Detailed understanding of protein function and malfunction hinges on the ability to characterize transiently populated states and the transitions between them. Here, we use 15 N, 1 H N , and 13 CO NMR R 2 relaxation dispersion to investigate spontaneous unfolding and refolding events of native apomyoglobin. Above pH 5.0, dispersion is dominated by processes involving fluctuations of the F-helix region, which is invisible in NMR spectra. Measurements of R 2 dispersion for residues contacted by the F-helix region in the native (N W ithin the cell, unfolding and refolding of proteins occurs constantly, and spontaneous unfolding and misfolding processes play a central role in the formation of amyloid fibrils (1). In addition, fluctuations between native protein structure and partially or fully unfolded states have functional significance for binding, allosteric regulation, translocation across membranes, protein trafficking, secretion, and degradation (2). Despite the importance of spontaneous unfolding for protein function and cellular proteostasis, little is known about the transient, partially unfolded states that are formed. Detailed structural characterization of such states is difficult because of their inherently low populations and the conformational heterogeneity present when both native and partially unfolded states simultaneously exist in solution.Carr-Purcell-Meiboom-Gill (CPMG)-based R 2 relaxation dispersion experiments are unique in their ability to measure the kinetics of microsecond-millisecond exchange processes involving transient states with populations as sparse as 1%, and, in addition, allow determination of the associated NMR chemical shift changes (Δω) (3-5). In these experiments, the effective transverse relaxation rate R eff 2 is deconvoluted into the contribution from the exchange process, R ex , which varies with the pulsing frequency in the CPMG refocusing element, and the intrinsic relaxation rate R 0 2 . Here, we apply a series of amide and carbonyl R 2 dispersion experiments to investigate transient unfolding and refolding events within the native (N) state freeenergy landscape of apomyoglobin (apoMb).ApoMb is an ideal model system for studies of protein folding/ unfolding equilibria. The folded state is marginally stable at neutral pH, adopting a similar tertiary structure to the holoprotein except for disorder in the EF loop, F helix, FG loop, and N-terminal region of the G helix (residues 82-104), which appear to fluctuate between a folded native-like conformational substate and an ensemble of locally unfolded states (6, 7). ApoMb forms an equilibrium molten globule (MG) state at pH 4.1, amenable to direct investigation by NMR, which contains a significant number of native contacts and an overall compactness close to that of the N state (8-10). Kinetic refolding experiments show rapid formation of a burst-phase intermediate that resembles the equilibrium MG and contains native-like topology in the most structured regions (10-12). Folding to the N state from the MG is the overall ra...
Factor for inversion stimulation (FIS) is a 22 kDa homodimeric protein found in enteric bacteria that is involved in the stimulation of certain DNA recombination events and transcription regulation of many genes. FIS has a central helix with a 20 degrees kink, which is only reduced by 4 degrees after a proline 61 to alanine mutation (P61A). This mutation appears to have little effect on FIS function, yet it is striking that proline 61 is highly conserved among fis genes. Therefore, we studied the role of proline 61 on the stability and flexibility of FIS. The urea-induced equilibrium denaturation of P61A FIS was monitored by circular dichroism and fluorescence anisotropy. Despite the apparent two-state transition, the concentration dependence of the transition slope (m value) shows that a two-state model, as seen for wild-type (WT) FIS, did not adequately describe the denaturation of P61A FIS. Global fitting of the data indicates that the denaturation of P61A FIS occurs via a three-state process involving a dimeric intermediate and has an overall DeltaG(H2O) for unfolding of 18.6 kcal/mol, 4 kcal/mol higher than that for WT FIS. Limited trypsin proteolysis experiments show that the DNA binding C-terminus of P61A FIS is more labile to cleavage than that of WT FIS, suggesting an increased flexibility of this region in P61A FIS. In contrast, the resulting dimeric core (residues 6-71) of P61A FIS is more resistant to proteolysis, consistent with the presence of a dimeric intermediate not seen in WT FIS. Model transition curves generated using the parameters obtained by global fitting predicted a two-state-like transition at low P61A concentrations that becomes less cooperative with increasing protein concentration, as was experimentally observed. At concentrations of P61A FIS much higher than are experimentally feasible, a biphasic transition is predicted. Thus, this work demonstrates that a single mutation may be sufficient to alter a protein's denaturation mechanism and underscores the importance of analyzing the denaturation mechanism of oligomeric proteins over a wide concentration range. These results suggest that proline 61 in FIS may be conserved in order to optimize the global stability and the dynamics of the functionally important C-terminus.
Fis is a nucleoid-associated protein that interacts with poorly related DNA sequences with a high degree of specificity. A difference of more than 3 orders of magnitude in apparent K d values was observed between specific (K d , ϳ1 to 4 nM) and nonspecific (K d , ϳ4 M) DNA binding. To examine the contributions of Fis residues to the high-affinity binding at different DNA sequences, 13 alanine substitutions were generated in or near the Fis helix-turn-helix DNA binding motif, and the resulting proteins were purified. In vitro binding assays at three different Fis sites (fis P II, hin distal, and attR) revealed that R85, T87, R89, K90, and K91 played major roles in high-affinity DNA binding and that R85, T87, and K90 were consistently vital for binding to all three sites. Other residues made variable contributions to binding, depending on the binding site. N84 was required only for binding to the attR Fis site, and the role of R89 was dramatically altered by the attR DNA flanking sequence. The effects of Fis mutations on fis P II or hin distal site binding in vitro generally correlated with their abilities to mediate fis P repression or DNA inversion in vivo, demonstrating that the in vitro DNAbinding effects are relevant in vivo. The results suggest that while Fis is able to recognize a minimal common set of DNA sequence determinants at different binding sites, it is also equipped with a number of residues that contribute to the binding strength, some of which play variable roles.Fis (factor for inversion stimulation) is the most abundant nucleoid-associated protein during the logarithmic growth phase in rapidly growing Escherichia coli cells (3, 6). However, during mid-to late-logarithmic growth, the intracellular levels of Fis decrease over 500-fold and become nearly imperceptible during the stationary phase. The impact of Fis on cell physiology is widespread. As a nucleoid-associated protein, it is able to interact with a large number of DNA sites to alter DNA topology (66,67). Numerous genes are subject to positive or negative regulation by Fis directly or indirectly (6,10,19,22,30,49,56,61,(73)(74)(75). In the case of the promoters rrnB P1 and proP P2, Fis binding to sites centered at positions Ϫ71 and Ϫ41, respectively, directly stimulates transcription by contacting the C-terminal domain of the RNA polymerase ␣ subunit (␣-CTD) (9, 43). In the case of the fis promoter (fis P), Fis binding to sites I and II, centered at positions ϩ25 and Ϫ44, respectively, negatively autoregulates the fis operon by hindering RNA polymerase binding (6, 50, 58). As its name suggests, Fis also stimulates site-specific DNA inversion involving the Hin, Gin, and Cin family of recombinases (24,32,35). During Hin-mediated DNA inversion, Fis binds to two high-affinity DNA sites (hin-proximal and -distal sites) in a 60-bp recombinational enhancer region and interacts with two DNA-bound Hin recombinases to form a nucleoprotein complex intermediate required for efficient DNA strand cleavage and inversion (25,33). Bacteriophage DNA excis...
Two crossed-linked variants of the homodimeric DNA binding protein factor for inversion stimulation (FIS) were created via engineering of single intermolecular disulfide bonds. The conservative S30C and the nonconservative V58C FIS independent mutations resulted in FIS crossed-linked at the A helix (C30-C30) and at the middle of the B helix (C58-C58). This study sought to investigate how the location of an intermolecular disulfide bond may determine the effect on stability and its propagation through the structure to preserve or alter the denaturation cooperativity of FIS. The oxidized and reduced S30C and V58C FIS exhibited a far-UV CD spectrum and DNA binding affinities that were similar to WT FIS, indicating no significant changes in secondary and tertiary structure. However, the reduced and oxidized forms of the mutants revealed significant differences in the stability and equilibrium denaturation mechanism between the two mutants. In the reduced state, S30C FIS had very little effect on FIS stability, whereas V58C FIS was 2-3 kcal/mol less stable than WT FIS. Interestingly, while both disulfide bonds significantly increased the resistance to urea- and guanidine hydrochloride (GuHCl)-induced denaturation, oxidized V58C FIS exhibited a three-state GuHCl-induced transition. In contrast, oxidized S30C FIS displayed a highly cooperative WT-like transition with both denaturants. The three-state denaturation mechanism of oxidized V58C FIS induced by the GuHCl salt was reproduced by urea denaturation at pH 4, suggesting that disruption of a C-terminus salt-bridge network is responsible for the loss of denaturation cooperativity of V58C FIS in GuHCl or urea, pH 4. A second mutation on V58C FIS created to place a single tryptophan probe (Y95W) at the C-terminus further implies that the denaturation intermediate observed in disulfide crossed-linked V58C FIS results from a decoupling of the stabilities of the C-terminus and the rest of the protein. These results show that, unlike the C30-C30 intermolecular disulfide bond, the C58-C58 disulfide bond did not evenly stabilize the FIS structure, thereby highlighting the importance of the location of an engineered disulfide bond on the propagation of stability and the denaturation cooperativity of a protein.
The factor for inversion stimulation (FIS) is a homodimeric DNA-binding protein found in enteric bacteria. FIS consists of 98 residues and self-assembles into an entwined dimer containing a flexible and mostly disordered N-terminus followed by four alpha-helices. Proline 61, which is 100% conserved in FIS homologues, is located at the center of helix B, and its substitution for alanine (P61A) was previously shown to result in nonuniform stabilization of the protein, leading to the appearance of a marginally populated dimeric intermediate in urea denaturation equilibrium studies. Here we show that, in contrast to WT FIS, the thermal denaturation of P61A FIS was incomplete and yielded a transition curve that was independent of FIS concentration, suggesting the presence of a dimeric intermediate at 90 degrees C. In the presence of urea, the thermal denaturation of P61A FIS became concentration dependent, consistent with the denaturation of the dimeric intermediate. The existence of a thermostable dimeric intermediate of P61A FIS was further confirmed by glutaraldehyde cross-linking experiments at 95 degrees C. Urea denaturation experiments at 90 degrees C revealed a cooperative transition, indicating that the dimeric intermediate of P61A FIS has a solvent-protected hydrophobic core. P61A FIS, unlike the WT protein, was found to be resistant to denaturation by low pH, but its thermal denaturation at pH 3.5 revealed a biphasic transition, providing clues about the structure of the dimeric intermediate. From a functional perspective, it is plausible that the full conservation of proline 61 in FIS may serve to limit the stability and proteolytic resistance of this highly regulated transcription factor.
Factor for inversion stimulation (FIS), a 98-residue homodimeric protein, does not contain tryptophan (Trp) residues but has four tyrosine (Tyr) residues located at positions 38, 51, 69, and 95. The equilibrium denaturation of a P61A mutant of FIS appears to occur via a three-state (N 2 I 2 2U) process involving a dimeric intermediate (I 2 ). Although it was suggested that this intermediate had a denatured C-terminus, direct evidence was lacking. Therefore, three FIS double mutants, P61A/Y38W, P61A/Y69W, and P61A/Y95W were made, and their denaturation was monitored by circular dichroism and Trp fluorescence. Surprisingly, the P61A/Y38W mutant best monitored the N 2 I 2 transition, even though Trp38 is buried within the dimer removed from the C-terminus. In addition, although Trp69 is located on the protein surface, the P61A/Y69W FIS mutant exhibited clearly biphasic denaturation curves. In contrast, P61A/Y95W FIS was the least effective in decoupling the two transitions, exhibiting a monophasic fluorescence transition with modest concentration-dependence. When considering the local environment of the Trp residues and the effect of each mutation on protein stability, these results not only confirm that P61A FIS denatures via a dimeric intermediate involving a disrupted C-terminus but also suggest the occurrence of conformational changes near Tyr38. Thus, the P61A mutation appears to compromise the denaturation cooperativity of FIS by failing to propagate stability to those regions involved mostly in intramolecular interactions. Furthermore, our results highlight the challenge of anticipating the optimal location to engineer a Trp residue for investigating the denaturation mechanism of even small proteins.
Significant resource is spent by drug discovery project teams to generate numerous, yet unique target constructs for the multiple platforms used to drive drug discovery programs including: functional assays, biophysical studies, structural biology, and biochemical high throughput screening campaigns. To improve this process, we developed Modular Protein Ligation (MPL), a combinatorial reagent platform utilizing Expressed Protein Ligation to site-specifically label proteins at the C-terminus with a variety of cysteine-lysine dipeptide conjugates. Historically, such proteins have been chemically labeled non-specifically through surface amino acids. To demonstrate the feasibility of this approach, we first applied MPL to proteins of varying size in different target classes using different recombinant protein expression systems, which were then evaluated in several different downstream assays. A key advantage to the implementation of this paradigm is that one construct can generate multiple final products, significantly streamlining the reagent generation for multiple early drug discovery project teams.
The earliest events in the folding of a protein are in general poorly understood. We used NMR R 2 relaxation dispersion experiments to study transient local collapse events in the unfolded-state (U) conformational ensemble of apomyoglobin (apoMb). Local residual secondary structure (seen in regions corresponding to the A, D, E, and H helices of the folded protein) is largely unchanged over the pH range of 2.3−2.75, yet a significant pH-dependent increase in the conformational exchange contribution to the R 2 relaxation rate (R ex ) indicates that transient intramolecular contacts occur on a microsecond to millisecond time scale at pH 2.75. A comparison of 15 N and 13 CO relaxation dispersion data at pH 2.75 for residues in the A, B, G, and H regions, which participate in the earliest folding intermediates, indicates that chain collapse and secondary structure formation are rapid and concomitant. Increasingly stabilizing conditions (lower temperature, higher pH) result in the observation of a relaxation dispersion in the C, CD, and E regions of the protein, which are known to fold at later stages. Mutation of Trp14 in the A-helix region to Ala eliminates conformational exchange throughout the protein, and the mutation of hydrophobic residues in other regions results in the selective inhibition of conformational exchange in the B, G, or H regions. The R 2 dispersion data for WT apoMb at pH 2.75 and 10 °C are best fit to a four-state model ABGH ⇆ AGH ⇆ U ⇆ ABCD that includes on-pathway (AGH and ABGH) and off-pathway (ABCD) transiently folded states, both of which are required to explain the behavior of the mutant proteins. The off-pathway intermediate is destabilized at higher temperatures. Our analysis provides insights into the earliest stages of apoMb folding where the collapsing polypeptide chain samples both productive and nonproductive states with stabilized secondary structure.
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