Reactive-site cleavage residues confer target specificity to baculovirus P49, a dimeric member of the P35 family of caspase inhibitors

Baculovirus proteins P49 and P35 are potent suppressors of apoptosis in diverse organisms. Although related, P49 and P35 inhibit initiator and effector caspases, respectively, during infection of permissive insect cells. The molecular basis of this novel caspase specificity is unknown. To advance strategies for selective inhibition of the cell death caspases, we investigated biochemical differences between these baculovirus substrate inhibitors. We report here that P49 and P35 use similar mechanisms for stoichiometric inhibition that require caspase cleavage of their reactive site loops (RSL) and chemical contributions of a conserved N-terminal cysteine to stabilize the resulting inhibitory complex. Our data indicated that P49 functions as a homodimer that simultaneously binds two caspases. In contrast, P35 is a monomeric, monovalent inhibitor. P49 and P35 also differ in their RSL caspase recognition sequences. We tested the role of the P₄-P₁ recognition motif for caspase specificity by monitoring virus-induced proteolytic processing of Sf-caspase-1, the principal effector caspase of the host insect Spodoptera frugiperda. When P49's TVTD recognition motif was replaced with P35's DQMD motif, P49 was impaired for inhibition of the initiator caspase that cleaves and activates pro-Sf-caspase-1 and instead formed a stable inhibitory complex with active Sf-caspase-1. In contrast, the effector caspase specificity of P35 was unaltered when P35's DQMD motif was replaced with TVTD. We concluded that the TVTD recognition motif is required but not sufficient for initiator caspase inhibition by P49. Our findings demonstrate a critical role for the P₄-P₁ recognition site in caspase specificity by P49 and P35 and indicate that additional determinants are involved in target selection.     

Crystal structure of baculovirus P35: role of a novel reactive site loop in apoptotic caspase inhibition

The aspartate-specific caspases are critical protease effectors of programmed cell death and consequently represent important targets for apoptotic intervention. Baculovirus P35 is a potent substrate inhibitor of metazoan caspases, a property that accounts for its unique effectiveness in preventing apoptosis in phylogenetically diverse organisms. Here we report the 2.2 A resolution crystal structure of P35, the first structure of a protein inhibitor of the death caspases. The P35 monomer possesses a solvent-exposed loop that projects from the protein's main beta-sheet core and positions the requisite aspartate cleavage site at the loop's apex. Distortion or destabilization of this reactive site loop by site-directed mutagenesis converted P35 to an efficient substrate which, unlike wild-type P35, failed to interact stably with the target caspase or block protease activity. Thus, cleavage alone is insufficient for caspase inhibition. These data are consistent with a new model wherein the P35 reactive site loop participates in a unique multi-step mechanism in which the spatial orientation of the loop with respect to the P35 core determines post-cleavage association and stoichiometric inhibition of target caspases.     

Crystal structure of an invertebrate caspase

Caspases play an essential role in the execution of apoptosis. These cysteine proteases are highly conserved among metazoans and are translated as inactive zymogens, which are activated by proteolytic cleavages to generate the large and small subunits and remove the N-terminal prodomain. The 2.3 A resolution crystal structure of active Sf-caspase-1, the principal effector caspase of the insect Spodoptera frugiperda, is presented here. The structure represents the first nonhuman caspase to be resolved. The structure of the cleaved and active protease was determined with the tetrapeptide inhibitor N-acetyl-Asp-Glu-Val-Asp-chloromethylketone covalently bonded to the active site cysteine. As expected, the overall fold of Sf-caspase-1 is exceedingly similar to that of the five active caspases from humans solved to date. The overall structure and active site arrangement of Sf-caspase-1 is most comparable with that of the human effector caspases, with which it shares highest sequence homology. The most prominent structural difference with Sf-caspase-1 is the position of the N-terminal region of the large subunit. Unlike the N terminus of human caspases, the N terminus of Sf-caspase-1 originates from the active site side where it interacts with active site loop L2 and then extends to the backside of the heterodimer. This unusual structural arrangement raises the possibility that the N-terminal prodomain plays a regulatory role during effector caspase activation or enzyme activity in insects.     

Mechanism-based inactivation of caspases by the apoptotic suppressor p35.

Caspases play a crucial role in the ability of animal cells to kill themselves by apoptosis. Caspase activity is regulated in vivo by members of three distinct protease inhibitor families, one of which--p35--has so far only been found in baculoviruses. P35 has previously been shown to rapidly form essentially irreversible complexes with its target caspases in a process that is accompanied by peptide bond cleavage. To determine the protease-inhibitory pathway utilized by this very selective protease inhibitor, we have analyzed the thermodynamic and kinetic stability of the protein. We show that the conformation of p35 is stabilized following cleavage within its reactive site loop. An inactive catalytic mutant of caspase 3 is bound by p35, but much less avidly than the wild-type enzyme, indicating that the protease catalytic nucleophile is required for stable complex formation. The inhibited protease is trapped as a covalent adduct, most likely with its catalytic Cys esterified to the carbonyl carbon of the scissile peptide bond. Together these data reveal that p35 is a mechanism-based inactivator that has adopted an inhibitory device reminiscent of the widely distributed serpin family, despite a complete lack of sequence or structural homology.     

The Global Conformation of an Active Hammerhead RNA during the Process of Self-cleavage

The RNA “hammerhead” domain is a small element of secondary structure found in the genomes of certain plant pathogens. It possesses a core of conserved sequence at the conjunction of three helix stems, and is capable of undergoing self-cleavage in the presence of divalent cations. Both crystallographic and solution studies suggest that the domain is highly structured, with the three stems assuming a Y-shaped global conformation; however, such studies have employed either RNA analogues that were catalytically inactive, or conditions of temperature and pH for which rates of self-cleavage are slow. Thus, it was unknown whether such species represented the principal conformers during the cleavage process itself. In order to address this issue, a series of time- resolved, transient electric birefringence measurements was conducted in an effort to define the global conformation of an RNA hammerhead in real time throughout the process of self-cleavage. The current study demonstrates that the angular relationship between the two helices that flank the cleavage center is essentially unchanged between the pre-cleavage and post-cleavage forms. Moreover, despite the fact that at least one kinetic intermediate is formed during the self-cleavage reaction, there is no evidence for the existence of a significant population of intermediates with altered global conformation during cleavage. Thus, any conformational isomerism that may occur is likely to be relatively localized to the active center. Finally, it was observed that sequence elements lying outside of the conserved region, at the base of stem I, influence interhelix geometry. The current results are consistent with a structural model in which the active center possesses similar conformations pre-cleavage and post-cleavage. Such a model would help to explain the significant rate of reversal of the cleavage reaction (self-ligation).     

Crystal structure of baculovirus P35 reveals a novel conformational change in the reactive site loop after caspase cleavage

Baculovirus P35 is a universal suppressor of apoptosis that stoichiometrically inhibits cellular caspases in a novel cleavage-dependent mechanism. Upon caspase cleavage at Asp-87, the 10- and 25-kDa cleavage products of P35 remain tightly associated with the inhibited caspase. Mutations in the alpha-helix of the reactive site loop preceding the cleavage site abrogate caspase inhibition and antiapoptotic activity. Substitution of Pro for Val-71, which is located in the middle of this alpha-helix, produces a protein that is cleaved at the requisite Asp-87 but does not remain bound to the caspase. This loss-of-function mutation provided the opportunity to structurally analyze the conformational changes of the P35 reactive site loop after caspase cleavage. We report here the 2.7 A resolution crystal structure of V71P-mutated P35 after cleavage by human caspase-3. The structure reveals a large movement in the carboxyl-terminal side of the reactive site loop that swings down and forms a new beta-strand that augments an existing beta-sheet. Additionally, the hydrophobic amino terminus releases and extends away from the protein core. Similar movements occur when P35 forms an inhibitory complex with human caspase-8. These findings suggest that the alpha-helix mutation may alter the sequential steps or kinetics of the conformational changes required for inhibition, thereby causing P35 loss of function.     

Conditional expression of anti-apoptotic protein p35 by Cre-mediated DNA recombination in cardiomyocytes from loxP-p35-transgenic mice

p35, a viral inhibitor of caspase, prevents cell death induced by various stimuli. We established an experimental system to study the involvement of caspases in cell death, using primary cultured cells from p35 transgenic mice in which the p35 open reading frame (ORF) had been disrupted by the insertion of a DNA segment flanked by loxP sites, the Cre recognition sites. In this system, p35 expression can be initiated by Cre recombinase. Cardiomyocytes, which are highly sensitive to hypoxic stress, were infected with an adenovirus carrying the cre gene (AxCANCre). Expression of p35 by infection with AxCANCre resulted in inhibition of caspase-3 activation and resistance to hypoxia-induced cell death. Hypoxia-induced cytochrome c release was also attenuated in p35-expressing cardiomyocytes. Our transgenic mice can be used as an experimental model for studying the involvement of caspases in various degenerative diseases as well as programmed cell death both in vitro and in vivo.     

Expression of the baculovirus p35 protein in tobacco affects cell death progression and compromises N gene-mediated disease resistance response to Tobacco mosaic virus

The p35 protein from baculovirus is a broad-range caspase inhibitor and suppresses programmed cell death in animals. We report here the effects of transgenic expression in tobacco of the p35 protein during the hypersensitive response (HR). Expression of p35 causes partial inhibition of nonhost HR triggered by bacteria and gene-for-gene HR triggered by virus. Infection of p35-expressing tobacco plants with Tobacco mosaic virus (TMV) disrupts N-mediated disease resistance, causing systemic spreading of the virus within a resistant background. Mutant variants altered in aspartate residues within the loop region of p35 are inefficient substrates for caspases in vitro, and they do not suppress caspase proteolytic activity in animal systems. Tobacco plants expressing these mutant variants of the p35 protein do not show inhibition of HR cell death or enhanced virus systemic movement. Thus, HR inhibition and TMV systemic spreading phenotype in p35-expressing plants correlate with the ability of the p35 protein to suppress caspase activity in animal systems. In addition, a C-terminal truncated variant of p35 is unable to suppress cell death in animals as well as HR cell death in transgenic tobacco. Our results provide evidence for the participation of caspase-like proteases during the HR. In addition, they suggest that timely activation of cell death is necessary for effective TMV containment within the primary infection site.     

Simple modifications of the serpin reactive site loop convert SCCA2 into a cysteine proteinase inhibitor: a critical role for the P3' proline in facilitating RSL cleavage

The human squamous cell carcinoma antigens (SCCA) 1 and 2 are members of the serpin family that are 92% identical in their amino acid sequence. Despite this similarity, they inhibit distinct classes of proteinases. SCCA1 neutralizes the papain-like cysteine proteinases, cathepsins (cat) S, L, and K; and SCCA2 inhibits the chymotrypsin-like serine proteinases, catG and human mast cell chymase. SCCA2 also can inhibit catS, as well as other papain-like cysteine proteinases, albeit at a rate 50-fold less than that of SCCA1. Analysis of the mechanism of inhibition by SCCA1 revealed that the reactive site loop (RSL) is important for cysteine proteinase inhibition. The inhibition of catS by a mutant SCCA2 containing the RSL of SCCA1 is comparable to that of wild-type SCCA1. This finding suggested that there were no motifs outside and only eight residues within the RSL that were directing catS-specific inhibition. The purpose of this study was to determine which of these residues might account for the marked difference in the ability of SCCA1 and SCCA2 to inhibit papain-like cysteine proteinases. SCCA2 molecules containing different RSL mutations showed that no single amino acid substitution could convert SCCA2 into a more potent cysteine proteinase inhibitor. Rather, different combinations of mutations led to incremental increases in catS inhibitory activity with residues in four positions (P1, P3', P4', and P11') accounting for 80% of the difference in activity between SCCA1 and SCCA2. Interestingly, the RSL cleavage site differed between wild-type SCCA2 and this mutant. Moreover, these data established the importance of a Pro residue in the P3' position for efficient inhibition of catS by both wild-type SCCA1 and mutated SCCA2. Molecular modeling studies suggested that this residue might facilitate positioning of the RSL within the active site of the cysteine proteinase.     

Caspase inhibitors of the P35 family are more active when purified from yeast than bacteria

Many insect viruses express caspase inhibitors of the P35 superfamily, which prevent defensive host apoptosis to enable viral propagation. The prototypical P35 family member, AcP35 from Autographa californica M nucleopolyhedrovirus, has been extensively studied. Bacterially purified AcP35 has been previously shown to inhibit caspases from insect, mammalian and nematode species. This inhibition occurs via a pseudosubstrate mechanism involving caspase-mediated cleavage of a "reactive site loop" within the P35 protein, which ultimately leaves cleaved P35 covalently bound to the caspase's active site. We observed that AcP35 purifed from Saccharomyces cerevisae inhibited caspase activity more efficiently than AcP35 purified from Escherichia coli. This differential potency was more dramatic for another P35 family member, MaviP35, which inhibited human caspase 3 almost 300-fold more potently when purified from yeast than bacteria. Biophysical assays revealed that MaviP35 proteins produced in bacteria and yeast had similar primary and secondary structures. However, bacterially produced MaviP35 possessed greater thermal stability and propensity to form higher order oligomers than its counterpart purified from yeast. Caspase 3 could process yeast-purified MaviP35, but failed to detectably cleave bacterially purified MaviP35. These data suggest that bacterially produced P35 proteins adopt subtly different conformations from their yeast-expressed counterparts, which hinder caspase access to the reactive site loop to reduce the potency of caspase inhibition, and promote aggregation. These data highlight the differential caspase inhibition by recombinant P35 proteins purified from different sources, and caution that analyses of bacterially produced P35 family members (and perhaps other types of proteins) may underestimate their activity.     

The p35 relative,p49, inhibits mammalian and Drosophila caspases including DRONC and protects against apoptosis

This study characterized the ability of a new member of the p35 family, p49, to inhibit a number of mammalian and insect caspases. p49 blocked apoptosis triggered by treatment with Fas ligand (FasL), Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or ultraviolet (UV) radiation but provided negligible protection against apoptosis induced by the chemotherapeutic drug cisplatin. The caspase cleavage site in p49 was determined, and mutation of the P1 residue of this site abolished the ability of p49 to inhibit caspases, implying that p49 inhibits caspases through an analogous suicide-substrate mechanism to p35. Unlike p35, p49 inhibited the upstream insect caspase DRONC.     

Linkage of catalysis and 5′ end recognition in ribonuclease RNase J

In diverse bacterial species, the turnover and processing of many RNAs is mediated by the ribonuclease RNase J, a member of the widely occurring metallo-β-lactamase enzyme family. We present crystal structures of Streptomyces coelicolor RNase J with bound RNA in pre- and post-cleavage states, at 2.27 Å and 2.80 Å resolution, respectively. These structures reveal snapshots of the enzyme cleaving substrate directionally and sequentially from the 5′ terminus. In the pre-cleavage state, a water molecule is coordinated to a zinc ion pair in the active site but is imperfectly oriented to launch a nucleophilic attack on the phosphate backbone. A conformational switch is envisaged that enables the in-line positioning of the attacking water and may be facilitated by magnesium ions. Adjacent to the scissile bond, four bases are stacked in a tightly sandwiching pocket, and mutagenesis results indicate that this organization helps to drive processive exo-ribonucleolytic cleavage. Like its numerous homologues, S. coelicolor RNase J can also cleave some RNA internally, and the structural data suggest how the preference for exo- versus endo-cleavage mode is linked with recognition of the chemical status of the substrate''s 5′ end.     

Specific pre-cleavage and post-cleavage complexes involved in the formation of SV40 late mRNA 3'' termini in vitro.

Complexes form between processing factors present in a crude nuclear extract from HeLa cells and a simian virus 40 (SV40) late pre-mRNA which spans the polyadenylation [poly(A)] site. A specific 'pre-cleavage complex' forms on the pre-mRNA before cleavage. Formation of this complex requires the highly conserved sequence AAUAAA: it is prevented by mutations in AAUAAA, and by annealing DNA oligonucleotides to that sequence. After cleavage, the 5' half-molecule is found in a distinct 'post-cleavage complex'. In contrast, the 3' half-molecule is released. After cleavage and polyadenylation, polyadenylated RNA also is released. De novo formation of the post-cleavage complex requires AAUAAA and a nearby 3' terminus. Competition experiments suggest that a component which recognizes AAUAAA is required for formation of both pre- and post-cleavage complexes.     

Isd11p protein activates the mitochondrial cysteine desulfurase Nfs1p protein

Cysteine desulfurases perform pyridoxal phosphate (PLP)-dependent desulfuration of cysteine. The key steps of the enzymatic cycle include substrate binding to PLP, formation of a covalent persulfide intermediate at the active site cysteine, and transfer of sulfur to recipients for use in various metabolic pathways. In Saccharomyces cerevisiae, the cysteine desulfurase Nfs1p and an accessory protein, Isd11p, are found primarily in mitochondria, and both are essential for cell viability. Although cysteine desulfurases are conserved from bacteria to humans, Isd11p is found only in eukaryotes and not in prokaryotes. Here we show that Isd11p activates Nfs1p. The enzyme without Isd11p was inactive and did not form the [(35)S]persulfide intermediate from the substrate [(35)S]cysteine. Addition of Isd11p to inactive Nfs1p induced formation of the persulfide. Remarkably, in a two-step assay, [(35)S]cysteine could be bound to the inactive Nfs1p in a PLP-dependent manner, and the enzyme could be subsequently induced to form the persulfide by addition of Isd11p. A mutant form of Isd11p with the (15)LYK(17) motif changed to (15)AAA(17) was able to bind but failed to activate Nfs1p, thus separating these two functions of Isd11p. Finally, compared with Nfs1p with or without the bound Isd11p mutant, the Nfs1p·Isd11p complex was more resistant to inactivation by an alkylating agent. On the basis of these novel findings, we propose that interaction of Isd11p with Nfs1p activates the enzyme by inducing a conformational change, thereby promoting formation of the persulfide intermediate at the active site cysteine. Such a conformational change may protect the active site cysteine from alkylating agents.     

Calmodulin binding and Cdk5 phosphorylation of p35 regulate its effect on microtubules

In the nervous system, Cdk5 and its neuronal activator p35 are involved in the control of various activities, including neuronal differentiation and migration. Recently, we have reported that p35 is a microtubule-associated protein that regulates microtubule dynamics ( Hou, Z., Li, Q., He, L., Lim, H. Y., Fu, X., Cheung, N. S., Qi, D. X., and Qi, R. Z. (2007) J. Biol. Chem. 282, 18666-18670 ). Here we present two regulatory modes of p35 function as a microtubule-associated protein. First, p35 is Ca(2+)-dependent calmodulin (CaM)-binding protein. The CaM- and microtubule binding domains are localized to overlapping regions at the N terminus of p35. Within the CaM-binding region, Ala substitution for Trp-52 abolishes the CaM-binding activity, corroborating specific CaM-binding of p35. Furthermore, CaM blocks p35 association with microtubules in a Ca(2+)-specific manner, suggesting that p35 may be involved in the Ca(2+)/CaM-mediated inhibition of microtubule assembly. Second, p35 phosphorylation by Cdk5 interferes with the microtubule-binding and polymerizing activities of p35. Using a mutational approach, we found that only phosphorylation at Thr-138, one of the two residues primarily phosphorylated in vivo, inhibits the polymerizing activity. In PC12 cells, expression of p35 promotes nerve growth factor-induced neurite outgrowth under a Cdk5 inhibitory condition. Such p35 activity is impaired by the phosphomimetic mutation of Thr-138. These data suggest that Thr-138 phosphorylation plays a critical role in the control of the p35 functions in microtubule assembly and neurite outgrowth.     

Blocked negative selection of developing T cells in mice expressing the baculovirus p35 caspase inhibitor.

Clonal deletion in the thymus by apoptosis is involved in purging the immune system of self-reactive T lymphocytes (negative selection). Cysteine proteases (caspases) belonging to the CPP32 family are activated during this process. We have produced transgenic mice expressing baculovirus p35, a broad-range caspase inhibitor. Thymocytes from p35 transgenic mice were resistant in vitro to several apoptosis-inducing agents; this resistance correlated with the inhibition of CPP32-like activity. Negative selection in vivo of thymocytes triggered by two exogenous antigens, staphylococcal enterotoxin B superantigen and an antigenic peptide in the F5 T-cell receptor transgenic model, was specifically inhibited in p35 transgenic mice. Our results provide direct evidence for caspase involvement in negative selection during thymocyte development.     

Pannexin 1, an ATP release channel, is activated by caspase cleavage of its pore-associated C-terminal autoinhibitory region

Pannexin 1 (PANX1) channels mediate release of ATP, a "find-me" signal that recruits macrophages to apoptotic cells; PANX1 activation during apoptosis requires caspase-mediated cleavage of PANX1 at its C terminus, but how the C terminus inhibits basal channel activity is not understood. Here, we provide evidence suggesting that the C terminus interacts with the human PANX1 (hPANX1) pore and that cleavage-mediated channel activation requires disruption of this inhibitory interaction. Basally silent hPANX1 channels localized on the cell membrane could be activated directly by protease-mediated C-terminal cleavage, without additional apoptotic effectors. By serial deletion, we identified a C-terminal region just distal to the caspase cleavage site that is required for inhibition of hPANX1; point mutations within this small region resulted in partial activation of full-length hPANX1. Consistent with the C-terminal tail functioning as a pore blocker, we found that truncated and constitutively active hPANX1 channels could be inhibited, in trans, by the isolated hPANX1 C terminus either in cells or when applied directly as a purified peptide in inside-out patch recordings. Furthermore, using a cysteine cross-linking approach, we showed that relief of inhibition following cleavage requires dissociation of the C terminus from the channel pore. Collectively, these data suggest a mechanism of hPANX1 channel regulation whereby the intact, pore-associated C terminus inhibits the full-length hPANX1 channel and a remarkably well placed caspase cleavage site allows effective removal of key inhibitory C-terminal determinants to activate hPANX1.     

The crystal structure of recombinant proDer p 1, a major house dust mite proteolytic allergen

Allergy to house dust mite is among the most prevalent allergic diseases worldwide. Most house dust mite allergic patients react to Der p 1 from Dermatophagoides pteronyssinus, which is a cysteine protease. To avoid heterogeneity in the sample used for crystallization, a modified recombinant molecule was produced. The sequence of the proDer p 1 allergen was modified to reduce glycosylation and to abolish enzymatic activity. The resulting rproDer p 1 preparation was homogenous and stable and yielded crystals diffracting to a resolution of 1.61 A. The active site is located in a large cleft on the surface of the molecule. The 80-aa pro-peptide adopts a unique fold that interacts with the active site cleft and a substantial adjacent area on the mature region, excluding access to the cleft and the active site. Studies performed using crossed-line immunoelectrophoresis and IgE inhibition experiments indicated that several epitopes are covered by the pro-peptide and that the epitopes on the recombinant mature molecule are indistinguishable from those on the natural one. The structure confirms previous results suggesting a preference for aliphatic residues in the important P2 position in substrates. Sequence variations in related species are concentrated on the surface, which explains the existence of cross-reacting and species-specific antibodies. This study describes the first crystal structure of one of the clinically most important house dust mite allergens, the cysteine protease Der p 1.     

Sufficiency of the reactive site loop of maspin for induction of cell-matrix adhesion and inhibition of cell invasion. Conversion of ovalbumin to a maspin-like molecule

Maspin, an ov-serpin, inhibits tumor invasion and induces cell adhesion to extracellular matrix molecules. Here, we use maspin/ovalbumin chimeric proteins and the maspin reactive site loop (RSL) peptide to characterize the role of the RSL in maspin-mediated functions. Replacement of the RSL plus the C-terminal region or the RSL alone of maspin with that of ovalbumin resulted in the loss of the stimulatory effect on adhesion of corneal stromal cells to type I collagen, fibronectin, and laminin and of mammary carcinoma MDA-MB-231 cells to fibronectin. Maspin with ovalbumin as the C-terminal region retained activity, suggesting the maspin C-terminal polypeptide is not required. An R340Q mutant retained full maspin activity; however, an R340A mutant lost activity. This indicates the arginine side chain at the putative P1 site forms a hydrogen bond and not an ionic bond. The RSL peptide (P10-P5', amino acids 330-345) alone induced cell-matrix adhesion of mammary carcinoma cells and corneal stromal cells and inhibited invasion of the carcinoma cells. Substitution of the RSL of ovalbumin with that of maspin converted inactive ovalbumin into a fully active molecule. Maspin bound specifically to the surface of the mammary carcinoma cells with a kd of 367 +/- 67 nM and 32.0 +/- 2.2 x 10(6) binding sites/cell. The maspin RSL peptide inhibited binding, suggesting the RSL is involved in maspin binding to cells. Sufficiency of the maspin RSL for activity suggests the mechanism by which maspin regulates cell-matrix adhesion and tumor cell invasion does not involve the serpin mechanism of protease inhibition.     

Determination of ultrafast protein folding rates from loop formation dynamics

Quenching of the triplet state of tryptophan by contact with cysteine can be used to measure the kinetics of loop formation in unfolded proteins. Here we show that cysteine quenching dynamics also provide a novel method for measuring folding rates when the exchange between folded and unfolded states is faster than the unquenched triplet lifetime (approximately 100 micros). We use this technique to investigate folding/unfolding kinetics of the 35 residue headpiece subdomain of the protein villin, which contains a single tryptophan residue and was engineered to contain a cysteine residue at the N terminus. At intermediate concentrations of denaturant the time-course of the triplet decay consists of two relaxations, the rates and amplitudes of which reveal the fast kinetics for folding and unfolding of this protein. The folding rates extracted using a simple kinetic model are close to those reported previously from laser-induced temperature-jump experiments that employ the change in tryptophan fluorescence as a probe. However, the results differ significantly from those reported from dynamic NMR line shape analysis on a variant with methionine at the N terminus, an issue that remains to be resolved. The analysis of the triplet quenching kinetics also shows that the quenching rates in the unfolded state increase with decreasing denaturant concentration, indicating a compaction of the unfolded protein.