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Escherichia coli malate dehydrogenase (EcMDH) and its eukaryotic counterpart, porcine mitochondrial malate dehydrogenase (PmMDH), are highly homologous proteins with significant sequence identity (60%) and virtually identical native structural folds. Despite this homology, EcMDH folds rapidly and efficiently in vitro and does not seem to interact with GroE chaperonins at physiological temperatures (37°C), whereas PmMDH folds much slower than EcMDH and requires these chaperonins to fold to the native state at 37°C. Double jump experiments indicate that the slow folding behavior of PmMDH is not limited by proline isomerization. Although the folding enhancer glycerol (<5 M) does not alter the renaturation kinetics of EcMDH, it dramatically accelerates the spontaneous renaturation of PmMDH at all temperatures tested. Kinetic analysis of PmMDH renaturation with increasing glycerol concentrations suggests that this osmolyte increases the onpathway kinetics of the monomer folding to assemblycompetent forms. Other osmolytes such as trimethylamine N-oxide, sucrose, and betaine also reactivate PmMDH at nonpermissive temperatures (37°C). Glycerol jump experiments with preformed GroEL⅐PmMDH complexes indicate that the shift between stringent (requires ATP and GroES) and relaxed (only requires ATP) complex conformations is rapid (<3-5 s). The similarity in irreversible misfolding kinetics of PmMDH measured with glycerol or the activated chaperonin complex (GroEL⅐GroES⅐ATP) suggests that these folding aids may influence the same step in the PmMDH folding reaction. Moreover, the interactions between glycerol-induced PmMDH folding intermediates and GroEL⅐GroES⅐ATP are diminished. Our results support the notion that the protein folding kinetics of sequentially and structurally homologous proteins, rather than the structural fold, dictates the GroE chaperonin requirement.GroEL is a complex, allosteric, protein folding machine whose function is controlled by associations with nucleotides, the co-chaperonin GroES, and substrate polypeptides (1-3). As with all allosteric proteins, ligand binding influences the structural constraints within the system, which in turn ultimately induce shifts between various functional states. Although detailed information is available on the structures of GroEL, with and without bound nucleotide, and of one GroEL⅐GroES complex, the exact mechanism(s) explaining chaperonin-assisted folding of substrate proteins remain(s) unclear. Differences in binding and conditions for productive release of substrate proteins are routinely observed, but the structural and energetic basis of these differences is not understood at the molecular level. Although molten globule folding intermediates have been suggested to be preferred substrates for chaperonins, the structures of these intermediate populations have broad distributions thus making it difficult to identify specific transient conformations that interact with the GroE chaperonins.In vitro and in vivo studies have shown that many proteins can interact with and...

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For the chaperonin substrates, rhodanese, malate dehydrogenase (MDH), and glutamine synthetase (GS), the folding efficiencies, and the lifetimes of folding intermediates were measured with either the nucleotide-free GroEL or the activated ATP⅐GroEL⅐GroES chaperonin complex. With both nucleotide-free and activated complex, the folding efficiency of rhodanese and MDH remained high over a large range of GroEL to substrate concentration ratios (up to 1:1). In contrast, the folding efficiency of GS began to decline at ratios lower than 8:1. At ratios where the refolding yields were initially the same, only a relatively small increase (1.6-fold) in misfolding kinetics of MDH was observed with either the nucleotide-free or activated chaperonin complex. For rhodanese, no change was detected with either chaperonin complex. In contrast, GS lost its ability to interact with the chaperonin system at an accelerated rate (8-fold increase) when the activated complex instead of the nucleotide-free complex was used to rescue the protein from misfolding. Our data demonstrate that the differences in the refolding yields are related to the intrinsic folding kinetics of the protein substrates. We suggest that the early kinetic events at the substrate level ultimately govern successful chaperonin-substrate interactions and play a crucial role in dictating polypeptide flux through the chaperonin system. Our results also indicate that an accurate assessment of the transient properties of folding intermediates that dictate the initial chaperonin-substrate interactions requires the use of the activated complex as the interacting chaperonin species.

Mouse-human chimeric antibodies (cAbs) against hepatitis C virus (HCV) core, NS3 (nonstructural), NS4, and NS5 antigens were developed as quality control (QC) reagents to replace the use of human sera/plasma for Abbott HCV immunoassays. The cAb retains the mouse monoclonal antibody (MAb) specificity and affinity but still reacts in the existing HCV assay format, which measures human anti-HCV immunoglobulin. Mouse heavy-chain (V H ) and light-chain (V L ) variable regions of anti-HCV core, NS3, NS4, and NS5 antigens were PCR amplified from hybridoma lines and then cloned with human IgG1 heavy-chain (C H ) and light-chain (C L ) constant regions, respectively. A single mammalian expression plasmid containing both heavy-chain and light-chain immunoglobulin genes was constructed and transfected into dihydrofolate reductase (DHFR)-deficient Chinese hamster ovary (CHO) cells. The transfected CHO cells were selected using hypoxanthineand thymidine-free medium and screened by an enzyme immunoassay (EIA). The clone secreting the highest level of antibody was isolated from the CHO transfectants and further subcloned. Each cAb-expressing CHO cell line was weaned into serum-free medium, and the cAb was purified by protein A affinity chromatography. The levels of cAb production for the various CHO cell lines varied from 10 to 20 mg/liter. Purified anti-HCV cAbs were tested with Abbott HCV immunoassays and showed reactivity. Moreover, yeast surface display combined with alanine-scanning mutagenesis was used to map the epitope at the individual amino acid level. Our results suggest that these HCV cAbs are ideal controls, calibrators, and/or QC reagents for HCV assay standardization.Infection with hepatitis C virus (HCV) causes an inflammation of the liver and is the most common chronic blood-borne infection in the United States. According to the U.S. Centers for Disease Control and Prevention, approximately 1.8% of the U.S. population, or 3.9 million Americans, have been infected with the virus. About 35,000 new cases of HCV are estimated to occur in the United States each year. Common routes of infection include needle stick accidents, blood transfusions, and injection drug use. Most individuals acutely infected with HCV become chronically infected. Once a person is chronically infected, the virus is almost never cleared without treatment (20).Abbott HCV immunoassays designed to detect anti-HCV antibodies in patient samples provide a fast and reliable serological diagnostic method. Typically, diagnostic kits contain one or more antibodies as calibrators/positive controls. Traditionally, these controls consist of human plasma and/or serum samples from infected individuals. The quality control (QC) reagents, such as assay sensitivity panels, are human plasma/ serum samples selected for antibodies against HCV core, NS3 (nonstructural), NS4, and NS5 antigen epitopes. However, the use of human serum/plasma has several significant disadvantages, including increasing regulatory concerns about patient sample drawing, sample storage a...