Stepwise Folding of an Autotransporter Passenger Domain Is Not Essential for Its Secretion

Autotransporters are a superfamily of virulence proteins produced by Gram-negative bacteria. They consist of an N-terminal β-helical domain (“passenger domain”) that is secreted into the extracellular space and a C-terminal β-barrel domain (“β-domain”) that anchors the protein to the outer membrane. Because the periplasm lacks ATP, vectorial folding of the passenger domain in a C-to-N-terminal direction has been proposed to drive the secretion reaction. Consistent with this hypothesis, mutations that disrupt the folding of the C terminus of the passenger domain of the Escherichia coli O157:H7 autotransporter EspP have been shown to cause strong secretion defects. Here, we show that point mutations introduced at specific locations near the middle or N terminus of the EspP β-helix that perturb folding also impair secretion, but to a lesser degree. Surprisingly, we found that even multiple mutations that potentially abolish the stability of several consecutive rungs of the β-helix only moderately reduce secretion efficiency. Although these results provide evidence that the free energy derived from passenger domain folding contributes to secretion efficiency, they also suggest that a significant fraction of the energy required for secretion is derived from another source.     

Bacterial Secretins Form Constitutively Open Pores Akin to General Porins

Proteins called secretins form large multimeric complexes that are essential for macromolecular transit across the outer membrane of Gram-negative bacteria. Evidence suggests that the channels formed by some secretin complexes are not tightly closed, but their permeability properties have not been well characterized. Here, we used cell-free synthesis coupled with spontaneous insertion into liposomes to investigate the permeability of the secretin PulD. Leakage assays using preloaded liposomes indicated that PulD allows the efflux of small fluorescent molecules with a permeation cutoff similar to that of general porins. Other secretins were also found to form similar pores. To define the polypeptide region involved in determining the pore size, we analyzed a collection of PulD variants and studied the roles of gates 1 and 2, which were previously reported to affect the pore size of filamentous phage f1 secretin pIV, in assembly and pore formation. Liposome leakage and a novel in vivo assay showed that replacement of the conserved proline residue at position 443 in PulD by leucine increased the apparent size of the pore. The in vitro approach described here could be used to study the pore properties of membrane proteins whose production in vivo is toxic.     

Artificial binding proteins (Affitins) as probes for conformational changes in secretin PulD

The DNA-binding protein Sac7d was previously modified to bind with high affinity to the N domain of the outer membrane secretin PulD from the bacterium Klebsiella oxytoca. Here, we show that binding of the Sac7d derivatives (affitins) to PulD is sensitive to conformational changes caused by denaturant and by the zwitterionic detergent Zwittergent 3-14 routinely used to extract secretins from outer membranes. This sensitivity to the conformational state of PulD allowed us to use the affitins as probes for the native structure of PulD and to devise protocols for examining in vitro synthesized protein in nonionic detergent and for the affinity purification of native PulD using affitins as ligands. When fused to periplasmic PhoA, three affitins inhibited PulD multimerization in vivo and caused loss of function. In two cases, this was likely to be due to dimerization of the affitin by the bound PhoA, as the effect was absent when the affitins were fused to monomeric MalE. In the third case, the MalE and PhoA moieties probably interfered sterically with PulD protomer interactions and, thereby, inhibited multimerization. None of the affitins tested interacted with PulD at sites of protomer interaction or blocked the secretin channel through which exoproteins cross the outer membrane in the Type II secretion pathway of which PulD is a key component.     

Malleability of the folding mechanism of the outer membrane protein PagP: parallel pathways and the effect of membrane elasticity

Understanding the interactions between membrane proteins and the lipid bilayer is key to increasing our ability to predict and tailor the folding mechanism, structure and stability of membrane proteins. Here, we have investigated the effects of changing the membrane composition and the relative concentrations of protein and lipid on the folding mechanism of the bacterial outer membrane protein PagP. The folding pathway, monitored by tryptophan fluorescence, was found to be characterized by a burst phase, representing PagP adsorption to the liposome surface, followed by a time course that reflects the folding and insertion of the protein into the membrane. In 1,2-dilauroyl-sn-glycero-3-phosphocholine (diC(12:0)PC) liposomes, the post-adsorption time course fits well to a single exponential at high lipid-to-protein ratios (LPRs), but at low LPRs, a second exponential phase with a slower folding rate constant is observed. Interrupted refolding assays demonstrated that the two exponential phases reflect the presence of parallel folding pathways. Partitioning between these pathways was found to be modulated by the elastic properties of the membrane. Folding into mixed 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine:diC(12:0)PC liposomes resulted in a decrease in PagP adsorption to the liposomes and a switch to the slower folding pathway. By contrast, inclusion of 1,2-dilauroyl-sn-glycero-3-phosphoserine into diC(12:0)PC liposomes resulted in a decrease in the folding rate of the fast pathway. The results highlight the effect of lipid composition in tailoring the folding mechanism of a membrane protein, revealing that membrane proteins have access to multiple, competing folding routes to a unique native structure.     

Degradation of Extracytoplasmic Catalysts for Protein Folding in Bacillus subtilis

The general protein secretion pathway of Bacillus subtilis has a high capacity for protein export from the cytoplasm, which is exploited in the biotechnological production of a wide range of enzymes. These exported proteins pass the membrane in an unfolded state, and accordingly, they have to fold into their active and protease-resistant conformations once membrane passage is completed. The lipoprotein PrsA and the membrane proteins HtrA and HtrB facilitate the extracytoplasmic folding and quality control of exported proteins. Among the native exported proteins of B. subtilis are at least 10 proteases that have previously been implicated in the degradation of heterologous secreted proteins. Recently, we have shown that these proteases also degrade many native membrane proteins, lipoproteins, and secreted proteins. The present studies were therefore aimed at assessing to what extent these proteases also degrade extracytoplasmic catalysts for protein folding. To this end, we employed a collection of markerless protease mutant strains that lack up to 10 different extracytoplasmic proteases. The results show that PrsA, HtrA, and HtrB are indeed substrates of multiple extracytoplasmic proteases. Thus, improved protein secretion by multiple-protease-mutant strains may be related to both reduced proteolysis and improved posttranslocational protein folding and quality control.     

Mechanisms Governing the Activation and Trafficking of Yeast G Protein-coupled Receptors

We have addressed the mechanisms governing the activation and trafficking of G protein-coupled receptors (GPCRs) by analyzing constitutively active mating pheromone receptors (Ste2p and Ste3p) of the yeast Saccharomyces cerevisiae. Substitution of the highly conserved proline residue in transmembrane segment VI of these receptors causes constitutive signaling. This proline residue may facilitate folding of GPCRs into native, inactive conformations, and/or mediate agonist-induced structural changes leading to G protein activation. Constitutive signaling by mutant receptors is suppressed upon coexpression with wild-type, but not G protein coupling-defective, receptors. Wild-type receptors may therefore sequester a limiting pool of G proteins; this apparent “precoupling” of receptors and G proteins could facilitate signal production at sites where cell surface projections form during mating partner discrimination. Finally, rather than being expressed mainly at the cell surface, constitutively active pheromone receptors accumulate in post-endoplasmic reticulum compartments. This is in contrast to other defective membrane proteins, which apparently are targeted by default to the vacuole. We suggest that the quality-control mechanism that retains receptors in post-endoplasmic reticulum compartments may normally allow wild-type receptors to fold into their native, fully inactive conformations before reaching the cell surface. This may ensure that receptors do not trigger a response in the absence of agonist.     

Co-factor Insertion and Disulfide Bond Requirements for Twin-arginine Translocase-dependent Export of the Bacillus subtilis Rieske Protein QcrA

The twin-arginine translocation (Tat) pathway can transport folded and co-factor-containing cargo proteins over bacterial cytoplasmic membranes. Functional Tat machinery components, a folded state of the cargo protein and correct co-factor insertion in the cargo protein are generally considered as prerequisites for successful translocation. The present studies were aimed at a dissection of these requirements with regard to the Rieske iron-sulfur protein QcrA of Bacillus subtilis. Notably, QcrA is a component of the cytochrome bc₁ complex, which is conserved from bacteria to man. Single amino acid substitutions were introduced into the Rieske domain of QcrA to prevent either co-factor binding or disulfide bond formation. Both types of mutations precluded QcrA translocation. Importantly, a proofreading hierarchy was uncovered, where a QcrA mutant defective in disulfide bonding was quickly degraded, whereas mutant QcrA proteins defective in co-factor binding accumulated in the cytoplasm and membrane. Altogether, these are the first studies on Tat-dependent protein translocation where both oxidative folding and co-factor attachment have been addressed in a single native molecule.     

Oxidation of an Exposed Methionine Instigates the Aggregation of Glyceraldehyde-3-phosphate Dehydrogenase

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous and abundant protein that participates in cellular energy production. GAPDH normally exists in a soluble form; however, following necrosis, GAPDH and numerous other intracellular proteins convert into an insoluble disulfide-cross-linked state via the process of “nucleocytoplasmic coagulation.” Here, free radical-induced aggregation of GAPDH was studied as an in vitro model of nucleocytoplasmic coagulation. Despite the fact that disulfide cross-linking is a prominent feature of GAPDH aggregation, our data show that it is not a primary rate-determining step. To identify the true instigating event of GAPDH misfolding, we mapped the post-translational modifications that arise during its aggregation. Solvent accessibility and energy calculations of the mapped modifications within the context of the high resolution native GAPDH structure suggested that oxidation of methionine 46 may instigate aggregation. We confirmed this by mutating methionine 46 to leucine, which rendered GAPDH highly resistant to free radical-induced aggregation. Molecular dynamics simulations suggest that oxidation of methionine 46 triggers a local increase in the conformational plasticity of GAPDH that likely promotes further oxidation and eventual aggregation. Hence, methionine 46 represents a “linchpin” whereby its oxidation is a primary event permissive for the subsequent misfolding, aggregation, and disulfide cross-linking of GAPDH. A critical role for linchpin residues in nucleocytoplasmic coagulation and other forms of free radical-induced protein misfolding should now be investigated. Furthermore, because disulfide-cross-linked aggregates of GAPDH arise in many disorders and because methionine 46 is irrelevant to native GAPDH function, mutation of methionine 46 in models of disease should allow the unequivocal assessment of whether GAPDH aggregation influences disease progression.     

Protein folding ‐ seeing is deceiving

This Perspective is intended to raise questions about the conventional interpretation of protein folding. According to the conventional interpretation, developed over many decades, a protein population can visit a vast number of conformations under unfolding conditions, but a single dominant native population emerges under folding conditions. Accordingly, folding comes with a substantial loss of conformational entropy. How is this price paid? The conventional answer is that favorable interactions between and among the side chains can compensate for entropy loss, and moreover, these interactions are responsible for the structural particulars of the native conformation. Challenging this interpretation, the Perspective introduces a proposal that high energy (i.e., unfavorable) excluding interactions winnow the accessible population substantially under physical–chemical conditions that favor folding. Both steric clash and unsatisfied hydrogen bond donors and acceptors are classified as excluding interactions, so called because conformers with such disfavored interactions will be largely excluded from the thermodynamic population. Both excluding interactions and solvent factors that induce compactness are somewhat nonspecific, yet together they promote substantial chain organization. Moreover, proteins are built on a backbone scaffold consisting of α‐helices and strands of β‐sheet, where the number of hydrogen bond donors and acceptors is exactly balanced. These repetitive secondary structural elements are the only two conformers that can be both completely hydrogen‐bond satisfied and extended indefinitely without encountering a steric clash. Consequently, the number of fundamental folds is limited to no more than ~10,000 for a protein domain. Once excluding interactions are taken into account, the issue of “frustration” is largely eliminated and the Levinthal paradox is resolved. Putting the “bottom line” at the top: it is likely that hydrogen‐bond satisfaction represents a largely under‐appreciated parameter in protein folding models.     

Understanding the Folding-Function Tradeoff in Proteins

When an amino-acid sequence cannot be optimized for both folding and function, folding can get compromised in favor of function. To understand this tradeoff better, we devise a novel method for extracting the “function-less” folding-motif of a protein fold from a set of structurally similar but functionally diverse proteins. We then obtain the β-trefoil folding-motif, and study its folding using structure-based models and molecular dynamics simulations. CompariA protein sequence serves two purpson with the folding of wild-type β-trefoil proteins shows that function affects folding in two ways: In the slower folding interleukin-1β, binding sites make the fold more complex, increase contact order and slow folding. In the faster folding hisactophilin, residues which could have been part of the folding-motif are used for function. This reduces the density of native contacts in functional regions and increases folding rate. The folding-motif helps identify subtle structural deviations which perturb folding. These may then be used for functional annotation. Further, the folding-motif could potentially be used as a first step in the sequence design of function-less scaffold proteins. Desired function can then be engineered into these scaffolds.     

A Two-step Mechanism for the Folding of Actin by the Yeast Cytosolic Chaperonin

Actin requires the chaperonin containing TCP1 (CCT), a hexadecameric ATPase essential for cell viability in eukaryotes, to fold to its native state. Following binding of unfolded actin to CCT, the cavity of the chaperone closes and actin is folded and released in an ATP-dependent folding cycle. In yeast, CCT forms a ternary complex with the phosducin-like protein PLP2p to fold actin, and together they can return nascent or chemically denatured actin to its native state in a pure in vitro folding assay. The complexity of the CCT-actin system makes the study of the actin folding mechanism technically challenging. We have established a novel spectroscopic assay through selectively labeling the C terminus of yeast actin with acrylodan and observe significant changes in the acrylodan fluorescence emission spectrum as actin is chemically unfolded and then refolded by the chaperonin. The variation in the polarity of the environment surrounding the fluorescent probe during the unfolding/folding processes has allowed us to monitor actin as it folds on CCT. The rate of actin folding at a range of temperatures and ATP concentrations has been determined for both wild type CCT and a mutant CCT, CCT4anc2, defective in folding actin in vivo. Binding of the non-hydrolysable ATP analog adenosine 5′-(β,γ-imino)triphosphate to the ternary complex leads to 3-fold faster release of actin from CCT following addition of ATP, suggesting a two-step folding process with a conformational change occurring upon closure of the cavity and a subsequent final folding step involving packing of the C terminus to the native-like state.     

Chaperones GroEL/GroES Accelerate the Refolding of a Multidomain Protein through Modulating On-pathway Intermediates

Despite a vast amount information on the interplay of GroEL, GroES, and ATP in chaperone-assisted folding, the molecular details on the conformational dynamics of folding polypeptide during its GroEL/GroES-assisted folding cycle is quite limited. Practically no such studies have been reported to date on large proteins, which often have difficulty folding in vitro. The effect of the GroEL/GroES chaperonin system on the folding pathway of an 82-kDa slow folding protein, malate synthase G (MSG), was investigated. GroEL bound to the burst phase intermediate of MSG and accelerated the slowest kinetic phase associated with the formation of native topology in the spontaneous folding pathway. GroEL slowly induced conformational changes on the bound burst phase intermediate, which was then transformed into a more folding-compatible form. Subsequent addition of ATP or GroES/ATP to the GroEL-MSG complex led to the formation of the native state via a compact intermediate with the rate several times faster than that of spontaneous refolding. The presence of GroES doubled the ATP-dependent reactivation rate of bound MSG by preventing multiple cycles of its GroEL binding and release. Because GroES bound to the trans side of GroEL-MSG complex, it may be anticipated that confinement of the substrate underneath the co-chaperone is not required for accelerating the rate in the assisted folding pathway. The potential role of GroEL/GroES in assisted folding is most likely to modulate the conformation of MSG intermediates that can fold faster and thereby eliminate the possibility of partial aggregation caused by the slow folding intermediates during its spontaneous refolding pathway.     

Structural Characteristic of the Initial Unfolded State on Refolding Determines Catalytic Efficiency of the Folded Protein in Presence of Osmolytes

Osmolytes are low molecular weight organic molecules accumulated by organisms to assist proper protein folding, and to provide protection to the structural integrity of proteins under denaturing stress conditions. It is known that osmolyte-induced protein folding is brought by unfavorable interaction of osmolytes with the denatured/unfolded states. The interaction of osmolyte with the native state does not significantly contribute to the osmolyte-induced protein folding. We have therefore investigated if different denatured states of a protein (generated by different denaturing agents) interact differently with the osmolytes to induce protein folding. We observed that osmolyte-assisted refolding of protein obtained from heat-induced denatured state produces native molecules with higher enzyme activity than those initiated from GdmCl- or urea-induced denatured state indicating that the structural property of the initial denatured state during refolding by osmolytes determines the catalytic efficiency of the folded protein molecule. These conclusions have been reached from the systematic measurements of enzymatic kinetic parameters (K _m and k _cat), thermodynamic stability (T _m and ΔH _m) and secondary and tertiary structures of the folded native proteins obtained from refolding of various denatured states (due to heat-, urea- and GdmCl-induced denaturation) of RNase-A in the presence of various osmolytes.     

Interaction of DnaK with native proteins and membrane proteins correlates with their accessible hydrophobicity

Molecular chaperones are involved in protein folding, protein targeting to membranes, and protein renaturation after stress. They interact specifically with hydrophobic sequences that are exposed in unfolded proteins, and buried in native proteins. We have studied the interaction of DnaK with native water-soluble proteins and membrane proteins. DnaK–native protein interactions are characterized by dissociation constants between 1 and 50 μM (compared with 0.01–1 μM for unfolded proteins). This affinity is within the range of most intracellular protein concentrations, suggesting that DnaK interacts with a greater number of native proteins than previously suspected. We found a correlation between the affinity of native proteins for DnaK and their affinity for hydrophobic-interaction chromatography adsorbents, suggesting that DnaK interacts with exposed hydrophobic groups in native proteins. The interaction between DnaK and membrane proteins is characterized by DnaK's high affinity for detergent-solubilized membrane proteins, and its lower affinity for membrane proteins inserted in lipid bilayers, suggesting that the chaperone can interact with the hydrophobic sequences of the former, while it cannot penetrate the hydrophobic core of lipid bilayers. Thus, the specificity of DnaK for hydrophobic sequences is involved in its interaction with not only unfolded proteins, but also native water-soluble proteins and membrane proteins. All proteins interact with DnaK according to their exposed hydrophobicity.     

The denatured state dictates the topology of two proteins with almost identical sequence but different native structure and function

The protein folding problem is often studied by comparing the mechanisms of proteins sharing the same structure but different sequence. The recent design of the two proteins G_A88 and G_B88, displaying different structures and functions while sharing 88% sequence identity (49 out of 56 amino acids), allows the unique opportunity for a complementary approach. At which stage of its folding pathway does a protein commit to a given topology? Which residues are crucial in directing folding mechanisms to a given structure? By using a combination of biophysical and computational techniques, we have characterized the folding of both G_A88 and G_B88. We show that, contrary to expectation, G_B88, characterized by a native α+β fold, displays in the denatured state a content of native-like helical structure greater than G_A88, which is all-α in its native state. Both experiments and simulations indicate that such residual structure may be tuned by changing pH. Thus, despite the high sequence identity, the folding pathways for these two proteins appear to diverge as early as in the denatured state. Our results suggest a mechanism whereby protein topology is committed very early along the folding pathway, being imprinted in the residual structure of the denatured state.     

The Effect of Electrostatics on the Marginal Cooperativity of an Ultrafast Folding Protein

Proteins fold up by coordinating the different segments of their polypeptide chain through a network of weak cooperative interactions. Such cooperativity results in unfolding curves that are typically sigmoidal. However, we still do not know what factors modulate folding cooperativity or the minimal amount that ensures folding into specific three-dimensional structures. Here, we address these issues on BBL, a small helical protein that folds in microseconds via a marginally cooperative downhill process (Li, P., Oliva, F. Y., Naganathan, A. N., and Muñoz, V. (2009) Proc. Natl. Acad. Sci. USA. 106, 103–108). Particularly, we explore the effects of salt-induced screening of the electrostatic interactions in BBL at neutral pH and in acid-denatured BBL. Our results show that electrostatic screening stabilizes the native state of the neutral and protonated forms, inducing complete refolding of acid-denatured BBL. Furthermore, without net electrostatic interactions, the unfolding process becomes much less cooperative, as judged by the broadness of the equilibrium unfolding curve and the relaxation rate. Our experiments show that the marginally cooperative unfolding of BBL can still be made twice as broad while the protein retains its ability to fold into the native three-dimensional structure in microseconds. This result demonstrates experimentally that efficient folding does not require cooperativity, confirming predictions from theory and computer simulations and challenging the conventional biochemical paradigm. Furthermore, we conclude that electrostatic interactions are an important factor in determining folding cooperativity. Thus, electrostatic modulation by pH-salt and/or mutagenesis of charged residues emerges as an attractive tool for tuning folding cooperativity.     

In vitro protein folding by E. coli ribosome: unfolded protein splitting 70S to interact with 50S subunit

Folding of unfolded protein on Escherichia coli 70S ribosome is accompanied by rapid dissociation of the ribosome into 50S and 30S subunits. The dissociation rate of 70S ribosome with unfolded protein is much faster than that caused by combined effect of translation and polypeptide release factors known to be involved in the dissociation of ribosome into subunits. The protein then reaches a “folding competent” state on 50S and is released to take up native conformation by itself. Release before attaining the folding competent state or prevention of release by cross-linking it with ribosome, would not allow the protein to get back to its native conformation.     

Isolation, folding and structural investigations of the amino acid transporter OEP16

Membrane proteins compose more than 30% of all proteins in the living cell. However, many membrane proteins have low abundance in the cell and cannot be isolated from natural sources in concentrations suitable for structure analysis. The overexpression, reconstitution, and stabilization of membrane proteins are complex and remain a formidable challenge in membrane protein characterization. Here we describe a novel, in vitro folding procedure for a cation-selective channel protein, the outer envelope membrane protein 16 (OEP16) of pea chloroplast, overexpressed in Escherichia coli in the form of inclusion bodies. The protein is purified and then folded with detergent on a Ni–NTA affinity column. Final concentrations of reconstituted OEP16 of up to 24 mg/ml have been achieved, which provides samples that are sufficient for structural studies by NMR and crystallography. Reconstitution of OEP16 in detergent micelles was monitored by circular dichroism, fluorescence, and NMR spectroscopy. Tryptophan fluorescence spectra of heterologous expressed OEP16 in micelles are similar to spectra of functionally active OEP16 in liposomes, which indicates folding of the membrane protein in detergent micelles. CD spectroscopy studies demonstrate a folded protein consisting primarily of α-helices. ¹⁵N-HSQC NMR spectra also provide evidence for a folded protein. We present here a convenient, effective and quantitative method to screen large numbers of conditions for optimal protein stability by using microdialysis chambers in combination with fluorescence spectroscopy. Recent collection of multidimensional NMR data at 500, 600 and 800 MHz demonstrated that the protein is suitable for structure determination by NMR and stable for weeks during data collection.     

The Mitochondrial Intermembrane Space Oxireductase Mia40 Funnels the Oxidative Folding Pathway of the Cytochrome c Oxidase Assembly Protein Cox19

Mia40-catalyzed disulfide formation drives the import of many proteins into the mitochondria. Here we characterize the oxidative folding of Cox19, a twin CX₉C Mia40 substrate. Cox19 oxidation is extremely slow, explaining the persistence of import-competent reduced species in the cytosol. Mia40 accelerates Cox19 folding through the specific recognition of the third Cys in the second helical CX₉C motif and the subsequent oxidation of the inner disulfide bond. This renders a native-like intermediate that oxidizes in a slow uncatalyzed reaction into native Cox19. The same intermediate dominates the pathway in the absence of Mia40, and chemical induction of an α-helical structure by trifluoroethanol suffices to accelerate productive folding and mimic the Mia40 folding template mechanism. The Mia40 role is to funnel a rough folding landscape, skipping the accumulation of kinetic traps, providing a rationale for the promiscuity of Mia40.     

Role of Protein Stabilizers on the Conformation of the Unfolded State of Cytochrome c and Its Early Folding Kinetics: INVESTIGATION AT SINGLE MOLECULAR RESOLUTION*

An insight into the conformation and dynamics of unfolded and early intermediate states of a protein is essential to understand the mechanism of its aggregation and to design potent inhibitor molecules. Fluorescence correlation spectroscopy has been used to study the effects of several model protein stabilizers on the conformation of the unfolded state and early folding dynamics of tetramethyl rhodamine-labeled cytochrome c from Saccharomyces cerevisiae at single molecular resolution. Special attention has been given to arginine, which is a widely used stabilizer for improving refolding yield of different proteins. The value of the hydrodynamic radius (r_H) obtained by analyzing the intensity fluctuations of the diffusing molecules has been found to increase in a two-state manner as the protein is unfolded by urea. The results further show that the presence of arginine and other protein stabilizers favors a relatively structured conformation of the unfolded states (r_H of 29 Å) over an extended one (r_H of 40 Å), which forms in their absence. Also, the time constant of a kinetic component (τ_R) of about 30 μs has been observed by analyzing the correlation functions, which represents formation of a collapsed state. This time constant varies with urea concentration representing an inverted Chevron plot that shows a roll-over and behavior in the absence of arginine. To the best of our knowledge, this is one of the first applications of fluorescence correlation spectroscopy to study direct folding kinetics of a protein.