An Overlapping Region between the Two Terminal Folding Units of the Outer Surface Protein A (OspA) Controls Its Folding Behavior

Although many naturally occurring proteins consist of multiple domains, most studies on protein folding to date deal with single-domain proteins or isolated domains of multi-domain proteins. Studies of multi-domain protein folding are required for further advancing our understanding of protein folding mechanisms. Borrelia outer surface protein A (OspA) is a β-rich two-domain protein, in which two globular domains are connected by a rigid and stable single-layer β-sheet. Thus, OspA is particularly suited as a model system for studying the interplays of domains in protein folding. Here, we studied the equilibria and kinetics of the urea-induced folding–unfolding reactions of OspA probed with tryptophan fluorescence and ultraviolet circular dichroism. Global analysis of the experimental data revealed compelling lines of evidence for accumulation of an on-pathway intermediate during kinetic refolding and for the identity between the kinetic intermediate and a previously described equilibrium unfolding intermediate. The results suggest that the intermediate has the fully native structure in the N-terminal domain and the single layer β-sheet, with the C-terminal domain still unfolded. The observation of the productive on-pathway folding intermediate clearly indicates substantial interactions between the two domains mediated by the single-layer β-sheet. We propose that a rigid and stable intervening region between two domains creates an overlap between two folding units and can energetically couple their folding reactions.     

Structural Interpretation of Paramagnetic Relaxation Enhancement-Derived Distances for Disordered Protein States

Paramagnetic relaxation enhancement (PRE) is a powerful technique for studying transient tertiary organizations of unfolded and partially folded proteins. The heterogeneous and dynamic nature of disordered protein states, together with the r⁻⁶ dependence of PRE, presents significant challenges for reliable structural interpretation of PRE-derived distances. Without additional knowledge of accessible conformational substates, ensemble-simulation-based protocols have been used to calculate structure ensembles that appear to be consistent with the PRE distance restraints imposed on the ensemble level with the proper r⁻⁶ weighting. However, rigorous assessment of the reliability of such protocols has been difficult without intimate knowledge of the true nature of disordered protein states. Here we utilize sets of theoretical PRE distances derived from simulated structure ensembles that represent the folded, partially folded and unfolded states of a small protein to investigate the efficacy of ensemble-simulation-based structural interpretation of PRE distances. The results confirm a critical limitation that, due to r⁻⁶ weighting, only one or a few members need to satisfy the distance restraints and the rest of the ensemble are essentially unrestrained. Consequently, calculated structure ensembles will appear artificially heterogeneous no matter whether the PRE distances are derived from the folded, partially unfolded or unfolded state. Furthermore, the nature of the heterogeneous ensembles is largely determined by the protein model employed in structure calculation and reflects little on the true nature of the underlying disordered state. These findings suggest that PRE measurements on disordered protein states alone generally do not contain enough information for a reliable structural interpretation and that the latter will require additional knowledge of accessible conformational substates. Interestingly, when a very large number of PRE measurements is available, faithful structural interpretation might be possible with intermediate ensemble sizes under ideal conditions.     

Characterization of amyloidogenic intermediate states through a combined use of CD and NMR spectroscopy

Characterization of amyloidogenic intermediate states is of central importance in understanding the molecular mechanism of amyloid formation. In this study, we utilized CD and NMR spectroscopy to investigate secondary structure of the monomeric amyloidogenic intermediate of a β-structured SH3 domain, which was induced by trifluoroethanol (TFE). The combined biophysical studies showed that the native state SH3 domain is gradually converted to the amyloidogenic intermediate state at TFE concentrations of 20-26% (v/v) and the aggregation-prone state contains substantial amount of the β-sheet conformation (∼ 30%) with disordered (54%) and some helical characters (16%). Under weaker amyloidogenic conditions of higher TFE concentrations (> 40%), the β-sheet structures were gradually changed to helical conformations and the relative content of the helical and β-sheet conformations was highly correlated with the aggregation propensity of the SH3 domain. This indicates that the β-sheet characters of the amyloidogenic states may be critical to the effective amyloid formation.     

NMR studies of the solution conformation of the sex peptide from Drosophila melanogaster

The insect sex peptide (SP) elicits a variety of biological responses upon transfer to the mated female. SP contains 36 amino acids, including a tryptophan-rich N-terminal region, a central region containing five hydroxyproline (Hyp) residues, and a C-terminal region enclosed by a disulfide bridge. The solution structure of SP, studied here using NMR spectroscopy, includes a motif WPWN that adopts a type I β-turn in the N-terminal Trp-rich region. This turn region is connected to the central Hyp-rich region, which adopts extended and/or PPII-like conformations. The C-terminal disulfide-bonded loop populates helical turns or nascent helical structure. Overall, the results reveal a rather flexible peptide that lacks a compact folded structure in solution.     

Temporary secondary structures in tau,an intrinsically disordered protein

The tau protein belongs to the category of intrinsically disordered proteins, which in their native state do not have an average stable structure and fluctuate between many conformations. In its physiological state, tau helps nucleating and stabilising the microtubules in the axons of the neurons. On the other hand, the same tau is involved in the development of Alzheimer disease, when it aggregates in paired helical filaments forming fibrils, which form insoluble tangles. The beginning of the pathological aggregation of tau has been attributed to a local transition of protein portions from random coil to a β-sheet. These structures would very likely be transient; therefore, we performed a molecular dynamics simulation of tau to gather information on the existence of segments of tau endowed with a secondary structure. We combined the results of our simulation with small-angle X-ray scattering experimental data to extract from the dynamics a set of most probable conformations of tau. The analysis of these conformations highlights the presence of transient secondary structures such as turns, β-bridges, β-sheets and α-helices. It also shows that a large segment of the N-terminal region is found near the repeats domain in a globular-like shape.     

Multistep denaturation of Borrelia burgdorferi OspA, a protein containing a single-layer beta-sheet

Outer surface protein A (OspA) from the Lyme disease spirochete, Borrelia burgdorferi, is a dumbbell-shaped protein in which two globular domains are connected by a three-stranded beta-sheet segment that is solvent-exposed on both faces. Previous studies showed that the whole protein, including the single-layer beta-sheet, is highly rigid. To elucidate the folding mechanism and the role of the central beta-sheet in the formation of the rigid molecule, we investigated the equilibrium thermal denaturation reaction of OspA. We applied differential scanning calorimetry, heteronuclear NMR spectroscopy, and solution small-angle X-ray scattering (SAXS) to characterize the reaction in detail. All three techniques revealed that OspA denatures in two separable cooperative transitions. NMR measurements on OspA specifically 15N-labeled at Lys residues identified the locations of the two folding units and revealed that the C-terminal segment is less stable than the remaining N-terminal segment. The boundary between the two folding units is located within the central beta-sheet. The interconversion among the three folding states (fully folded, C-terminus unfolded, and fully denatured) is slow relative to chemical shift differences (<24 Hz), indicating that there are significant kinetic barriers in the denaturation reactions. SAXS measurements determined the radius of gyration of the native protein to be 25.0 +/- 0.3 A, which increases to 34.4 +/- 1.0 A in the first transition, and then to 56.1 +/- 1.6 A in the second transition. Thus, the intermediate state, in which the C-terminal folding unit is already denatured, is still compact. These results provide a basis for elucidating the folding mechanism of OspA.     

Structures and free energy landscapes of aqueous zinc(II)-bound amyloid-β(1–40) and zinc(II)-bound amyloid-β(1–42) with dynamics

Binding of divalent metal ions with intrinsically disordered fibrillogenic proteins, such as amyloid-β (Aβ), influences the aggregation process and the severity of neurodegenerative diseases. The Aβ monomers and oligomers are the building blocks of the aggregates. In this work, we report the structures and free energy landscapes of the monomeric zinc(II)-bound Aβ40 (Zn:Aβ40) and zinc(II)-bound Aβ42 (Zn:Aβ42) intrinsically disordered fibrillogenic metallopeptides in an aqueous solution by utilizing an approach that employs first principles calculations and parallel tempering molecular dynamics simulations. The structural and thermodynamic properties, including the secondary and tertiary structures and conformational Gibbs free energies of these intrinsically disordered metallopeptide alloforms, are presented. The results show distinct differing characteristics for these metallopeptides. For example, prominent β-sheet formation in the N-terminal region (Asp1, Arg5, and Tyr10) of Zn:Aβ40 is significantly decreased or lacking in Zn:Aβ42. Our findings indicate that blocking multiple reactive residues forming abundant β-sheet structure located in the central hydrophobic core and C-terminal regions of Zn:Aβ42 via antibodies or small organic molecules might help to reduce the aggregation of Zn(II)-bound Aβ42. Furthermore, we find that helix formation increases but β-sheet formation decreases in the C-terminal region upon Zn(II) binding to Aβ. This depressed β-sheet formation in the C-terminal region (Gly33-Gly38) in monomeric Zn:Aβ42 might be linked to the formation of amorphous instead of fibrillar aggregates of Zn:Aβ42.     

Formation of the single-layer beta-sheet of Borrelia burgdorferi OspA in the absence of the C-terminal capping globular domain

Borrelia outer surface protein A (OspA) contains a unique single-layer beta-sheet that connects N and C-terminal globular domains. This single-layer beta-sheet segment (beta-strands 8-10) is highly stable in solution, although it is exposed to the solvent on both faces of the sheet and thus it does not contain a hydrophobic core. Here, we tested whether interactions with the C-terminal domain are essential for the formation of the single-layer beta-sheet. We characterized the solution structure, dynamics and stability of an OspA fragment corresponding to beta-strands 1-12 (termed OspA[27-163]), which lacks a majority of the C-terminal globular domain. Analyses of NMR chemical shifts and backbone nuclear Overhauser effect (NOE) connectivities showed that OspA[27-163] is folded except the 12th and final beta-strand. (1)H-(15)N heteronuclear NOE measurements and amide H-(2)H exchange revealed that the single-layer beta-sheet in this fragment is more flexible than the corresponding region in full-length OspA. Thermal-denaturation experiments using differential scanning calorimetry and NMR spectroscopy revealed that the N-terminal globular domain in the fragment has a conformational stability similar to that of the same region in the full-length protein, and that the single-layer beta-sheet region also has a modest thermal stability. These results demonstrate that the unique single-layer beta-sheet retains its conformation in the absence of its interactions with the C-terminal domain. This fragment is significantly smaller than the full-length OspA, and thus it is expected to facilitate studies of the folding mechanism of this unusual beta-sheet structure.     

Characterizing a partially folded intermediate of the villin headpiece domain under non-denaturing conditions: contribution of His41 to the pH-dependent stability of the N-terminal subdomain

The contribution of interactions involving the imidazole ring of His41 to the pH-dependent stability of the villin headpiece (HP67) N-terminal subdomain has been investigated by nuclear magnetic resonance (NMR) spin relaxation. NMR-derived backbone N-H order parameters (S2) for wild-type (WT) HP67 and H41Y HP67 indicate that reduced conformational flexibility of the N-terminal subdomain in WT HP67 is due to intramolecular interactions with the His41 imidazole ring. These interactions, together with desolvation effects, contribute to significantly depress the pKa of the buried imidazole ring in the native state. 15N R1rho relaxation dispersion data indicate that WT HP67 populates a partially folded intermediate state that is 10.9 kJ mol(-1) higher in free energy than the native state under non-denaturing conditions at neutral pH. The partially folded intermediate is characterized as having an unfolded N-terminal subdomain while the C-terminal subdomain retains a native-like fold. Although the majority of the residues in the N-terminal subdomain sample a random-coil distribution of conformations, deviations of backbone amide 1H and 15N chemical shifts from canonical random-coil values for residues within 5A of the His41 imidazole ring indicate that a significant degree of residual structure is maintained in the partially folded ensemble. The pH-dependence of exchange broadening is consistent with a linear three-state exchange model whereby unfolding of the N-terminal subdomain is coupled to titration of His41 in the partially folded intermediate with a pKa,I=5.69+/-0.07. Although maintenance of residual interactions with the imidazole ring in the unfolded N-terminal subdomain appears to reduce pKa,I compared to model histidine compounds, protonation of His41 disrupts these interactions and reduces the difference in free energy between the native state and partially folded intermediate under acidic conditions. In addition, chemical shift changes for residues Lys70-Phe76 in the C-terminal subdomain suggest that the HP67 actin binding site is disrupted upon unfolding of the N-terminal subdomain, providing a potential mechanism for regulating the villin-dependent bundling of actin filaments.     

A delicate interplay of structure, dynamics, and thermodynamics for function: a high pressure NMR study of outer surface protein A

Outer surface protein A (OspA) is a crucial protein in the infection of Borrelia burgdorferi causing Lyme disease. We studied conformational fluctuations of OspA with high-pressure ¹⁵N/¹H two-dimensional NMR along with high-pressure fluorescence spectroscopy. We found evidence within folded, native OspA for rapid local fluctuations of the polypeptide backbone in the nonglobular single layer β-sheet connecting the N- and C-terminal domains with τ << ms, which may give the two domains certain independence in mobility and thermodynamic stability. Furthermore, we found that folded, native OspA is in equilibrium (τ >> ms) with a minor conformer I, which is almost fully disordered and hydrated for the entire C-terminal part of the polypeptide chain from β8 to the C-terminus. Conformer I is characterized with ΔG⁰ = 32 ± 9 kJ/mol and ΔV⁰ = −140 ± 40 mL/mol, populating only ∼0.001% at 40°C at 0.1 MPa, pH 5.9. Because in the folded conformer the receptor binding epitope of OspA is buried in the C-terminal domain, its transition into conformer I under in vivo conditions may be critical for the infection of B. burgdorferi. The formation and stability of the peculiar conformer I are apparently supported by a large packing defect or cavity located in the C-terminal domain.     

Structural characterization of an intrinsically unfolded mini-HBX protein from hepatitis B virus

The hepatitis B virus x protein (HBX) is expressed in HBVinfected liver cells and can interact with a wide range of cellular proteins. In order to understand such promiscuous behavior of HBX we expressed a truncated mini-HBX protein (named Tr-HBX) (residues 18-142) with 5 Cys → Ser mutations and characterized its structural features using circular dichroism (CD) spectropolarimetry, NMR spectroscopy as well as bioinformatics tools for predicting disorder in intrinsically unstructured proteins (IUPs). The secondary structural content of Tr-HBX from CD data suggests that Tr-HBX is only partially folded. The protein disorder prediction by IUPred reveals that the unstructured region encompasses its N-terminal ～30 residues of Tr-HBX. A two-dimensional (1)H-(15)N HSQC NMR spectrum exhibits fewer number of resonances than expected, suggesting that Tr-HBX is a hybrid type IUP where its folded C-terminal half coexists with a disordered N-terminal region. Many IUPs are known to be capable of having promiscuous interactions with a multitude of target proteins. Therefore the intrinsically disordered nature of Tr-HBX revealed in this study provides a partial structural basis for the promiscuous structure-function behavior of HBX.     

Impact of N-glycosylation site variants during human PrP aggregation and fibril nucleation

Misfolding and aggregation of the human prion protein (PrP) cause neurodegenerative transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease. Mature native PrP is composed of 209 residues and is folded into a C-terminal globular domain (residues 125–209) comprising a small two-stranded β-sheet and three α-helices. The N-terminal domain (residues 23–124) is intrinsically disordered. Expression of truncated PrP (residues 90–231) is sufficient to cause prion disease and residues 90/100–231 is comprising the amyloid-like fibril core of misfolded infectious PrP. During PrP fibril formation under native conditions in vitro, the disordered N-terminal domain slows down fibril formation likely due to a mechanism of initial aggregation forming morphologically disordered aggregates. The morphological disordered aggregate is a transient phase. Nucleation of fibrils occurs from this initial aggregate. The aggregate phase is largely circumvented by seeding with preformed PrP fibrils. In vivo PrP is N-glycosylated at positions Asn181 and Asn197. Little is known about the importance of these positions and their glycans for PrP stability, aggregation and fibril formation. We have in this study taken a step towards that goal by mutating residues 181 and 197 for cysteines to study the positional impact on these processes. We have further by organic synthetic chemistry and chemical modification generated synthetic glycosylations in these positions. Our data shows that residue 181 when mutated to a cysteine is a key residue for self-chaperoning, rendering a trap in the initial aggregate preventing conformational changes towards amyloid fibril formation. Position 197 is less involved in the aggregate trapping and is more geared towards β-sheet structure conversion within amyloid fibrils. As expected, synthetic glycosylated 197 is less affected towards fibril formation compared to glycosylated 181. Our data are rather compatible with the parallel in-register intermolecular β-sheet model structure of the PrP90–231 fibril and sheds light on the misfolding transitions of PrP in vitro. We hypothesize that glycosylation of position 181 is a key site for prion strain differentiation in vivo.     

Characterization of the folding kinetics of a three-helix bundle protein via a minimalist Langevin model.

We use a simple off-lattice Langevin model of protein folding to characterize the folding and unfolding of a fast-folding, 46 residue three-helix bundle. Under conditions at which the C-terminal helix is 30 % stable, we observe a clear three-state folding mechanism. In the on-pathway intermediate state, the middle and C-terminal helices are folded and in contact with each other, while the N-terminal region remains disordered. Nevertheless, under these conditions this intermediate is thermodynamically unstable relative to its unfolded state. The first and highest folding barrier corresponds to the organization of the hinge between the middle and C-terminal helices. A subsequent major barrier corresponds to the organization of the hinge between the middle and N-terminal helices. Hyperstabilizing the hinge regions leads to twice the folding rate that is obtained from hyperstabilizing the helices, even though much fewer contacts are involved in hinge hyperstabilization than in helix hyperstabilization. Unfolding follows single-exponential kinetics, even at temperatures only slightly above the folding transition temperature.     

Ethanol and acetonitrile induces conformational changes in porcine pepsin at alkaline denatured state

Pepsin, a member of the aspartate protease family, exists in a partially unfolded state at alkaline pH where the N-terminal domain of pepsin has a flexible structure while the C-terminal domain has a highly folded structure. In this work, the conformational stability of porcine pepsin in an alkaline denatured (A(D)) state against acetonitrile and ethanol solvents was studied using a combination of electronic circular dichroism (ECD) and fluorescence techniques. The ECD results demonstrate that both ethanol and acetonitrile induce secondary structural changes in pepsin at A(D) state. However, the minimum concentration required to induce significant secondary structural changes in pepsin varies for ethanol (>30%, v/v) and acetonitrile (>60%, v/v) solvents. At maximum concentration used (90%, v/v), both solvents induce predominantly β-sheet conformation. Unlike acetonitrile, ethanol induces significant amount of non-native α-helical conformations at the intermediate concentrations (50-80%). The tryptophan fluorescence results demonstrate that both acetonitrile and ethanol induce substantial changes in the tertiary structure of pepsin in the A(D) state above certain concentrations. The current results have important implications in understanding the effect of co-solvents on the conformation of proteins in the "denatured state".     

A PixD--PapB Chimeric Protein Reveals the Function of the BLUF Domain C-Terminal α-Helices for Light Signal Transduction

Blue light-using flavin (BLUF) proteins form a subfamily of blue light photoreceptors, are found in many bacteria and algae, and are further classified according to their structures. For one type of BLUF-containing protein, e.g. PixD, the central axes of its two C-terminal α-helices are perpendicular to the β-sheet of its N-terminal BLUF domain. For another type, e.g. PapB, the central axes of its two C-terminal α-helices are parallel to its BLUF domain β-sheet. However, the functional significance of the different orientations with respect to phototransduction is not clear. For the study reported herein, we constructed a chimeric protein, Pix0522, containing the core of the PixD BLUF domain and the C-terminal region of PapB, including the two α-helices, and characterized its biochemical and spectroscopic properties. Fourier transform infrared spectroscopy detected similar light-induced conformational changes in the C-terminal α-helices of Pix0522 and PapB. Pix0522 interacts with and activates the PapB-interacting enzyme, PapA, demonstrating the functionality of Pix0522. These results provide direct evidence that the BLUF C-terminal α-helices function as an intermediary that accepts the flavin-sensed blue light signal and transmits it downstream during phototransduction.     

The group II chaperonin Mm-Cpn binds and refolds human γD crystallin

Chaperonins assist in the folding of nascent and misfolded proteins, though the mechanism of folding within the lumen of the chaperonin remains poorly understood. The archeal chaperonin from Methanococcus marapaludis, Mm-Cpn, shares the eightfold double barrel structure with other group II chaperonins, including the eukaryotic TRiC/CCT, required for actin and tubulin folding. However, Mm-Cpn is composed of a single species subunit, similar to group I chaperonin GroEL, rather than the eight subunit species needed for TRiC/CCT. Features of the β-sheet fold have been identified as sites of recognition by group II chaperonins. The crystallins, the major components of the vertebrate eye lens, are β-sheet proteins with two homologous Greek key domains. During refolding in vitro a partially folded intermediate is populated, and partitions between productive folding and off-pathway aggregation. We report here that in the presence of physiological concentrations of ATP, Mm-Cpn suppressed the aggregation of HγD-Crys by binding the partially folded intermediate. The complex was sufficiently stable to permit recovery by size exclusion chromatography. In the presence of ATP, Mm-Cpn promoted the refolding of the HγD-Crys intermediates to the native state. The ability of Mm-Cpn to bind and refold a human β-sheet protein suggests that Mm-Cpn may be useful as a simplified model for the substrate recognition mechanism of TRiC/CCT.     

Calorimetric dissection of thermal unfolding of OspA,a predominantly beta-sheet protein containing a single-layer beta-sheet

Outer surface protein A (OspA) from Borrelia burgdorferi is a predominantly beta-sheet protein comprised of beta-strands beta1-beta21 and a short C-terminal alpha-helix. It contains two globular domains (N and C-terminal domains) and a unique single-layer beta-sheet (central beta-sheet) that connects the two domains. OspA contains an unusually large number of charged amino acid residues. To understand the mechanism of stabilization of this unique beta-sheet protein, thorough thermodynamic investigations of OspA and its truncated mutant lacking a part of the C-terminal domain were conducted using calorimetry and circular dichroism. The stability of OspA was found to be sensitive to pH and salt concentration. The heat capacity curve clearly consisted of two components, and all the thermodynamic parameters were obtained for each step. The thermodynamic parameters associated with the two transitions are consistent with a previously proposed model, in which the first transition corresponds to the unfolding of the C-terminal domain and the last two beta-strands of the central beta-sheet, and the second transition corresponds to that of the N-terminal domain and the first beta-strand of the central beta-sheet in the second peak. The ratio of calorimetric and van't Hoff enthalpies indicates that the first peak includes another thermodynamic intermediate state. Large heat capacity changes were observed for both transitions, indicative of large changes in the exposure of hydrophobic surfaces associated with the transitions. This observation demonstrates that hydrophobic parts are buried efficiently in the native structure in spite of the low content of hydrophobic residues in OspA. By decomposing the enthalpy, entropy, and Gibbs free energy into contributions from different interactions, we found that the enthalpy changes for hydrogen bonding and polar interactions are exceptionally large, indicating that OspA maintains its stability by making full use of its unique beta-sheet and high content of polar residues. These thermodynamic analyses demonstrated that it is possible to maintain protein tertiary structure by making effective use of an unusual amino acid composition.     

Paramagnetic relaxation enhancement‐assisted structural characterization of a partially disordered conformation of ubiquitin

Nuclear magnetic resonance (NMR) is a powerful tool to study three‐dimensional structures as well as protein conformational fluctuations in solution, but it is compromised by increases in peak widths and missing signals. We previously reported that ubiquitin has two folded conformations, N₁ and N₂ and plus another folded conformation, I, in which some amide group signals of residues 33–41 almost disappeared above 3 kbar at pH 4.5 and 273 K. Thus, well‐converged structural models could not be obtained for this region owing to the absence of distance restraints. Here, we reexamine the problem using the ubiquitin Q41N variant as a model for this locally disordered conformation, I. We demonstrate that the variant shows pressure‐induced loss of backbone amide group signals at residues 28, 33, 36, and 39–41 like the wild‐type, with a similar but smaller effect on CαH and CβH signals. In order to characterize this I structure, we measured paramagnetic relaxation enhancement (PRE) under high pressure to obtain distance restraints, and calculated the structure assisted by Bayesian inference. We conclude that the more disordered I conformation observed at pH 4.0, 278 K, and 2.5 kbar largely retained the N₂ conformation, although the amide groups at residues 33–41 have more heterogeneous conformations and more contact with water, which differ from the N₁ and N₂ states. The PRE‐assisted strategy has the potential to improve structural characterization of proteins that lack NMR signals, especially for relatively more open and hydrated protein conformations.     

pH-induced conformational transitions of a molten-globule-like state of the inhibitory prodomain of furin: implications for zymogen activation

The endoprotease furin, which belongs to the family of mammalian proprotein convertase (PC), is synthesized as a zymogen with an N-terminal, 81-residue inhibitory prodomain. It has been shown that the proenzyme form of furin undergoes a multistep 'autocatalytic' removal of the prodomain at the C-terminal side of the two consensus sites, R(78)-T-K-R(81) approximately and R(44)-G-V-T-K-R(49) approximately. The furin-mediated cleavage at R(44)-G-V-T-K-R(49) approximately, in particular, is significantly accelerated in an 'acidic' environment. Here, we show that under neutral pH conditions, the inhibitory prodomain of furin is partially folded and undergoes conformational exchanges as indicated by extensive broadening of the NMR spectra. Presence of many ring-current shifted methyl resonances suggests that the partially folded state of the prodomain may still possess a 'semirigid' protein core with specific packing interactions among amino acid side chains. Measurements of the hydrodynamic radii and compaction factors indicate that this partially folded state is significantly more compact than a random chain. The conformational stability of the prodomain appears to be pH sensitive, in that the prodomain undergoes an unfolding transition towards acidic conditions. Our NMR analyses establish that the acid-induced unfolding is mainly experienced by the residues from the C-terminal half of the prodomain (residues R(44)-R(81)) that contains the two furin cleavage sites. A 38-residue peptide fragment derived from the entire pH-sensitive C-terminal region (residues R(44)-R(81)) does not exhibit any exchange-induced line broadening and adopts flexible conformations. We propose that at neutral pH, the cleavage site R(44)-G-V-T-K-R(49) approximately is buried within the protein core that is formed in part by residues from the N-terminal region, and that the cleavage site becomes exposed under acidic conditions, leading to a facile cleavage by the furin enzyme.     

Exploring subdomain cooperativity in T4 lysozyme II: uncovering the C-terminal subdomain as a hidden intermediate in the kinetic folding pathway

Intermediates along a protein's folding pathway can play an important role in its biology. Previous kinetics studies have revealed an early folding intermediate for T4 lysozyme, a small, well-characterized protein composed of an N-terminal and a C-terminal subdomain. Pulse-labeling hydrogen exchange studies suggest that residues from both subdomains contribute to the structure of this intermediate. On the other hand, equilibrium native state hydrogen experiments have revealed a high-energy, partially unfolded form of the protein that has an unstructured N-terminal subdomain and a structured C-terminal subdomain. To resolve this discrepancy between kinetics and equilibrium data, we performed detailed kinetics analyses of the folding and unfolding pathways of T4 lysozyme, as well as several point mutants and large-scale variants. The data support the argument for the presence of two distinct intermediates, one present on each side of the rate-limiting transition state barrier. The effects of circular permutation and site-specific mutations in the wild-type and circular permutant background, as well as a fragment containing just the C-terminal subdomain, support a model for the unfolding intermediate with an unfolded N-terminal and a folded C-terminal subdomain. Our results suggest that the partially unfolded form identified by native state hydrogen exchange resides on the folded side of the rate-limiting transition state and is, therefore, under most conditions, a "hidden" intermediate.