Dynamics of thermodynamically stable, kinetically trapped, and inhibitor-bound states of pepsin

The pepsin folding mechanism involves a prosegment (PS) domain that catalyzes folding, which is then removed, resulting in a kinetically trapped native state. Although native pepsin (Np) is kinetically stable, it is irreversibly denatured due to a large folding barrier, and in the absence of the PS it folds to a more thermodynamically stable denatured state, termed refolded pepsin (Rp). This system serves as a model to understand the nature of kinetic barriers and folding transitions between compact states. Quasielastic neutron scattering (QENS) was used to characterize and compare the flexibility of Np, as a kinetically trapped state, with that of Rp, as a thermodynamically stable fold. Additionally, the dynamics of Np were compared with those of a partially unfolded form and a thermally stabilized, inhibitor-bound form. QENS revealed length-scale-dependent differences between Np and Rp on a picosecond timescale and indicated greater flexibility in Np, leading to the conclusion that kinetic stabilization likely does not correspond to reduced internal dynamics. Furthermore, large differences were observed upon inhibition, indicating that QENS of proteins in solution may prove useful for examining the role of conformational entropy changes in ligand binding.     

The native conformation of plasmepsin II is kinetically trapped at neutral pH

Plasmepsin II (PMII), an aspartic protease from the malarial parasite Plasmodium falciparum, represents a model for understanding protease structure/function relationships due to its unique structure and properties. The present study undertook a thermodynamic and kinetic analysis of the PMII folding mechanism and a pH stability profile. Differential scanning calorimetry revealed that the native state of PMII (Np) was irreversibly unfolded, and in the pH range of 6.5–8.0, PMII refolds to a denatured state (Rp) with higher thermal stability than Np. Rp could also be formed upon partially unfolding PMII at pH 11.0 and 37 °C for 2 h, followed by adjustment to a pH in the range of 6.5–8.0. While Rp could be folded/unfolded reversibly, Np was shown to exist as a kinetically trapped state. By examining the unfolding kinetics of Np and the kinetics of Rp folding to Np at 25 °C, it was found that Np is kinetically trapped by an unfolding barrier of 25.5 kcal/mol, and yet once unfolded, is prevented from folding by a comparable folding barrier. The folding mechanism of PMII is similar to that reported for pepsin. It is hypothesized that the PMII zymogen also utilizes a prosegment-catalyzed folding mechanism.     

Understanding the Mechanism of Prosegment-catalyzed Folding by Solution NMR Spectroscopy

Multidomain protein folding is often more complex than a two-state process, which leads to the spontaneous folding of the native state. Pepsin, a zymogen-derived enzyme, without its prosegment (PS), is irreversibly denatured and folds to a thermodynamically stable, non-native conformation, termed refolded pepsin, which is separated from native pepsin by a large activation barrier. While it is known that PS binds refolded pepsin and catalyzes its conversion to the native form, little structural details are known regarding this conversion. In this study, solution NMR was used to elucidate the PS-catalyzed folding mechanism by examining the key equilibrium states, e.g. native and refolded pepsin, both in the free and PS-bound states, and pepsinogen, the zymogen form of pepsin. Refolded pepsin was found to be partially structured and lacked the correct domain-domain structure and active-site cleft formed in the native state. Analysis of chemical shift data revealed that upon PS binding refolded pepsin folds into a state more similar to that of pepsinogen than to native pepsin. Comparison of pepsin folding by wild-type and mutant PSs, including a double mutant PS, indicated that hydrophobic interactions between residues of prosegment and refolded pepsin lower the folding activation barrier. A mechanism is proposed for the binding of PS to refolded pepsin and how the formation of the native structure is mediated.     

1H, 13C,and 15N backbone resonance assignments of the porcine pepsin and porcine pepsin complexed with pepstatin

Pepsin is formed as the zymogen, pepsinogen, which includes an additional 44 residue prosegment (PS) on the N-terminus. Upon acidification (pH <3) the PS is removed, yielding active pepsin. The PS is critical to such processes as the initiation of correct folding and protein stability. In the present study, the NMR assignments of the 34.6 kDa native porcine pepsin and porcine pepsin complexed with pepstatin are reported in order to obtain structural information regarding PS-catalyzed protein folding. Such information would contribute to a better understanding of the nature of folding/unfolding energy barrier of pepsin and other aspartic proteases.     

Comparison of solution structures and stabilities of native, partially unfolded and partially refolded pepsin

A zymogen-derived protein, pepsin, appears to be incapable of folding to the native state without the presence of the prosegment. To better understand the nature of the irreversible denaturation of pepsin, the present study reports on the characterization of the stability and low-resolution tertiary and secondary structures of native, alkaline unfolded and acid refolded porcine pepsin. Through a combination of small-angle neutron scattering (SANS), CD, and DSC, acid refolded pepsin (Rp) was shown to have secondary and tertiary structures intermediate between the alkaline denatured and native forms but was found to be thermodynamically stable relative to the native state. It was also observed that the acid refolded state of pepsin was dependent on the protein concentration during refolding because CD and SANS data revealed that both the secondary and tertiary structures of concentrated-refolded pepsin (>10 mg/mL) (CRp) were native-like, in contrast to the intermediate nature of Rp, refolded under dilute concentration (<10 mg/mL). Despite a native-like conformation, CRp was more stable and had substantially reduced activity compared to that of the native state, suggesting that the protein was misfolded. It is proposed that the stable but misfolded, acid-refolded states are evidence that pepsin in its native conformation was metastable. Furthermore, the disruption of the active site cleft in the denatured states could be discerned by modeling of the SANS data.     

Statistical optimization and multiple objective programming of lysozyme refolding catalyzed by recombinant DsbA in vitro

DsbA (disulfide bond formation protein A) is essential for disulfide bond formation directly affecting the nascent peptides folding to the correct conformation in vivo. In this paper, recombinant DsbA protein was employed to catalyze denatured lysozyme refolding and inhibit the aggregation of folding intermediates in vitro. Statistical methods, i.e., Plackett–Burman design and small central composite design, were adopted to screen out important factors affecting the refolding process and correlating these parameters with the refolding efficiency including both protein recovery and specific activity of refolded lysozyme. Four important parameters: initial lysozyme concentration, urea concentration, KCl concentration and GSSG (glutathione disulfide) concentration were picked out and operating conditions were optimized by introducing the effectiveness coefficient method and transforming the multiple objective programming into an ordinary constrained optimization issue. Finally, 99.7% protein recovery and 25,600 U/mg specific activity of lysozyme were achieved when 281.35 μg/mL denatured lysozyme refolding was catalyzed by an equivalent molar of DsbA at the optimal settings. The results indicated that recombinant DsbA protein could effectively catalyze the oxidized formation and reduced isomerization of intramolecular disulfide bonds in the refolding of lysozyme in vitro.     

Clinical mutants of human glucose 6-phosphate dehydrogenase: Impairment of NADP+ binding affects both folding and stability

Human glucose 6-phosphate dehydrogenase (G6PD) has both the “catalytic” NADP⁺ site and a “structural” NADP⁺ site where a number of severe G6PD deficiency mutations are located. Two pairs of G6PD clinical mutants, G6PD_Wisconsin (R393G) and G6PD_Nashville (R393H), and G6PD_Fukaya (G488S) and G6PD_Campinas (G488V), in which the mutations are in the vicinity of the “structural” NADP⁺ site, showed elevated K_d values of the “structural” NADP⁺, ranging from 53 nM to 500 nM compared with 37 nM for the wild-type enzyme. These recombinant enzymes were denatured by Gdn-HCl and refolded by rapid dilution in the presence of l-Arg, NADP⁺ and DTT at 25 °C. The refolding yields of the mutants exhibited strong NADP⁺-dependence and ranged from 1.5% to 59.4% with 1000 μM NADP⁺, in all cases lower than the figure of 72% for the wild-type enzyme. These mutant enzymes also displayed decreased thermostability and high susceptibility to chymotrypsin digestion, in good agreement with their corresponding melting temperatures in CD experiments. Taken together, the results support the view that impaired binding of “structural” NADP⁺ can hinder folding as well as cause instability of these clinical mutant enzymes in the fully folded state.     

Catalytic activity and chaperone function of human protein-disulfide isomerase are required for the efficient refolding of proinsulin.

Protein-disulfide isomerase (PDI) catalyzes the formation, rearrangement, and breakage of disulfide bonds and is capable of binding peptides and unfolded proteins in a chaperone-like manner. In this study we examined which of these functions are required to facilitate efficient refolding of denatured and reduced proinsulin. In our model system, PDI and also a PDI mutant having only one active site increased the rate of oxidative folding when present in catalytic amounts. PDI variants that are completely devoid of isomerase activity are not able to accelerate proinsulin folding, but can increase the yield of refolding, indicating that they act as a chaperone. Maximum refolding yields, however, are only achieved with wild-type PDI. Using genistein, an inhibitor for the peptide-binding site, the ability of PDI to prevent aggregation of folding proinsulin was significantly suppressed. The present results suggest that PDI is acting both as an isomerase and as a chaperone during folding and disulfide bond formation of proinsulin.     

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.     

Conserved Prosegment Residues Stabilize a Late-Stage Folding Transition State of Pepsin Independently of Ground States

The native folding of certain zymogen-derived enzymes is completely dependent upon a prosegment domain to stabilize the folding transition state, thereby catalyzing the folding reaction. Generally little is known about how the prosegment accomplishes this task. It was previously shown that the prosegment catalyzes a late-stage folding transition between a stable misfolded state and the native state of pepsin. In this study, the contributions of specific prosegment residues to catalyzing pepsin folding were investigated by introducing individual Ala substitutions and measuring the effects on the bimolecular folding reaction between the prosegment peptide and pepsin. The effects of mutations on the free energies of the individual misfolded and native ground states and the transition state were compared using measurements of prosegment-pepsin binding and folding kinetics. Five out of the seven prosegment residues examined yielded relatively large kinetic effects and minimal ground state perturbations upon mutation, findings which indicate that these residues form strengthened and/or non-native contacts in the transition state. These five residues are semi- to strictly conserved, while only a non-conserved residue had no kinetic effect. One conserved residue was shown to form native structure in the transition state. These results indicated that the prosegment, which is only 44 residues long, has evolved a high density of contacts that preferentially stabilize the folding transition state over the ground states. It is postulated that the prosegment forms extensive non-native contacts during the process of catalyzing correct inter- and intra-domain contacts during the final stages of folding. These results have implications for understanding the folding of multi-domain proteins and for the evolution of prosegment-catalyzed folding.     

The specificity of some pig and human pepsins towards synthetic peptide substrates.

1. The peptidase activities of pig pepsins A and C and human pepsin and gastricsin were compared. 2. The peptides studied had the general formula A Leu Val-His-B. Hydrolysis at 37 degrees C and pH 2.07 occurred at the amino side of the leucine residue for all the enzymes and all the peptides. 3. When A was Ac-Ala the peptides were hydrolysed under these conditions slowly by pig pepsin C only. 4. Pig pepsin A and human pepsin were unable to hydrolyse the tyrosine-containing peptides under the conditions tested. Gastricsin (human pepsin C) had about one-third of the activity of pig pepsin C with these substrates. 5. The increase in the rate of hydrolysis caused by the extension of the chain by a single alanine residue was most marked for pig pepsin A and human pepsin.     

Reversible association of proteins into sub-visible amorphous aggregates using short solubility controlling peptide tags

Careful analysis of sub-visible amorphous aggregates, where proteins associate non-covalently in either native or denatured states without forming a specific quaternary structure, may shed insight into the mechanisms of protein aggregation and solubility. Here we report a biophysical and biochemical analysis of our model protein, a bovine pancreatic trypsin inhibitor variant (BPTI-19A), whose oligomerization were controlled by attaching solubility controlling peptide tags (SCP tags) to its C terminus, which are short peptides composed of a single type of amino acid that modulate protein solubility. The dynamic light scattering and static light scattering at 25 °C indicated that 11 out of 15 SCP tags merely affected the hydrodynamic radius and light scattering intensity of our reference variants BPTI-19A and BPTI-C2G. On the other hand, hydrophobic SCP tags composed of 5 Ile (C5I) or 5 Leu (C5L) were associated into sub-visible aggregates. Circular dichroism indicated that all tagged BPTI variants had the same secondary structure contents as the reference BPTI-19A at 25 °C, suggesting that BPTI-C5I and C5L kept their native structure upon association. Furthermore, the thermal denaturation of all of the BPTI variants was fully reversible and typical of natively folded small globular proteins, as monitored by CD at 222 nm. However, the thermal stability of BPTI-19A tagged with hydrophobic residues decreased with increasing protein concentration and tag's hydrophobicity, and BPTI-C5I and C5L were partially denatured at 37 °C. Biochemical stability assessed by limited proteolysis with pepsin correlated with the extent of the variants' aggregation, and the large sub-visible aggregates formed by BPTI-C5I and C5L significantly increased their resistance to pepsin proteolysis. Altogether, these observations indicated that hydrophobic SCP tags led to the reversible association of native-like proteins into sub-visible soluble amorphous aggregates resistant to pepsin digestion.     

Uncovering Specific Electrostatic Interactions in the Denatured States of Proteins

The stability and folding of proteins are modulated by energetically significant interactions in the denatured state that is in equilibrium with the native state. These interactions remain largely invisible to current experimental techniques, however, due to the sparse population and conformational heterogeneity of the denatured-state ensemble under folding conditions. Molecular dynamics simulations using physics-based force fields can in principle offer atomistic details of the denatured state. However, practical applications are plagued with the lack of rigorous means to validate microscopic information and deficiencies in force fields and solvent models. This study presents a method based on coupled titration and molecular dynamics sampling of the denatured state starting from the extended sequence under native conditions. The resulting denatured-state pK_as allow for the prediction of experimental observables such as pH- and mutation-induced stability changes. I show the capability and use of the method by investigating the electrostatic interactions in the denatured states of wild-type and K12M mutant of NTL9 protein. This study shows that the major errors in electrostatics can be identified by validating the titration properties of the fragment peptides derived from the sequence of the intact protein. Consistent with experimental evidence, our simulations show a significantly depressed pK_a for Asp⁸ in the denatured state of wild-type, which is due to a nonnative interaction between Asp⁸ and Lys¹². Interestingly, the simulation also shows a nonnative interaction between Asp⁸ and Glu⁴⁸ in the denatured state of the mutant. I believe the presented method is general and can be applied to extract and validate microscopic electrostatics of the entire folding energy landscape.     

Conformational and thermodynamic characterization of the premolten globule state occurring during unfolding of the molten globule state of cytochrome c

It has already been shown that the mutant Leu94Gly of horse cytochrome c exists in a molten globule (MG) state. We have carried out studies of reversible folding and unfolding induced by LiCl of this mutant at pH 6.0 and 25 °C by observing changes in the difference molar absorption coefficient at 402 nm, the mean residue ellipticity at 222 nm, and the difference mean residue ellipticity at 409 nm. This process is a three-state process when measured by these probes. The stable folding intermediate state has been characterized by far- and near-UV circular dichroism, tryptophan fluorescence, 8-anilino-1-naphthalenesulfonic acid binding, and dynamic light scattering measurements, which led us to conclude that the intermediate is a premolten globule (PMG). Analysis of the reversible unfolding transition curves for the stability of different states in terms of the Gibbs free energy change at pH 6.0 and 25 °C led us to conclude that the MG state is more stable than the PMG state by 5.4 ± 0.1 kcal mol⁻¹, whereas the PMG state is more stable than the denatured (D) state by only 1.1 ± 0.1 kcal mol⁻¹. A comparison of the conformational and thermodynamic properties of the LiCl-induced PMG state at pH 6.0 with those of the PMG state induced by NaCl at pH 2.0 suggests that a similar PMG state is obtained under both denaturing conditions. Differential scanning calorimetry measurements suggest that heat induces a reversible two-state transition between MG and D states.     

Effects of heme on the structure of the denatured state and folding kinetics of cytochrome b562

Heme-linked proteins, such as cytochromes, are popular subjects for protein folding studies. There is the underlying question of whether the heme affects the structure of the denatured state by cross-linking it and forming other interactions, which would perturb the folding pathway. We have studied wild-type and mutant cytochrome b562 from Escherichia coli, a 106 residue four-alpha-helical bundle. The holo protein apparently refolds with a half-life of 4 micros in its ferrous state. We have analysed the folding of the apo protein using continuous-flow fluorescence as well as stopped-flow fluorescence and CD. The apo protein folded much more slowly with a half-life of 270 micros that was unaffected by the presence of exogenous heme. We examined the nature of the denatured states of both holo and apo proteins by NMR methods over a range of concentrations of guanidine hydrochloride. The starting point for folding of the holo protein in concentrations of denaturant around the denaturation transition was a highly ordered native-like species with heme bound. Fully denatured holo protein at higher concentrations of denaturant consisted of denatured apo protein and free heme. Our results suggest that the very fast folding species of denatured holo protein is in a compact state, whereas the normal folding pathway from fully denatured holo protein consists of the slower folding of the apo protein followed by the binding of heme. These data should be considered in the analysis of folding of heme proteins.     

Common Structural Transitions in Explicit-Solvent Simulations of Villin Headpiece Folding

Molecular dynamics simulations of protein folding can provide very high-resolution data on the folding process; however, due to computational challenges most studies of protein folding have been limited to small peptides, or made use of approximations such as Gō potentials or implicit solvent models. We have performed a set of molecular dynamics simulations totaling >50 μs on the villin headpiece subdomain, one of the most stable and fastest-folding naturally occurring proteins, in explicit solvent. We find that the wild-type villin headpiece reliably folds to a native conformation on timescales similar to experimentally observed folding, but that a fast folding double-norleucine mutant shows significantly more heterogeneous behavior. Along with other recent simulation studies, we note the occurrence of nonnative structures intermediates, which may yield a nativelike signal in the fluorescence measurements typically used to study villin folding. Based on the wild-type simulations, we propose alternative approaches to measure the formation of the native state.     

Thermodynamically stable aggregation-resistant antibody domains through directed evolution

Protein aggregates are usually formed by interactions between unfolded or partially unfolded species, and often occur when a protein is denatured by, for example, heat or low pH. In earlier work, we used a Darwinian selection strategy to create human antibody variable domains that resisted heat aggregation. The repertoires of domains were displayed on filamentous phage and denatured (at 80 °C in pH 7.4), and folded domains were selected by binding to a generic ligand after cooling. This process appeared to select for domains with denatured states that resisted aggregation, but the domains only had low free energies of folding (ΔG_N-D^o = 15-20 kJ/mol at 25 °C in pH 7.4). Here, using the same phage repertoire, we have extended the method to the selection of domains resistant to acid aggregation. In this case, however, the thermodynamic stabilities of selected domains were higher than those selected by thermal denaturation (under both neutral and acidic conditions; ΔG_N-D^o = 26-47 kJ/mol at 25 °C in pH 7.4, or ΔG_N-D^o = 27-34 kJ/mol in pH 3.2). Furthermore, we identified a key determinant (Arg28) that increased the aggregation resistance of the denatured states of the domains at low pH without compromising their thermodynamic stabilities. Thus, the selection process yielded domains that combined thermodynamic stability and aggregation-resistant unfolded states. We suggest that changes to these properties are controlled by the extent to which the folding equilibrium is displaced during the process of selection.     

Familial Alzheimer disease-linked presenilin 1 variants enhance production of both Abeta 1-40 and Abeta 1-42 peptides that are only partially sensitive to a potent aspartyl protease transition state inhibitor of "gamma-secretase"

Presenilin 1 (PS1) plays an essential role in intramembranous "gamma-secretase" processing of several type I membrane proteins, including the beta-amyloid precursor proteins (APP) and Notch1. In this report, we examine the activity of two familial Alzheimer's disease-linked PS1 variants on the production of secreted Abeta peptides and the effects of L-685,458, a potent gamma-secretase inhibitor, on inhibition of Abeta peptides from cells expressing these PS1 variants. We now report that PS1 variants enhance the production and secretion of both Abeta1-42 and Abeta1-40 peptides. More surprisingly, whereas the IC(50) for inhibition of Abeta1-40 peptide production from cells expressing wild-type PS1 is approximately 1.5 microm, cells expressing the PS1deltaE9 mutant PS1 exhibit an IC(50) of approximately 4 microm. Immunoprecipitation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry reveal that the levels of Abeta1-43 peptides are elevated in medium of PS1deltaE9 cells treated with higher concentrations of inhibitor. The differential effects of wild-type and mutant PS1 on gamma-secretase production of Abeta peptides and the disparity in sensitivity of these peptides to a potent gamma-secretase suggest that PS may be necessary, but not sufficient, to catalyze hydrolysis at the scissile bonds that generate the termini of Abeta1-40 and Abeta1-42 peptides.     

Vascular Ehlers-Danlos Syndrome Mutations in Type III Collagen Differently Stall the Triple Helical Folding

Vascular Ehlers-Danlos syndrome (EDS) type IV is the most severe form of EDS. In many cases the disease is caused by a point mutation of Gly in type III collagen. A slower folding of the collagen helix is a potential cause for over-modifications. However, little is known about the rate of folding of type III collagen in patients with EDS. To understand the molecular mechanism of the effect of mutations, a system was developed for bacterial production of homotrimeric model polypeptides. The C-terminal quarter, 252 residues, of the natural human type III collagen was attached to (GPP)₇ with the type XIX collagen trimerization domain (NC2). The natural collagen domain forms a triple helical structure without 4-hydroxylation of proline at a low temperature. At 33 °C, the natural collagenous part is denatured, but the C-terminal (GPP)₇-NC2 remains intact. Switching to a low temperature triggers the folding of the type III collagen domain in a zipper-like fashion that resembles the natural process. We used this system for the two known EDS mutations (Gly-to-Val) in the middle at Gly-910 and at the C terminus at Gly-1018. In addition, wild-type and Gly-to-Ala mutants were made. The mutations significantly slow down the overall rate of triple helix formation. The effect of the Gly-to-Val mutation is much more severe compared with Gly-to-Ala. This is the first report on the folding of collagen with EDS mutations, which demonstrates local delays in the triple helix propagation around the mutated residue.