Two distinct intermediates of trigger factor are populated during guanidine denaturation

Trigger factor (TF) is an important catalyst of nascent peptide folding and possesses both peptidyl-prolyl cis-trans isomerase (PPIase) and chaperone activities. TF has a modular structure, containing three domains with distinct structural and functional properties. The guanidine hydrochloride (GuHCl) induced unfolding of TF was investigated by monitoring Trp fluorescence, far-UV CD, second-derivative UV absorption, enzymatic and chaperone activities, chemical crosslinking and binding of the hydrophobic dye, 1-anilinonaphthalene-8-sulfonate (ANS); and was compared to the urea induced unfolding. The native state of TF was found to bind ANS in 1:1 stoichiometry with a K(d) of 84 microM. A native-like state, N', is stable around 0.5 M GuHCl, and shows increased ANS binding, while retaining PPIase activity and most secondary and tertiary structure, but loses chaperone and dimerization activities, consistent with slight conformational rearrangement. A compact denatured state, I, is populated around 1.0 M GuHCl, is inactive and does not show significant binding to ANS. The data suggest that TF unfolds in a stepwise manner, consistent with its modular structure. The ability of TF to undergo structural rearrangement to maintain enzymatic activity while reducing chaperone and dimerization abilities may be related to the physiological function of TF.     

Identification of a potential hydrophobic peptide binding site in the C-terminal arm of trigger factor

Trigger factor (TF) is the first chaperone to interact with nascent chains and facilitate their folding in bacteria. Escherichia coli TF is 432 residues in length and contains three domains with distinct structural and functional properties. The N-terminal domain of TF is important for ribosome binding, and the M-domain carries the PPIase activity. However, the function of the C-terminal domain remains unclear, and the residues or regions directly involved in substrate binding have not yet been identified. Here, a hydrophobic probe, bis-ANS, was used to characterize potential substrate-binding regions. Results showed that bis-ANS binds TF with a 1:1 stoichiometry and a K(d) of 16 microM, and it can be covalently incorporated into TF by UV-light irradiation. A single bis-ANS-labeled peptide was obtained by tryptic digestion and identified by MALDI-TOF mass spectrometry as Asn391-Lys392. In silico docking analysis identified a single potential binding site for bis-ANS on the TF molecule, which is adjacent to this dipeptide and lies in the pocket formed by the C-terminal arms. The bis-ANS-labeled TF completely lost the ability to assist GAPDH or lysozyme refolding and showed increased protection toward cleavage by alpha-chymotrypsin, suggesting blocking of hydrophobic residues. The C-terminal truncation mutant TF389 also showed no chaperone activity and could not bind bis-ANS. These results suggest that bis-ANS binding may mimic binding of a substrate peptide and that the C-terminal region of TF plays an important role in hydrophobic binding and chaperone function.     

Thermal unfolding of Escherichia coli trigger factor studied by ultra-sensitive differential scanning calorimetry

Temperature-induced unfolding of Escherichia coli trigger factor (TF) and its domain truncation mutants, NM and MC, were studied by ultra-sensitive differential scanning calorimetry (UC-DSC). Detailed thermodynamic analysis showed that thermal induced unfolding of TF and MC involves population of dimeric intermediates. In contrast, the thermal unfolding of the NM mutant involves population of only monomeric states. Covalent cross-linking experiments confirmed the presence of dimeric intermediates during thermal unfolding of TF and MC. These data not only suggest that the dimeric form of TF is extremely resistant to thermal unfolding, but also provide further evidence that the C-terminal domain of TF plays a vital role in forming and stabilizing the dimeric structure of the TF molecule. Since TF is the first molecular chaperone that nascent polypeptides encounter in eubacteria, the stable dimeric intermediates of TF populated during thermal denaturation might be important in responding to stress damage to the cell, such as heat shock.     

Functional dissection of Escherichia coli trigger factor: unraveling the function of individual domains

In Escherichia coli, the ribosome-associated chaperone Trigger Factor (TF) promotes the folding of newly synthesized cytosolic proteins. TF is composed of three domains: an N-terminal domain (N), which mediates ribosome binding; a central domain (P), which has peptidyl-prolyl cis/trans isomerase activity and is involved in substrate binding in vitro; and a C-terminal domain (C) with unknown function. We investigated the contributions of individual domains (N, P, and C) or domain combinations (NP, PC, and NC) to the chaperone activity of TF in vivo and in vitro. All fragments comprising the N domain (N, NP, NC) complemented the synthetic lethality of Deltatig DeltadnaK in cells lacking TF and DnaK, prevented protein aggregation in these cells, and cross-linked to nascent polypeptides in vitro. However, DeltatigDeltadnaK cells expressing the N domain alone grew more slowly and showed less viability than DeltatigDeltadnaK cells synthesizing either NP, NC, or full-length TF, indicating beneficial contributions of the P and C domains to TF's chaperone activity. In an in vitro system with purified components, none of the TF fragments assisted the refolding of denatured d-glyceraldehyde-3-phosphate dehydrogenase in a manner comparable to that of wild-type TF, suggesting that the observed chaperone activity of TF fragments in vivo is dependent on their localization at the ribosome. These results indicate that the N domain, in addition to its function to promote binding to the ribosome, has a chaperone activity per se and is sufficient to substitute for TF in vivo.     

PPIase domain of trigger factor acts as auxiliary chaperone site to assist the folding of protein substrates bound to the crevice of trigger factor

Trigger factor (TF) is the first chaperone encountered by nascent chains in bacteria, which consists of two modules: peptidyl-prolyl-cis/trans-isomerase (PPIase) domain and a crevice built by both N- and C-terminal domains. While the crevice is suggested to provide a protective space over the peptide exit site of ribosome for nascent polypeptides to fold, it remains unclear whether PPIase domain is directly involved in assisting protein folding. Here, we introduced structural change into different regions of TF, and investigated their influence on the chaperone function of TF in assisting the folding of various substrate proteins, including oligomeric glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and monomeric carbonic anhydrase II (CA II) and lysozyme. Results showed that structural disturbances by site-specific mutations in the PPIase active site or by deletion of the PPIase domain from TF affected the chaperone activity of TF toward CA II and GAPDH but had no effect on TF-assisted lysozyme refolding, suggesting PPIase domain is involved in assisting the folding of substrates larger than lysozyme. Mutants with the structural disturbances in the crevice totally lost the chaperone activity toward all the substrates we used in this investigation. These results provide further evidence to confirm that the crevice is the major chaperone site of TF, and the hydrophobic pocket in PPIase domain acts as an auxiliary site to assist the folding of substrate proteins bound to the crevice in a substrate-dependent manner, which is beneficial for TF to provide appropriate assistance for protein folding by changing protective space and binding affinity.     

The folding of an enzyme. III. Structure of the transition state for unfolding of barnase analysed by a protein engineering procedure.

The structure of the first significant transition state on the unfolding pathway of barnase has been analysed in detail by protein engineering methods. Over 50 mutations placed strategically over the whole protein have been used as probes to report on the local structure in the transition state. Several different probes for many regions of the protein give consistent results as do multiple probes at the same site. The overall consistency of phi values indicates that the mutations have not produced changes in the protein that significantly alter the transition state for unfolding. A fine-structure analysis of interactions has also been conducted by removing different parts of the same side-chains. Many of the results of simple mutations fall nicely into the two clear-cut cases of phi = 1 or 0, indicating that the local noncovalent bonds are either fully broken or fully made in the transition state. Much of the structure of barnase in the transition state for unfolding is very similar to that in the folded protein. Both major alpha-helices fray at the N terminus. The last two turns in helix1 are certainly intact, as is the C terminus of helix2. The general picture of the beta-sheet is that the three central beta-strands are completely intact while the two edge beta-strands are mainly present but certainly weakened. The first five residues of the protein unwind but the C terminus remains folded. Three of the five loops are unfolded. The edges of the main hydrophobic core (core1) are significantly weakened, however, and their breaking appears partly rate determining. The centre of the small hydrophobic core3 remains intact. Core2 is completely disrupted. The first events in unfolding are thus: the unfolding of several loops, the unwinding of the helices from the N termini, and the weakening and disruption of the hydrophobic cores. The values of phi are found to be substantially the same under conditions that favour folding as under conditions that are highly denaturing, and so the structure of the unfolding transition state is substantially the same in water as in the presence of denaturant. The structure of the final kinetically significant transition state for refolding is identical to that for unfolding. The final events in refolding are, accordingly, the consolidation of the hydrophobic cores, the closing of many loops and the capping of the N termini of the helices.     

Detection and structure determination of an equilibrium unfolding intermediate of Rd-apocytochrome b562: native fold with non-native hydrophobic interactions

The absence of detectable kinetic and equilibrium folding intermediates by optical probes is commonly taken to indicate that protein folding is a two-state process. However, for some small proteins with apparent two-state behavior, unfolding intermediates have been identified in native-state hydrogen exchange or kinetic unfolding experiments monitored by nuclear magnetic resonance. Rd-apocytochrome b(562), a four-helix bundle, is one such protein. Here, we found another unfolding intermediate for Rd-apocytochrome b(562). It is based on a cooperative transition of (15)N chemical shifts of amide protons as a function of urea concentrations before the global unfolding. We have solved the high-resolution structure of the protein at 2.8 M urea, which is after this cooperative transition but before the global unfolding. All four helices remained intact, but a number of hydrophobic core residues repacked. This intermediate provides a possible structural interpretation for the kinetic unfolding intermediates observed using nuclear magnetic resonance methods for several proteins and has important implications for theoretical studies of protein folding.     

Elucidation of stable intermediates in urea-induced unfolding pathway of human carbonic anhydrase IX

Human carbonic anhydrase IX (CAIX) has evolved as a promising biomarker for cancer prognosis, due to its overexpression in various cancers and restricted expression in normal tissue. However, limited information is available on its biophysical behavior. The unfolding of CAIX in aqueous urea solution was studied using all-atom molecular dynamics simulation approach. The results of this study revealed a stable intermediate state along the unfolding pathway of CAIX. At intermediate concentrations of urea (2.0–4.0 M), the protein displays a native-like structure with a large population of its secondary structure and hydrophobic contacts remaining intact in addition to small confined overall motions. Beyond 4.0 M urea, the unfolding is more gradual and at 8.0 M urea the structure is largely collapsed due to the solvent effect. The hydrophobic contact analysis suggests that the contact in terminal α-helices is separated initially which propagates in the loss of contacts from centrally located β-sheets. The reduction of 60–65% tertiary contacts in 7.0–8.0 M urea suggested the presence of residual structure in unfolded state and is confirmed with structural snap shot. Free energy landscape analysis suggested that unfolding of CAIX exists through the different intermediate states.     

Calreticulin is a thermostable protein with distinct structural responses to different divalent cation environments

Calreticulin is a soluble calcium-binding chaperone of the endoplasmic reticulum (ER) that is also detected on the cell surface and in the cytosol. Calreticulin contains a single high affinity calcium-binding site within a globular domain and multiple low affinity sites within a C-terminal acidic region. We show that the secondary structure of calreticulin is remarkably thermostable at a given calcium concentration. Rather than corresponding to complete unfolding events, heat-induced structural transitions observed for calreticulin relate to tertiary structural changes that expose hydrophobic residues and reduce protein rigidity. The thermostability and the overall secondary structure content of calreticulin are impacted by the divalent cation environment, with the ER range of calcium concentrations enhancing stability, and calcium-depleting or high calcium environments reducing stability. Furthermore, magnesium competes with calcium for binding to calreticulin and reduces thermostability. The acidic domain of calreticulin is an important mediator of calcium-dependent changes in secondary structure content and thermostability. Together, these studies indicate interactions between the globular and acidic domains of calreticulin that are impacted by divalent cations. These interactions influence the structure and stability of calreticulin, and are likely to determine the multiple functional activities of calreticulin in different subcellular environments.     

Unfolding and Refolding of Bovine α-Crystallin in Urea and Its Chaperone Activity

We undertook an unfolding and refolding study of α_L-crystallin in presence of urea to explore the breakdown and formation of various levels of structure and to find out whether the breakdown of various levels of structure occurs simultaneously or in a hierarchal manner. We used various techniques such as circular dichroism, fluorescence spectroscopy, light scattering, polarization to determine the changes in secondary, tertiary, and quaternary structure. Unfolding and refolding occurred through a number of intermediates. The results showed that all levels of structure in α_L-crystallin collapsed or reformed simultaneously. The intermediates that occurred in the 2–4 M urea concentration range during unfolding and refolding differed from each other in terms of the polarity of the tryptophan environment. The ANS binding experiments revealed that refolded α_L-crystallin had higher number of hydrophobic pockets compared to native one. On the other hand, polarity of these pockets remained same as that of the native protein. Both light scattering and polarization measurements showed smaller oligomeric size of refolded α_L-crystallin. Thus, although the secondary structural changes were almost reversible, the tertiary and quaternary structural changes were not. The refolded α_L-crystallin had more exposed hydrophobic sites with increased binding affinity. The refolded form also showed higher chaperone activity than native one. Since the refolded form was smaller in oligomeric size, some buried hydrophobic sites were available. The higher chaperone activity of lower sized oligomer of α_L-crystallin again revealed that chaperone activity was dependent on hydrophobicity and not on oligomeric size.     

Ion-induced conformational and stability changes in Nereis sarcoplasmic calcium binding protein: evidence that the APO state is a molten globule

Nereis sarcoplasmic Ca(2+)-binding protein (NSCP) is a calcium buffer protein that binds Ca(2+) ions with high affinity but is also able to bind Mg(2+) ions with high positive cooperativity. We investigated the conformational and stability changes induced by the two metal ions. The thermal reversible unfolding, monitored by circular dichroism spectroscopy, shows that the thermal stability is maximum at neutral pH and increases in the order apo < Mg(2+) < Ca(2+). The stability against chemical denaturation (urea, guanidinium chloride) studied by circular dichroism or intrinsic fluorescence was found to have a similar ion dependence. To explore in more detail the structural basis of stability, we used the fluorescent probes to evaluate the hydrophobic surface exposure in the different ligation states. The apo-NSCP exhibits accessible hydrophobic surfaces, able to bind fluorescent probes, in clear contrast with denatured or Ca(2+)/Mg(2+)-bound states. Gel filtration experiments showed that, although the metal-bound NSCP has a hydrodynamic volume in agreement with the molecular mass, the volume of the apo form is considerably larger. The present results demonstrate that the apo state has many properties in common with the molten globule. The possible factors of the metal-dependent structural changes and stability are discussed.     

Structural reorganization triggered by charging of Lys residues in the hydrophobic interior of a protein

Structural consequences of ionization of residues buried in the hydrophobic interior of proteins were examined systematically in 25 proteins with internal Lys residues. Crystal structures showed that the ionizable groups are buried. NMR spectroscopy showed that in 2 of 25 cases studied, the ionization of an internal Lys unfolded the protein globally. In five cases, the internal charge triggered localized changes in structure and dynamics, and in three cases, it promoted partial or local unfolding. Remarkably, in 15 proteins, the ionization of the internal Lys had no detectable structural consequences. Highly stable proteins appear to be inherently capable of withstanding the presence of charge in their hydrophobic interior, without the need for specialized structural adaptations. The extent of structural reorganization paralleled loosely with global thermodynamic stability, suggesting that structure-based pK(a) calculations for buried residues could be improved by calculation of thermodynamic stability and by enhanced conformational sampling.     

Effect of C-terminal truncation on the molecular chaperone function and dimerization of Escherichia coli trigger factor

To examine the role of the C-terminal domain in the chaperone function of trigger factor (TF), a number of truncation mutants were constructed, namely: TF419, TF389, TF380, TF360, TF344, and TF251, in which the C-terminal 13, 43, 52, 72, 88 residues or the entire C-domain were deleted, respectively. Co-expression of mutant chicken adenylate kinase (AK) with TF and the C-terminal truncation mutants was achieved using a plasmid pBVAT that allows expression of TF and AK from a single plasmid. The results show that truncation of the C-terminus of TF has only minor effect on its ability to assist AK refolding in vivo. Further, ribosome-binding experiments indicate that C-terminal truncation mutants can still bind to the ribosome and the presence of the C-terminus may in fact lower the affinity of TF for the ribosome in vivo. This indicates that the C-domain of trigger factor may not be essential for the ribosome-associated molecular chaperone function of TF. However, the purified TF C-terminal truncation mutants had a dramatically reduced ability to assist rabbit muscle GAPDH refolding in vitro and a reduced tendency to dimerize. This shows that the structural integrity of the C-terminus contributes to both the chaperone function of TF and the stability of the dimeric form.     

Inactivation and conformational changes in methyl parathion hydrolase in 2,2,2-trifluoroethanol solutions: Inactivation kinetics and molecular dynamics simulation

Many improvements have been made in the understanding of functional and structural characteristics of proteins in a denaturant-based microenvironment. This study reports the chemical denaturation of methyl parathion hydrolase (MPH, EC 3.1.8.1) using 2,2,2-trifluoroethanol (TFE). MPH is an important enzyme that catalyzes the hydrolysis of organophosphorus agents. However, the regulation of MPH activity and structural changes during unfolding are not well studied, particularly for TFE unfolding. We investigated MPH unfolding with TFE for the first time. In this study, changes in enzymatic activity and unfolding of MPH at different TFE concentrations were investigated by enzyme activity measurements, intrinsic fluorescence and by 1-anilino-8-naphthalenesulfonate (ANS) fluorescence emission spectral scans. The results showed TFE inactivated MPH in a dose-dependent manner. A Lineweaver–Burk plot analysis revealed that the type of inhibition was reversible noncompetitive inhibition. Intrinsic fluorescence and ANS-binding fluorescence showed that TFE induced obvious tertiary structural changes in MPH by exposing hydrophobic groups. Furthermore, we conducted a docking simulation between MPH and TFE. The computer simulation successfully showed the binding structure and we estimated stability by calculating the binding energy (lowest binding energy: -3.18 kcal/mol). The results demonstrate that MPH can be inactivated by TFE, and provide new insights into the mechanism of TFE-induced unfolding of MPH and inhibition of ligand binding.     

Structural alteration of Escherichia coli Hsp31 by thermal unfolding increases chaperone activity

Escherichia coli Hsp31, encoded by hchA, is a heat-inducible molecular chaperone. We found that Hsp31 undergoes a conformational change via temperature-induced unfolding, generating a high molecular weight (HMW) form with enhanced chaperone activity. Although it has previously been reported that some subunits of the Hsp31 crystal structure show structural heterogeneity with increased hydrophobic surfaces, Hsp31 basically forms a dimer. We found that a C-terminal deletion (CΔ19) of Hsp31 exhibited structurally and functionally similar characteristics to that of the HMW form. Both the CΔ19 and HMW forms achieved a structure with considerably more β-sheets and less α-helices than the native dimeric form, exposing a portion of its hydrophobic surfaces. The structural alterations were determined from its spectral changes in circular dichroism, intrinsic fluorescence of tryptophan residues, and fluorescence of bis-ANS binding to a hydrophobic surface. Interestingly, during thermal transition, the dimeric Hsp31 undergoes a conformational change to the HMW species via the CΔ19 structure, as monitored with near-UV CD spectrum, implying that the CΔ19 resembles an intermediate state between the dimer and the HMW form. From these results, we propose that Hsp31 transforms itself into a fully functional chaperone by altering its tertiary and quaternary structures.     

Hydrophobic Collapse of Trigger Factor Monomer in Solution

Trigger factor (TF) is a chaperone, found in bacterial cells and chloroplasts, that interacts with nascent polypeptide chains to suppress aggregation. While its crystal structure has been resolved, the solution structure and dynamics are largely unknown. We performed multiple molecular dynamics simulations on Trigger factor in solution, and show that its tertiary domains display collective motions hinged about inter-domain linkers with minimal or no loss in secondary structure. Moreover, we find that isolated TF typically adopts a collapsed state, with the formation of domain pairs. This collapse of TF in solution is induced by hydrophobic interactions and stabilised by hydrophilic contacts. To determine the nature of the domain interactions, we analysed the hydrophobicity of the domain surfaces by using the hydrophobic probe method of Acharya et al. [1], [2], as the standard hydrophobicity scales predictions are limited due to the complex environment. We find that the formation of domain pairs changes the hydrophobic map of TF, making the N-terminal and arm2 domain pair more hydrophilic and the head and arm1 domain pair more hydrophobic. These insights into the dynamics and interactions of the TF domains are important to eventually understand chaperone-substrate interactions and chaperone function.     

Effects of congenital cataract mutation R116H on αA-crystallin structure,function and stability

α-crystallin is a molecular chaperone that maintains the optical properties of the lens and delays the onset scattering caused by aging-related protein aggregation. In this research, we found that the missense mutation R116H resulted in an altered size distribution, impaired packing of the secondary structures and modified quaternary structure with great hydrophobic exposure. The mutant exhibited a substrate-dependent chaperone (aggregation–inhibition) or anti-chaperone (aggregation–promotion) effect. Equilibrium unfolding experiments indicated that the mutation stabilized an aggregation-prone intermediate which was not populated during the unfolding of the wild-type protein. The accumulation of this intermediate greatly promoted the formation of non-native large oligomers or aggregates during unfolding. These results suggested that both the aggregation of the mutant upon stress and co-deposition with the target proteins were likely to be responsible for the onset of cataract.     

Recombinant expression and purification of T4 phage Hoc, Soc, gp23, gp24 proteins in native conformations with stability studies

Understanding the biological activity of bacteriophage particles is essential for rational design of bacteriophages with defined pharmacokinetic parameters and to identify the mechanisms of immunobiological activities demonstrated for some bacteriophages. This work requires highly purified preparations of the individual phage structural proteins, possessing native conformation that is essential for their reactivity, and free of incompatible biologically active substances such as bacterial lipopolysaccharide (LPS). In this study we describe expression in E. coli and purification of four proteins forming the surface of the bacteriophage T4 head: gp23, gp24, gphoc and gpsoc. We optimized protein expression using a set of chaperones for effective production of soluble proteins in their native conformations. The assistance of chaperones was critical for production of soluble gp23 (chaperone gp31 of T4 phage) and of gpsoc (chaperone TF of E. coli). Phage head proteins were purified in native conditions by affinity chromatography and size-exclusion chromatography. Two-step LPS removal allowed immunological purity grade with the average endotoxin activity less than 1 unit per ml of protein preparation. The secondary structure and stability of the proteins were studied using circular dichroism (CD) spectrometry, which confirmed that highly purified proteins preserve their native conformations. In increasing concentration of a denaturant (guanidine hydrochloride), protein stability was proved to increase as follows: gpsoc, gp23, gphoc. The denaturation profile of gp24 protein showed independent domain unfolding with the most stable larger domain. The native purified recombinant phage proteins obtained in this work were shown to be suitable for immunological experiments in vivo and in vitro.     

Trigger factor assisted folding of green fluorescent protein

Guanidine induced equilibrium and kinetic folding of a variant of green fluorescent protein (F99S/M153T/V163A, GFPuv) was studied. Using manual mixing and stopped-flow techniques, we combined different probes, including tryptophan fluorescence, chromophore fluorescence and reactivity with DTNB, to trace the spontaneous and TF-assisted folding of guanidine denatured GFPuv. We found that both unfolding and refolding of GFPuv occurred in a stepwise manner and a stable intermediate was populated under equilibrium conditions. The thermodynamic parameters obtained show that the intermediate state of GFPuv is quite compact compared to the denatured state and most of the green fluorescence is retained in this state. By studying GFPuv folding assisted by TF and a number of TF mutants, we found that wild-type TF catalyzes proline isomerization and accelerates the folding rate at low TF concentrations, but retards GFPuv folding and decelerates the folding rate at high TF concentrations. This reflects the two activities of TF, as an enzyme and as a chaperone. A general mechanism of TF assisted protein folding is discussed.     

Appendant structure plays an important role in amyloidogenic cystatin dimerization prior to domain swapping

It has been hypothesized that prior to protein domain swapping, unfolding occurs in regions important for the stability of the native monomeric structure, which probably increases the possibility of intermolecular interaction. In order to explore the detailed information of the important unfolding regions in cystatin prior to domain swapping, 20?ns molecular dynamic simulations were performed at atomic level with typical amyloidogenic chicken cystatin (cC) mutant I66Q monomer under conditions that enable forming amyloid fibrils in biological experiments. Our results showed that I66Q mutant exhibited relatively large secondary structure changes and obvious expanding tendency of hydrophobic core compared to wild-type cC. More importantly, the appendant structure (AS) showed a large displacement and distortion towards the hydrophobic core in amyloidogenic cystatin. The structural analysis on cystatin monomer suggested that structural changes of the AS might make the hydrophobic core expand more easily. In addition, analysis on docking dimer has shown that the distorted AS was favor to intermolecular interactions between two cystatin monomers. Data from an independent theoretical derived algorithm as well as biological experiments also support this hypothesis.