Nonuniform chain collapse during early stages of staphylococcal nuclease folding detected by fluorescence resonance energy transfer and ultrarapid mixing methods.
ABSTRACT: The development of tertiary structure during folding of staphylococcal nuclease (SNase) was studied by time-resolved fluorescence resonance energy transfer measured using continuous- and stopped-flow techniques. Variants of this two-domain protein containing intradomain and interdomain fluorescence donor/acceptor pairs (Trp and Cys-linked fluorophore or quencher) were prepared to probe the intradomain and interdomain structural evolution accompanying SNase folding. The intra-domain donor/acceptor pairs are within the β-barrel domain (Trp27/Cys64 and Trp27/Cys97) and the interdomain pair is between the α-helical domain and the β-barrel domain (Trp140/Cys64). Time-resolved energy transfer efficiency accompanying folding and unfolding at different urea concentrations was measured over a time range from 30 μs to ≈ 10 s. Information on average donor/acceptor distances at different stages of the folding process was obtained by using a quantitative kinetic modeling approach. The average distance for the donor/acceptor pairs in the β-barrel domain decreases to nearly native values whereas that of the interdomain donor/acceptor pairs remains unchanged in the earliest intermediate (<500 μs of refolding). This indicates a rapid nonuniform collapse resulting in an ensemble of heterogeneous conformations in which the central region of the β-barrel domain is well developed while the C-terminal α-helical domain remains disordered. The distance between Trp140 and Cys64 decreases to native values on the 100-ms time scale, indicating that the α-helical domain docks onto the preformed β-barrel at a late stage of the folding. In addition, the unfolded state is found to be more compact under native conditions, suggesting that changes in solvent conditions may induce a nonspecific hydrophobic collapse.
Project description:Folding stability and cooperativity of the three forms of 1-110 residues fragment of staphylococcal nuclease (SNase110) have been studied by various biophysical and NMR methods. Samples of G-88W- and V-66W-mutant SNase110, namely G-88W110 and V-66W110, in aqueous solution and SNase110 in 2.0 M TMAO are adopted in this study. The unfolding transitions and folded conformations of the three SNase fragments were detected by far- and near-ultraviolet circular dichroism and intrinsic tryptophan fluorescence measurements. The tertiary structures and internal motions of the fragments were determined by NMR spectroscopy. Both G-88W and V-66W single mutations as well as a small organic osmolyte (Trimethylamine N-oxide, TMAO) can fold the fragment into a native-like conformation. However, the tertiary structures of the three fragments exhibit different degrees of folding stability and compactness. G-88W110 adopts a relatively rigid structure representing a most stable native-like beta-subdomain conformation of the three fragments. V-66W110- and TMAO-stabilized SNase110 produce less compact structures having a less stable "beta-barrel" structural region. The different folding status accounts for the different backbone dynamic and urea-unfolding transition features of the three fragments. The G-20I/G-29I-mutant variants of the three fragments have provided the evidence that the folding status is correlated closely to the packing of the beta-strands in the beta-barrel of the fragments. The native-like beta-barrel structural region acts as a nonlocal nucleus for folding the fragment. The tertiary folding of the three fragments is initiated by formation of the local nucleation sites at two beta-turn regions, I-18-D-21 and Y-27-Q-30, and developed by the formation of a nonlocal nucleation site at the beta-barrel region. The formation of beta-barrel and overall structure is concerted, but the level of cooperativity is different for the three 1-110 residues SNase fragments.
Project description:The investigation and understanding of the folding mechanism of multidomain proteins is still a challenge in structural biology. The use of single-molecule Förster resonance energy transfer offers a unique tool to map conformational changes within the protein structure. Here, we present a study following denaturant-induced unfolding transitions of yeast phosphoglycerate kinase by mapping several inter- and intradomain distances of this two-domain protein, exhibiting a quite heterogeneous behavior. On the one hand, the development of the interdomain distance during the unfolding transition suggests a classical two-state unfolding behavior. On the other hand, the behavior of some intradomain distances indicates the formation of a compact and transient molten globule intermediate state. Furthermore, different intradomain distances measured within the same domain show pronounced differences in their unfolding behavior, underlining the fact that the choice of dye attachment positions within the polypeptide chain has a substantial impact on which unfolding properties are observed by single-molecule Förster resonance energy transfer measurements. Our results suggest that, to fully characterize the complex folding and unfolding mechanism of multidomain proteins, it is necessary to monitor multiple intra- and interdomain distances because a single reporter can lead to a misleading, partial, or oversimplified interpretation.
Project description:Folding of small proteins often occurs in a two-state manner and is well understood both experimentally and theoretically. However, many proteins are much larger and often populate misfolded states, complicating their folding process significantly. Here we study the complete folding and assembly process of the 1,418 amino acid, dimeric chaperone Hsp90 using single-molecule optical tweezers. Although the isolated C-terminal domain shows two-state folding, we find that the isolated N-terminal as well as the middle domain populate ensembles of fast-forming, misfolded states. These intradomain misfolds slow down folding by an order of magnitude. Modeling folding as a competition between productive and misfolding pathways allows us to fully describe the folding kinetics. Beyond intradomain misfolding, folding of the full-length protein is further slowed by the formation of interdomain misfolds, suggesting that with growing chain lengths, such misfolds will dominate folding kinetics. Interestingly, we find that small stretching forces applied to the chain can accelerate folding by preventing the formation of cross-domain misfolding intermediates by leading the protein along productive pathways to the native state. The same effect is achieved by cotranslational folding at the ribosome in vivo.
Project description:Effector-induced allosteric transitions in cytochrome P450 3A4 (CYP3A4) were investigated by luminescence resonance energy transfer (LRET) between two SH-reactive probes attached to various pairs of distantly located cysteine residues, namely the double-cysteine mutants CYP3A4(C64/C468), CYP3A4(C377/C468) and CYP3A4(C64/C121). Successive equimolar labeling of these proteins with the phosphorescent probe erythrosine iodoacetamide (donor) and the near-infrared fluorophore DY-731 maleimide (acceptor) allowed us to establish donor/acceptor pairs sensitive to conformational motions. The interactions of all three double-labeled mutants with the allosteric activators ?-naphthoflavone and testosterone resulted in an increase in the distance between the probes. A similar effect was elicited by cholesterol. These changes in distance vary from 1.3 to 8.5 Å, depending on the position of the donor/acceptor pair and the nature of the effector. In contrast, the changes in the interprobe distance caused by such substrates as bromocriptine or 1-pyrenebutanol were only marginal. Our results provide a decisive support to the paradigm of allosteric modulation of CYP3A4 and indicate that the conformational transition caused by allosteric effectors increases the spatial separation between the beta-domain of the enzyme (bearing residues Cys64 and Cys377) and the alpha-domain, where Cys121 and Cys468 are located.
Project description:The LDL receptor internalizes circulating LDL and VLDL particles for degradation. Its extracellular binding domain contains ten (seven LA and three EGF) cysteine-rich modules, each bearing three disulfide bonds. Despite the enormous number of disulfide combinations possible, LDLR oxidative folding leads to a single native species with 30 unique intradomain disulfides. Previous folding studies of the LDLR have shown that non native disulfides are initially formed that lead to compact species. Accordingly, the folding of the LDLR has been described as a "coordinated nonvectorial" reaction, and it has been proposed that early compaction funnels the reaction toward the native structure. Here we analyze the oxidative folding of LA4 and LA5, the modules critical for ApoE binding, isolated and in the LA45 tandem. Compared to LA5, LA4 folding is slow and inefficient, resembling that of LA5 disease-linked mutants. Without Ca++, it leads to a mixture of many two-disulfide scrambled species and, with Ca++, to the native form plus two three-disulfide intermediates. The folding of the LA45 tandem seems to recapitulate that of the individual repeats. Importantly, although the folding of the LA45 tandem takes place through formation of scrambled isomers, no interdomain disulfides are detected, i.e. the two adjacent modules fold independently without the assistance of interdomain covalent interactions. Reduction of incredibly large disulfide combinatorial spaces, such as that in the LDLR, by intradomain confinement of disulfide bond formation might be also essential for the efficient folding of other homologous disulfide-rich receptors.
Project description:Bacterial autotransporters comprise a C-terminal β-barrel domain, which must be correctly folded and inserted into the outer membrane to facilitate translocation of the N-terminal passenger domain to the cell exterior. Once at the surface, the passenger domains of most autotransporters are folded into an elongated β-helix. In a cellular context, key molecules catalyze the assembly of the autotransporter β-barrel domain. However, how the passenger domain folds into its functional form is poorly understood. Here we use mutational analysis on the autotransporter Pet to show that the β-hairpin structure of the fifth extracellular loop of the β-barrel domain has a crucial role for passenger domain folding into a β-helix. Bioinformatics and structural analyses, and mutagenesis of a homologous autotransporter, suggest that this function is conserved among autotransporter proteins with β-helical passenger domains. We propose that the autotransporter β-barrel domain is a folding vector that nucleates folding of the passenger domain.
Project description:BamA interacts with the BamBCDE lipoproteins, and together they constitute the essential β-barrel assembly machine (BAM) of Escherichia coli. The simultaneous absence of BamB and BamE confers a conditional lethal phenotype and a severe β-barrel outer membrane protein (OMP) biogenesis defect. Without BamB and BamE, wild-type BamA levels are significantly reduced, and the folding of the BamA β-barrel, as assessed by the heat-modifiability assay, is drastically compromised. Single-amino-acid substitutions in the β-barrel domain of BamA improve both bacterial growth and OMP biogenesis in a bamB bamE mutant and restore BamA levels close to the BamB(+) BamE(+) level. The substitutions alter BamA β-barrel folding, and folding in the mutants becomes independent of BamB and BamE. Remarkably, BamA β-barrel alterations also improve OMP biogenesis in cells lacking the major periplasmic chaperone, SurA, which, together with BamB, is thought to facilitate the transfer of partially folded OMPs to the soluble POTRA (polypeptide-transport-associated) domain of BamA. Unlike the bamB bamE mutant background, the absence of BamB or SurA does not affect BamA β-barrel folding. Thus, substitutions in the outer membrane-embedded BamA β-barrel domain overcome OMP biogenesis defects that occur at the POTRA domain of BamA in the periplasm. Based on the structure of FhaC, the altered BamA residues are predicted to lie on a highly conserved loop that folds inside the β-barrel and in regions pointing outside the β-barrel, suggesting that they influence BamA function by both direct and indirect mechanisms.
Project description:A 3-ns molecular dynamics simulation in explicit solvent was performed to examine the inter- and intradomain motions of the two-domain enzyme yeast phosphoglycerate kinase without the presence of substrates. To elucidate contributions from individual domains, simulations were carried out on the complete enzyme as well as on each isolated domain. The enzyme is known to undergo a hinge-bending type of motion as it cycles from an open to a closed conformation to allow the phosphoryl transfer occur. Analysis of the correlation of atomic movements during the simulations confirms hinge bending in the nanosecond timescale: the two domains of the complete enzyme exhibit rigid body motions anticorrelated with respect to each other. The correlation of the intradomain motions of both domains converges, yielding a distinct correlation map in the enzyme. In the isolated domain simulations-in which interdomain interactions cannot occur-the correlation of domain motions no longer converges and shows a very small correlation during the same simulation time. This result points to the importance of interdomain contacts in the overall dynamics of the protein. The secondary structure elements responsible for interdomain contacts are also discussed.
Project description:The deletion of phenylalanine 508 in the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator is directly associated with >90% of cystic fibrosis cases. This mutant protein fails to traffic out of the endoplasmic reticulum and is subsequently degraded by the proteasome. The effects of this mutation may be partially reversed by the application of exogenous osmolytes, expression at low temperature, and the introduction of second site suppressor mutations. However, the specific steps of folding and assembly of full-length cystic fibrosis transmembrane conductance regulator (CFTR) directly altered by the disease-causing mutation are unclear. To elucidate the effects of the ?F508 mutation, on various steps in CFTR folding, a series of misfolding and suppressor mutations in the nucleotide binding and transmembrane domains were evaluated for effects on the folding and maturation of the protein. The results indicate that the isolated NBD1 responds to both the ?F508 mutation and intradomain suppressors of this mutation. In addition, identification of a novel second site suppressor of the defect within the second transmembrane domain suggests that ?F508 also effects interdomain interactions critical for later steps in the biosynthesis of CFTR.
Project description:Proteins with similar crystal structures can have dissimilar rates of substrate binding and catalysis. Here we used molecular dynamics simulations and biochemical analysis to determine the role of intradomain and interdomain motions in conferring distinct activation rates to two G? proteins, G?(i1) and GPA1. Despite high structural similarity, GPA1 can activate itself without a receptor, whereas G?(i1) cannot. We found that motions in these proteins vary greatly in type and frequency. Whereas motion is greatest in the Ras domain of G?(i1), it is greatest in helices ?A and ?B from the helical domain of GPA1. Using protein chimeras, we show that helix ?A from GPA1 is sufficient to confer rapid activation to G?(i1). G?(i1) has less intradomain motion than GPA1 and instead displays interdomain displacement resembling that observed in a receptor-heterotrimer crystal complex. Thus, structurally similar proteins can have distinct atomic motions that confer distinct activation mechanisms.