The cyanobacterial clock proteins KaiA, KaiB and KaiC interact with each other to generate circadian oscillations. We have identified the residues of the KaiA homodimer affected through association with hexameric KaiC (KaiC6mer) using a spin-label-tagged KaiA C-terminal domain protein (KaiAc) and performing electron spin resonance (ESR) analysis. Cys substitution and/or the attachment of a spin label to residues located at the bottom area of the KaiAc concave surface, a KaiC-binding groove, hindered the association of KaiAc with KaiC6mer, suggesting that the groove likely mediates the interaction with KaiC6mer. The residues affected by KaiC6mer association were concentrated in the three areas: the concave surface, a lobe-like structure (a mobile lobe near the concave surface) and a region adjacent to both the concave surface and the mobile lobe. The distance between the two E254, D255, L258 and R252 residues located on the mobile lobe decreased after KaiC association, suggesting that the two mobile lobes approach each other during the interaction. Analyzing the molecular dynamics of KaiAc showed that these structural changes suggested by ESR analysis were possible. Furthermore, the analyses identified three asymmetries in KaiAc dynamic structures, which gave us a possible explanation of an asymmetric association of KaiAc with KaiC6mer.
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 ?-barrel domain (Trp27/Cys64 and Trp27/Cys97) and the interdomain pair is between the ?-helical domain and the ?-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 ?s 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 ?-barrel domain decreases to nearly native values whereas that of the interdomain donor/acceptor pairs remains unchanged in the earliest intermediate (<500 ?s of refolding). This indicates a rapid nonuniform collapse resulting in an ensemble of heterogeneous conformations in which the central region of the ?-barrel domain is well developed while the C-terminal ?-helical domain remains disordered. The distance between Trp140 and Cys64 decreases to native values on the 100-ms time scale, indicating that the ?-helical domain docks onto the preformed ?-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.
We studied the role of the 2 salt bridges (Asp143-Arg147 and Asp146-Arg150) in helix 1 of mouse prion protein (PrP) on the formation of the complex between PrP and the monoclonal antibody T2. We introduced 6 charge-changing mutations to the amino acid residues associated with the salt bridges. Analysis of the circular dichroism spectra of the mutant PrPs showed that the salt bridge mutations did not change the secondary structures. We analyzed the kinetics of the association and dissociation of the PrPs with the T2 antibody. The results showed that the association kinetics were not significantly different among the variants except Arg150Lys, while the dissociation rate of the neutralized-charge variants was 2 orders of magnitude higher than that of the wild type. These results indicate that salt bridges make the interaction of PrP with T2 tighter by slowing down dissociation.
Is the folding pathway conserved in homologous proteins? To address this question, we compared the folding pathways of goat alpha-lactalbumin and canine milk lysozyme using equilibrium and kinetic circular dichroism spectroscopy. Both Ca(2+)-binding proteins have 41% sequence identity and essentially identical backbone structures. The Phi-value analysis, based on the effect of Ca(2+) on the folding kinetics, showed that the Ca(2+)-binding site was well organized in the transition state in alpha-lactalbumin, although it was not yet organized in lysozyme. Equilibrium unfolding and hydrogen-exchange 2D NMR analysis of the molten globule intermediate also showed that different regions were stabilized in the two proteins. In alpha-lactalbumin, the Ca(2+)-binding site and the C-helix were weakly organized, whereas the A- and B-helices, both distant from the Ca(2+)-binding site, were well organized in lysozyme. The results thus provide an example of highly homologous proteins taking different folding pathways. To understand the molecular origin of this difference, we investigated the native three-dimensional structures of the proteins in terms of non-local contact clusters, a parameter based on the residue-residue contact map and known to be well correlated with the folding rate of non-two-state proteins. There were remarkable differences between the proteins in the distribution of the non-local contact clusters, and these differences provided a reasonable explanation of the observed difference in the folding initiation sites. In conclusion, the protein folding pathway is determined not only by the backbone topology but also by the specific side-chain interactions of contacting residues.
Tear lipocalin and beta-lactoglobulin are members of the lipocalin superfamily. They have similar tertiary structures but unusually low overall sequence similarity. Non-native helical structures are formed during the early stage of beta-lactoglobulin folding. To address whether the non-native helix formation is found in the folding of other lipocalin superfamily proteins, the folding kinetics of a tear lipocalin variant were investigated by stopped-flow methods measuring the time-dependent changes in circular dichroism (CD) spectrum and small-angle X-ray scattering (SAXS). CD spectrum showed that extensive secondary structures are not formed during a burst-phase (within a measurement dead time). The SAXS data showed that the radius of gyration becomes much smaller than in the unfolded state during the burst-phase, indicating that the molecule is collapsed during an early stage of folding. Therefore, non-native helix formation is not general for folding of all lipocalin family members. The non-native helix content in the burst-phase folding appears to depend on helical propensities of the amino acid sequence.
The acid transition of ?(2)-microglobulin (?2m) was studied by tryptophan fluorescence, peptide circular dichroism, and NMR spectroscopy. The protein exhibits a three-state transition with an equilibrium intermediate accumulated at pH4 (25°C). The pH4 intermediate has typical characteristics of the molten globule (MG) state; it showed a native-like secondary structure without specific side-chain tertiary structure, and the hydrodynamic radius determined by pulse field gradient NMR was only 20% larger than that of the native state. The accumulation of the pH4 intermediate is very analogous to the behavior of apomyoglobin, for which the pH4 MG has been well characterized, although ?2m, a ?-protein, is structurally very different from ?-helical apomyoglobin. NMR pH titration of histidine residues of ?2m has also indicated that His84 has an abnormally low pK(a) value in the native state. From the pH dependence of the unfolding transition, the protonations of this histidine and 10 weakly abnormal carboxylates triggered the transition from the native to the MG state. This behavior is again analogous to that of apomyoglobin, suggesting a common mechanism of production of the pH4 MG. In contrast to the folding of apomyoglobin, in which the MG was equivalent to the burst-phase kinetic folding intermediate, the burst-phase refolding intermediate of ?2m, detected by stopped-flow circular dichroism, was significantly more structured than the pH4 intermediate. It is proposed that the folding of ?2m from its acid-denatured state takes place in the following order: denatured state?MG?burst-phase intermediate?native state.
The kinetic folding of ?(2)-microglobulin from the acid-denatured state was investigated by interrupted-unfolding and interrupted-refolding experiments using stopped-flow double-jump techniques. In the interrupted unfolding, we first unfolded the protein by a pH jump from pH7.5 to pH2.0, and the kinetic refolding assay was carried out by the reverse pH jump by monitoring tryptophan fluorescence. Similarly, in the interrupted refolding, we first refolded the protein by a pH jump from pH2.0 to pH7.5 and used a guanidine hydrochloride (GdnHCl) concentration jump as well as the reverse pH jump as unfolding assays. Based on these experiments, the folding is represented by a parallel-pathway model, in which the molecule with the correct Pro32 cis isomer refolds rapidly with a rate constant of 5-6 s(-1), while the molecule with the Pro32 trans isomer refolds more slowly (pH7.5 and 25°C). At the last step of folding, the native-like trans conformer produced on the latter pathway isomerizes very slowly (0.001-0.002 s(-1)) into the native cis conformer. In the GdnHCl-induced unfolding assays in the interrupted refolding, the native-like trans conformer unfolded remarkably faster than the native cis conformer, and the direct GdnHCl-induced unfolding was also biphasic, indicating that the native-like trans conformer is populated at a significant level under the native condition. The one-dimensional NMR and the real-time NMR experiments of refolding further indicated that the population of the trans conformer increases up to 7-9% under a more physiological condition (pH7.5 and 37°C).
Equilibria and kinetics of folding/unfolding of ?-lactalbumin and its two N-terminal variants were studied by circular dichroism spectroscopy. The two variants were wild-type recombinant and Glu1-deletion (E1M) variants expressed in Escherichia coli. The presence of an extra methionine at the N terminus in recombinant ?-lactalbumin destabilized the protein by 2 kcal/mol, while the stability was recovered in the E1M variant in which Glu1 was replaced by Met1. Kinetic folding/unfolding reactions of the proteins, induced by stopped-flow concentration jumps of guanidine hydrochloride, indicated the presence of a burst-phase in refolding, and gave chevron plots with significant curvatures in both the folding and unfolding limbs. The folding-limb curvature was interpreted in terms of accumulation of the burst-phase intermediate. However, there was no burst phase observed in the unfolding kinetics to interpret the unfolding-limb curvature. We thus assumed a sequential four-state mechanism, in which the folding from the burst-phase intermediate takes place via two transition states separated by a high-energy intermediate. We estimated changes in the free energies of the burst-phase intermediate and two transition states, caused by the N-terminal variations and also by the presence of stabilizing calcium ions. The ? values at the N terminus and at the Ca(2+)-binding site thus obtained increased successively during folding, demonstrating the validity of the sequential mechanism. The stability and the folding behavior of the E1M variant were essentially identical to those of the authentic protein, allowing us to use this variant as a pseudo-wild-type ?-lactalbumin in future studies.
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