Recognition of nascent polypeptides for targeting and folding.

A major difference between the refolding of proteins in vitro and the in vivo folding process, in which we include localization and assembly, is the need for additional factors in vivo, apart from the protein product itself. Thus, the amino acid sequence of a naturally selected protein contains not only the information specifying its three-dimensional structure, but also the information that enables these factors to recognize the nascent polypeptide. In this review, we consider how this latter information may be encoded and, in turn, interpreted by binding species.

The ins and outs of a molecular chaperone machine.

Genetic and biochemical work has highlighted the biological importance of the GroEL/GroES (Hsp60/Hsp10; cpn60/cpn10) chaperone machine in protein folding. GroEL's donut-shaped structure has attracted the attention of structural biologists because of its elegance as well as the secrets (substrates) it can hide. The recent determination of the GroES and GroEL/GroES structures provides a glimpse of their plasticity, revealing dramatic conformational changes that point to an elaborate mechanism, coupling ATP hydrolysis to substrate release by GroEL.

Domain-specific spectroscopy of 5-hydroxytryptophan-containing variants of Escherichia coli DnaJ.

Tryptophan-containing variants of Escherichia coli DnaJ protein were constructed in order to examine the hypothetical domain structure by fluorescence quenching and denaturant-induced unfolding. Two residues in the J-domain and one in the Gly/Phe-rich region were targeted for replacement and the proteins were expressed in a tryptophan auxotrophic strain in the presence of 5-hydroxytryptophan (5-HW). Fluorescence quenching with iodide of 5-HW in the variant proteins suggests that the Gly/Phe-rich region is more accessible to solvent than the J-domain. This is consistent with the proposal that the Gly/Phe-rich region is unstructured. Unfolding of the 5-HW-containing variants was monitored by fluorescence, and the results showed that the unfolding of the J-domain is cooperative and the unfolding of the Gly/Phe-rich region is not cooperative.

Renaturation of citrate synthase: influence of denaturant and folding assistants.

Citrate synthase (CS), which has been denatured in either guanidine hydrochloride (GdnHCl) or urea can be assisted in its renaturation in a variety of ways. The addition of each of the assistants--bovine serum albumin (BSA), oxaloacetate (OAA), and glycerol--promotes renaturation. In combination, the effect of these substances is additive with respect to the yield of folded CS. The report of Buchner et al. (Buchner, J., Schmidt, M., Fuchs, M., Jaenicke, R., Rudolph, R., Schmid, F.X., & Kiefhaber, T., 1991, Biochemistry 30, 1586-1591) that refolding of CS is facilitated by the GroE system (an Escherichia coli chaperonin [cpn] that is composed of GroEL [cpn60] and GroES [cpn10]) has been confirmed. However, we observed substantially higher yield of reactivated CS, 82%, and almost no reactivation in the absence of GroES, < 5%, whereas Buchner et al. reported 28% and 16%, respectively. In addition, we find that GroE-assisted refolding is more efficient for CS denatured in GdnHCl than for CS denatured in urea. This result is discussed in light of the known difference in the denatured states generated in GdnHCl and urea. Because GroEL inhibits the BSA/glycerol/OAA-assisted refolding, this system will be useful in future studies on the mechanism of GroE-facilitated refolding.

Reversible denaturation of oligomeric human chaperonin 10: denatured state depends on chemical denaturant.

Chaperonins cpn60/cpn10 (GroEL/GroES in Escherichia coli) assist folding of nonnative polypeptides. Folding of the chaperonins themselves is distinct in that it entails assembly of a sevenfold symmetrical structure. We have characterized denaturation and renaturation of the recombinant human chaperonin 10 (cpn10), which forms a heptamer. Denaturation induced by chemical denaturants urea and guanidine hydrochloride (GuHCl) as well as by heat was monitored by tyrosine fluorescence, far-ultraviolet circular dichroism, and cross-linking; all denaturation reactions were reversible. GuHCl-induced denaturation was found to be cpn10 concentration dependent, in accord with a native heptamer to denatured monomer transition. In contrast, urea-induced denaturation was not cpn10 concentration dependent, suggesting that under these conditions cpn10 heptamers denature without dissociation. There were no indications of equilibrium intermediates, such as folded monomers, in either denaturant. The different cpn10 denatured states observed in high [GuHCl] and high [urea] were supported by cross-linking experiments. Thermal denaturation revealed that monomer and heptamer reactions display the same enthalpy change (per monomer), whereas the entropy-increase is significantly larger for the heptamer. A thermodynamic cycle for oligomeric cpn10, combining chemical denaturation with the dissociation constant in absence of denaturant, shows that dissociated monomers are only marginally stable (3 kJ/mol). The thermodynamics for co-chaperonin stability appears conserved; therefore, instability of the monomer could be necessary to specify the native heptameric structure.

Growth performance, carcass and pork quality of finisher pigs fed oat-based diets containing different levels of beta-glucans.

A study was undertaken to investigate the growth performance and carcass and meat quality of pigs (BW = 52 to 108 kg) fed oat-based (Avena sativa L.) diets containing four levels of mixed-linkage (1 --> 3), (1 --> 4)-beta-D-glucans. One hundred sixty pigs-80 barrows and 80 gilts (average starting BW = 52.7 kg)--were allocated to one of five diets: a wheat-barley-based control diet and four experimental diets. The groats of Marion, a covered oat, and OT789, a hulless oat, were used to formulate four isonitrogenous and isocaloric diets to achieve 4.1,3.3, 2.1, or 1.6% total /beta-glucans (as fed). Growth performance (daily gain and gain to feed ratio) was not affected (P > 0.05) by the different levels of beta-glucans. Carcass yield, although lower (P < or = 0.05) for pigs fed the control diet, was similar (P > 0.05) for pigs fed any of the experimental diets. Cutout yields were also alike (P > 0.05) across the five diets. Beta-glucan content had no effect (P > 0.05) on the longissimus muscle area, or, by and large, on the proportions of commercial cuts; the only exceptions were a commercial picnic from pigs fed the 2.1% diet lower (P < 0.05) relative to all other diets and a lower (P < 0.5) commercial loin from pigs fed diets 4.1 or 3.3% relative to the control diet. Furthermore, the relative proportions oftotal lean, total bone, and total dissectable fat in the four lean cuts (picnic, butt, loin, and ham) were not different (P > 0.05) among the five diets. For pigs fed 4.1% beta-glucans, the proportion of lean in each of the four major cuts was lower (P < 0.05). No differences (P > 0.05) associated with the level of beta-glucans were detected for either the initial or ultimate pH mean values, the subjective assessment of color or structure of the longissimus muscle, or the instrumentally measured color (L value). Similarly, drip loss was not influenced (P > 0.05) by the level of beta-glucans in the diets. Soluble protein did differ (P < 0.05) among the high- to low-beta-glucans diets. No differences (P > 0.05) associated with diets were found for fat hardness and shear values of grilled pork chops. Chemical fat of the longissimus muscle from pigs fed 4.1, 3.3, or 2.1% beta-glucans was lower (P < 0.05) compared to pigs fed the control or 1.6% beta-glucans diets. In summary, no evidence of detrimental effect of beta-glucans in oat-based diets, particularly at levels below 4%, was detected, lending support for the inclusion of oat into finisher diets.

A novel approach to grading pork carcasses: computer vision and ultrasound.

A Computer Vision System prototype for grading pork carcasses was developed at the Lacombe Research System. The system consists of two components: ultrasound imaging to scan a cross-section of the loin muscle and video imaging to capture two-dimensional (2D) and three-dimensional (3D) images of the carcass. For each of the 241 carcasses (114 barrows and 127 gilts), salable meat yield was determined from a full cutout. Linear, two- and three-dimensional, angular and curvature measurements and carcass volume were derived from each image. Muscle area and fat thickness (7 cm off the mid-line) measured by ultrasound at the next to last rib site, together with 2D and 3D measurements provided the most accurate model for estimating salable meat yield (R(2)=0.82 and RSD=1.68). Models incorporating fat thickness and muscle depth measured at the Canadian grading site (3/4 last rib, 7 cm off the mid-line) with the Destron PG-100 probe, had the lowest R(2) and highest residual standard deviation (RSD) values (R(2)=0.66 and RSD=2.15). Cross-validation demonstrated the reliability and stability of the models; hence conferring them good industry applicability. The Lacombe Computer Vision System prototype appears to offer a marked improvement over probes currently used by the Canadian pork industry.

Local protein instability predictive of helper T-cell epitopes.

Although selectivity at the levels of peptide binding to major histocompatibility complex (MHC) class II and recognition by T cells may partially account for immunodominance patterns, it is clear that differential antigen processing also exerts a strong effect. Here, Sam Landry correlates immunodominant epitopes with nearby structurally unstable segments, as identified by hydrogen-deuterium exchange nuclear magnetic resonance (NMR), and suggests that epitope presentation is directed by preferential proteolytic cleavage at the unstable sites.

Helper T-cell epitope immunodominance associated with structurally stable segments of hen egg lysozyme and HIV gp120.

Although many antigen sequences potentially can bind to the MHCII proteins, only a small number of epitopes dominate the immune response. Additional mechanisms of processing, presentation or recognition must restrict the immune response against a large portion of the antigen. A highly significant correlation is found between epitope immunodominance and local structural stability in hen egg lysozyme. Since antigen proteins are likely to retain substantial native-like structure in the processing compartment, protease action may be focused on regions that are most readily accommodated in the protease active sites, and thus, the intervening sequence are preferentially presented. Immunodominance also correlates with sequence conservation as expected from the constraints imposed by structure. These results suggest that the three-dimensional structure of the antigen limits the immune response against some antigen segments. For HIV gp120, a substantial improvement in the accuracy of epitope prediction is obtained by combining rules for MHCII binding with a restriction of the predicted epitopes to well-conserved sequences.

The small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and its precursor expressed in Escherichia coli are associated with groEL protein.

The small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase is synthesized in the cytoplasm as a precursor which is transported into the chloroplast. During or after transport the precursor is processed to its mature size by removal of an amino-terminal transit peptide. Eight small subunits and eight large subunits (synthesized in the chloroplast) assemble to form the holoenzyme. We have expressed the precursor of the small subunit in Escherichia coli as a fusion to the carboxyl terminus of staphylococcal protein A'. The fusion protein was recovered from the bacterial lysate by chromatography on IgG-agarose. A 58-kDa protein copurified with the fusion protein in approximately equal amounts. Much less of the 58-kDa protein copurified with a fusion in which the transit peptide was deleted, and it did not copurify with protein A'. The 58-kDa protein was identified as the E. coli groEL gene product with antibodies directed against a homologous mitochondrial heat shock protein. This finding is particularly interesting because a chloroplast protein involved in the assembly of ribulose-1,5-bisphosphate carboxylase/oxygenase also is homologous to the groEL protein. These homologs could modulate protein-protein interactions during folding and assembly of subunits into native complexes.

The chaperonin GroEL binds a polypeptide in an alpha-helical conformation.

Chaperones facilitate folding and assembly of nascent polypeptides in vivo and prevent aggregation in refolding assays in vitro. A given chaperone acts on a number of different proteins. Thus, chaperones must recognize features present in incompletely folded polypeptide chains and not strictly dependent on primary structural information. We have used transferred nuclear Overhauser effects to demonstrate that the Escherichia coli chaperonin GroEL binds to a peptide corresponding to the N-terminal alpha-helix in rhodanese, a mitochondrial protein whose in vitro refolding is facilitated by addition of GroEL, GroES, and ATP. Furthermore, the peptide, which is unstructured when free in aqueous solution, adopts an alpha-helical conformation upon binding to GroEL. Modification of the peptide to reduce its intrinsic propensity to take up alpha-helical structure lowered its affinity for GroEL, but, nonetheless, it could be bound and took up a helical conformation when bound. We propose that GroEL interacts with sequences in an incompletely folded chain that have the potential to adopt an amphipathic alpha-helix and that the chaperonin binding site promotes formation of a helix.

Temperature dependence of backbone dynamics in loops of human mitochondrial heat shock protein 10.

A highly flexible, yet conserved polypeptide loop of Hsp10 mediates binding to Hsp60 in the course of chaperonin-dependent protein folding. Previous transferred nuclear Overhauser effect (trNOE) studies with peptides based on the mobile loop of the Escherichiacoli and bacteriophage T4 Hsp10s suggested that the mobile loop adopts a characteristic hairpin turn upon binding to the E. coli Hsp60 GroEL. In this paper, we identify the sequence and characterize the nascent structure and dynamics of the 18-residue mobile loop in the 15N-enriched human Hsp10. We also identify four residues of another flexible loop, the roof beta hairpin. The mobile loop and/or roof beta hairpin of several subunits are absent from the X-ray crystal structure of human Hsp10. NMR data suggest that the mobile loop of Hsp10 preferentially samples a hairpin conformation despite the fact that the backbone motion resembles that of a disordered polypeptide. Analysis of backbone dynamics by measurement of 15N relaxation times, T1 and T2, and the 1H-15N nuclear Overhauser effect (1H-15N NOE) indicates that motion is greatest near the center of the loop. Inversion of the temperature dependence of the T1 near the center of the loop marks a transition to motion with a dominant time scale of less than 3 ns. Analysis of the relaxation data by spectral density mapping shows that subnanosecond motion increases uniformly along the loop at elevated temperatures, whereas nanosecond motion increases near the ends of the loop and decreases near the center of the mobile loop. The transition to dominance by fast motion in the center of the loop occurs at a distance from the well-structured part of Hsp10 that is equal to the persistence length of an unstructured polypeptide. Simulation of the spectral density function for the 15N resonance and its temperature dependence using the Lipari-Szabo formalism suggests that the dominant time scales of loop motion range from 0.6 to 18 ns. For comparison, the time scale for molecular rotation of the 70 kDa Hsp10 heptamer is estimated to be 37 ns. Complex behavior of the T2 relaxation time indicates that motion also occurs on longer time scales. All of the modes of loop motion are likely to have an impact on Hsp10/Hsp60 interaction and therefore affect Hsp10/Hsp60 function as a chaperonin.

Allocation of helper T-cell epitope immunodominance according to three-dimensional structure in the human immunodeficiency virus type I envelope glycoprotein gp120.

The specificity and intensity of CD4(+) helper T-cell responses determine the effectiveness of immune effector functions. Promiscuously immunodominant helper T-cell epitopes in the human immunodeficiency virus (HIV) envelope glycoprotein gp120 could be important in the development of broadly protective immunity, but the underlying mechanisms of immunodominance and promiscuity remain poorly defined. In this study, gp120 helper T-cell epitopes were systematically mapped in CBA/J and BALB/c mice by restimulation assays using a set of overlapping peptides spanning the entire sequence of the gp120 encoded by HIV strain 89.6. The results were analyzed in the context of the HIV gp120 structure determined by x-ray crystallography. One major finding was that all of the promiscuously immunodominant gp120 sequences are located in the outer domain. Further analyses indicated that epitope immunogenicity in the outer domain correlates with structural disorder in adjacent N-terminal segments, as indicated by crystallographic B-factors or sequence divergence. In contrast, the correlation was poor when the analysis encompassed the entire gp120 sequence or was restricted to only the inner domain. These findings suggest that local disorder promotes the processing and presentation of adjacent epitopes in the outer domain of gp120 and therefore reveal how three-dimensional structure shapes the profile of helper T-cell epitope immunogenicity.

The disordered mobile loop of GroES folds into a defined beta-hairpin upon binding GroEL.

The GroES mobile loop is a stretch of approximately 16 amino acids that exhibits a high degree of flexible disorder in the free protein. This loop is responsible for the interaction between GroES and GroEL, and it undergoes a folding transition upon binding to GroEL. Results derived from a combination of transferred nuclear Overhauser effect NMR experiments and molecular dynamics simulations indicate that the mobile loop adopts a beta-hairpin structure with a Type I, G1 Bulge turn. This structure is distinct from the conformation of the loop in the co-crystal of GroES with GroEL-ADP but identical to the conformation of the bacteriophage-panned "strongly binding peptide" in the co-crystal with GroEL. Analysis of sequence conservation suggests that sequences of the mobile loop and strongly binding peptide were selected for the ability to adopt this hairpin conformation.

Role of the J-domain in the cooperation of Hsp40 with Hsp70.

The Escherichia coli Hsp40 DnaJ and Hsp70 DnaK cooperate in the binding of proteins at intermediate stages of folding, assembly, and translocation across membranes. Binding of protein substrates to the DnaK C-terminal domain is controlled by ATP binding and hydrolysis in the N-terminal ATPase domain. The interaction of DnaJ with DnaK is mediated at least in part by the highly conserved N-terminal J-domain of DnaJ that includes residues 2-75. Heteronuclear NMR experiments with uniformly 15N-enriched DnaJ2-75 indicate that the chemical environment of residues located in helix II and the flanking loops is perturbed on interaction with DnaK or a truncated DnaK molecule, DnaK2-388. NMR signals corresponding to these residues broaden and exhibit changes in chemical shifts in the presence of DnaK(MgADP). Addition of MgATP largely reversed the broadening, indicating that NMR signals of DnaJ2-75 respond to ATP-dependent changes in DnaK. The J-domain interaction is localized to the ATPase domain of DnaK and is likely to be dominated by electrostatic interactions. The results suggest that the J-domain tethers DnaK to DnaJ-bound substrates, which DnaK then binds with its C-terminal peptide-binding domain.

The importance of a mobile loop in regulating chaperonin/ co-chaperonin interaction: humans versus Escherichia coli.

Chaperonins are universally conserved proteins that nonspecifically facilitate the folding of a wide spectrum of proteins. While bacterial GroEL is functionally promiscuous with various co-chaperonin partners, its human homologue, Hsp60 functions specifically with its co-chaperonin partner, Hsp10, and not with other co-chaperonins, such as the bacterial GroES or bacteriophage T4-encoded Gp31. Co-chaperonin interaction with chaperonin is mediated by the co-chaperonin mobile loop that folds into a beta-hairpin conformation upon binding to the chaperonin. A delicate balance of flexibility and conformational preferences of the mobile loop determines co-chaperonin affinity for chaperonin. Here, we show that the ability of Hsp10, but not GroES, to interact specifically with Hsp60 lies within the mobile loop sequence. Using mutational analysis, we show that three substitutions in the GroES mobile loop are necessary and sufficient to acquire Hsp10-like specificity. Two of these substitutions are predicted to preorganize the beta-hairpin turn and one to increase the hydrophobicity of the GroEL-binding site. Together, they result in a GroES that binds chaperonins with higher affinity. It seems likely that the single ring mitochondrial Hsp60 exhibits intrinsically lower affinity for the co-chaperonin that can be compensated for by a higher affinity mobile loop.

Characterization of a functionally important mobile domain of GroES.

Although genetic and biochemical evidence has established that GroES is required for the full function of the molecular chaperone, GroEL, little is known about the molecular details of their interaction. GroES enhances the cooperativity of ATP binding and hydrolysis by GroEL (refs 4, 5) and is necessary for release and folding of several GroEL substrates. Here we report that native GroES has a highly mobile and accessible polypeptide loop whose mobility and accessibility are lost upon formation of the GroES/GroEL complex. In addition, lesions present in eight independently isolated mutant groES alleles map in the mobile loop. Studies with synthetic peptides suggest that the loop binds in a hairpin conformation at a site on GroEL that is distinct from the substrate-binding site. Flexibility may be required in the mobile loops on the GroES seven-mer to allow them to bind simultaneously to sites on seven GroEL subunits, which may themselves be able to adopt different arrangements, and thus to modulate allosterically GroEL/substrate affinity.