical cross-linking studies indicated that all three HA cleavage site mutants could oligomerize to a trimeric species, like WT HA. The three HA cleavage site mutant proteins were efficiently transported to the cell surface and bound erythrocytes in hemadsorption assays. The mutants were cleaved at a low level to HA1and HA2by an endogenous host cell protease and cleavage could be increased somewhat by addition of exogenous trypsin. The fusogenic activities of the HA cleavage site mutants were assessed in comparison to the WT HA protein by determining their syncytium formation ability and by using an R18 lipid-mixing assay and a NBD-taurine aqueous-content mixing assay. While the fusion activity of the WT HA protein was dependent on exogenous trypsin to activate HA, the three HA cleavage site mutant proteins were able to induce fusion in the absence of trypsin when assayed with the R18 lipid-mixing and NBD-taurine aqueous-content mixing assays, but were unable to induce syncytium formation in either the presence or absence of exogenous trypsin. Our results suggest that while the presence of a subtilisin-like protease cleavage sequence at the influenza B virus HA1/HA2boundary does enable some HA0molecules to be cleaved intracellularly, it alone is not sufficient for efficient cleavage.
Self-association of truncated forms of HIV-1 gp120
1997, Virus ResearchHIV-1 gp120 and truncated forms were expressed in HeLa T4 cells by vaccinia recombinant viruses. The truncated gp120 molecules consisted of N-terminal overlapping envelope proteins of 204, 287 and 393 amino acids respectively. Immunoprecipitation with specific monoclonal antibodies and SDS–PAGE analyses of HIV-1 gp120 revealed bands corresponding to low amounts of secreted and cell-bound stable dimers. In contrast, the truncated forms of gp120 expressed larger amounts of SDS–stable putative dimers and the amounts observed were inversely proportional to their size. The shortest gp120 mutant (204 aa) was found to be secreted almost exclusively as a dimer. The processing of gp120 and its truncated forms was further investigated in the presence of inhibitors of N-glycosylation. Monomers and dimers migrated on gels with the same relative changes, confirming that the protein with the higher molecular weight is a multimer of the smaller one. The putative dimeric form of the truncated gp120s could be stabilized by chemical cross-linking. Finally, the possible existence of an association domain in the N-terminal 204 amino acids (aa) of gp120 is discussed.
Purification and renaturation of japanese encephalitis virus nonstructural glycoprotein ns1 overproduced by insect cells
1995, Protein Expression and PurificationThe nonstructural protein NS1 of Japanese encephalitis virus is a major immunogen produced during flavivirus infection. However, the function of this protein has not been identified. To analyze its biochemical properties and evaluate its potential activity in the virus life cycle, the protein was produced in Spodoptera frugiperda insect cells (Sf9), using a recombinant baculovirus, and purified. As described previously by M. Flamand, V. Deubel, and M. Girard (1992, Virology 191, 826-836), a small fraction of the synthesized recombinant protein could mature into a dimer, whereas the major part was retained in intracellular aggregates. This insolubility was used to recover the protein in a purified form using a two-step procedure. Isolated inclusion bodies, in which NS1 constituted over 60% of the protein, were solubilized in 8 M urea. NS1 was further purified by reverse-phase HPLC and recovered at over 90% purity with an overall yield of over 60%. Conditions promoting reoxidation-renaturation of the purified protein were then investigated at a concentration of 100 μg/ml at pH 8. The presence of 8 M urea during reoxidation of NS1 with oxidized glutathione was essential prior to renaturation by dialysis to avoid reaggregation, the main side pathway of refolding in vitro. Three major species, all monomeric, were resolved by nonreducing SDS-PAGE. The form showing the lowest apparent molecular weight comigrated with native unreduced NS1 and was recognized by a monoclonal antibody directed against a conformational epitope strictly dependent on the native structure of the protein. Thus, this form, representing over 30% of the renatured products, may have reached the native conformation of the protein. This simple renaturation-reoxidation procedure may be applicable to other disulfide bond-containing proteins to be recovered in the native state from inclusion bodies.
Individual Roles of N-Linked Oligosaccharide Chains in Intracellular Transport of the Paramyxovirus SV5 Fusion Protein
1995, VirologyThe role of N-linked glycosylation in the assembly, intracellular transport, and fusion activity of the paramyxovirus SV5 fusion (F) protein was examined. Each of the six potential glycosylation sites in the F protein was individually removed by oligonucleotide-directed mutagenesis on a cDNA clone encoding the SV5 F protein. When the mutant F proteins were expressed in eukaryotic cells using the vaccinia virus-T7 transient expression system they all had a significant change in gel mobility, indicating that all six sites in the F protein are used for the addition of N-linked oligosaccharides. All of the mutant F proteins could form a homooligomer. Removal of individual carbohydrate chains from the F2 subunit had little effect on the surface expression of the F protein. However, removal of individual carbohydrate chains from the F1 subunit had deleterious effects, which ranged from a partial delay in intracellular transport and decreased stability of the protein to severe transport delays and acute instability of the F protein.