Hydrogen deuterium exchange mass spectrometry applied to chaperones and chaperone-assisted protein folding

Introduction: Hydrogen–deuterium exchange (HDX) mass spectrometry (MS) is ideal for monitoring the protein folding and unfolding. The exchange of a deuterium in solution for an amide hydrogen in a protein can be very different depending on the degree of folding and protection of backbone amide positions. Molecular chaperones that assist with protein folding in vivo are necessary for folding of many substrate (client) proteins. HDX MS provides valuable insight into what chaperones are doing in protein folding and how they are doing it.

Areas covered: Application of HDX MS to the protein folding problem was desirable from the outset of the technique, but technical issues prohibited many studies. In the last 20 years, conformational changes of chaperones themselves (e.g., GroEL/GroES, Hsp70, and Hsp90) have been studied. Studies of interactions between chaperones, co-chaperones, and substrate proteins have revealed binding interfaces, allosteric conformational changes, and remodeling of components during various chaperone cycles. Experiments elucidating how chaperones contribute to and enhance the folding pathway of substrate proteins have been demonstrated.

Expert opinion: Technical issues that once prevented the analysis of chaperones have largely been resolved, permitting exciting comprehensive HDX MS studies of folding pathways during chaperone-assisted protein folding.     

GroEL-Mediated Protein Folding: Making the Impossible,Possible

ABSTRACT

Protein folding is a spontaneous process that is essential for life, yet the concentrated and complex interior of a cell is an inherently hostile environment for the efficient folding of many proteins. Some proteins—constrained by sequence, topology, size, and function—simply cannot fold by themselves and are instead prone to misfolding and aggregation. This problem is so deeply entrenched that a specialized family of proteins, known as molecular chaperones, evolved to assist in protein folding. Here we examine one essential class of molecular chaperones, the large, oligomeric, and energy utilizing chaperonins or Hsp60s. The bacterial chaperonin GroEL, along with its co-chaperonin GroES, is probably the best-studied example of this family of protein-folding machine. In this review, we examine some of the general properties of proteins that do not fold well in the absence of GroEL and then consider how folding of these proteins is enhanced by GroEL and GroES. Recent experimental and theoretical studies suggest that chaperonins like GroEL and GroES employ a combination of protein isolation, unfolding, and conformational restriction to drive protein folding under conditions where it is otherwise not possible.     

The ins and outs of GroEL-mediated protein folding.

Two papers recently published in Cell investigate the role of protein encapsulation by GroEL in assisting folding. The first shows how encapsulation can "smooth" the folding landscape, accelerating folding of some proteins. The second defines a confinement-independent pathway, which allows GroEL to assist folding of substrates too large to be encapsulated.     

GroEL/GroES: structure and function of a two-stroke folding machine

Recent structural and functional studies have greatly advanced our understanding of the mechanism by which chaperonins (Cpn60) mediate protein folding, the final step in the accurate expression of genetic information. Escherichia coli GroEL has a symmetric double-toroid architecture, which binds nonnative polypeptide substrates on the hydrophobic walls of its central cavity. The asymmetric binding of ATP and cochaperonin GroES to GroEL triggers a major conformational change in the cis ring, creating an enlarged chamber into which the bound nonnative polypeptide is released. The structural changes that create the cis assembly also change the lining of the cavity wall from hydrophobic to hydrophilic, conducive to folding into the native state. ATP hydrolysis in the cis ring weakens it and primes the release of products. When ATP and GroES bind to the trans ring, it forms a stronger assembly, which disassembles the cis complex through negative cooperativity between rings. The opposing function of the two rings operates as if the system had two cylinders, one expelling the products of the reaction as the other loads up the reactants. One cycle of the reaction gives the polypeptide about 15 s to fold at the cost of seven ATP molecules. For some proteins, several cycles of GroEL assistance may be needed in order to achieve their native states.     

The effect of C-terminal mutations of HSP60 on protein folding

HSP60 is an essential gene in Saccharomyces cerevisiae. The protein forms homotetradecameric double toroid complexes. The flexible C-terminal end of each subunit, which is hydrophobic in nature, protrudes inside the central cavity where protein folding occurs. In order to study the functional role of the C-terminus of Hsp60, we generated and characterized yeast strains expressing mutants of Hsp60 proteins. Most of the yeast strains expressing Hsp60 with C-terminal deletions grew normally, unless the deletion impaired the interaction between neighboring subunits. The cells carrying Hsp60 mutants with an epitope of influenza hemagglutinin (HA) and T7 alone in the C-terminal region grew normally, but the mutant containing both HA and T7 was unable to grow in nonfermentable carbon sources. In vitro biochemical assays were performed using purified Hsp60 proteins. All the mutants examined remained capable of interacting with Hsp10 in a nucleotide-dependent manner. However, binding and/or refolding of denatured rhodanese became defective in most of the hsp60 mutants. Therefore, the hydrophobic C-terminal tail of Hsp60 plays an important role in the refolding of protein substrates, although it is flexible in structure.     

Isolation and characterisation of mutants of GroEL that are fully functional as single rings

A key aspect of the reaction mechanism for the molecular chaperone GroEL is the transmission of an allosteric signal between the two rings of the GroEL complex. Thus, the single-ring mutant SR1 is unable to act as a chaperone as it cannot release bound substrate or GroES. We used a simple selection procedure to identify mutants of SR1 that restored chaperone activity in vivo. A large number of single amino acid changes, mapping at diverse positions throughout the protein, enabled SR1 to regain its ability to act as a chaperone while remaining as a single ring. In vivo assays were used to identify the proteins that had regained maximal activity. In some cases, no difference could be detected between strains expressing wild-type GroEL and those expressing the mutated proteins. Three of the most active proteins where the mutations were in distinct parts of the protein were purified to homogeneity and characterised in vitro. All were capable of acting efficiently as chaperones for two different GroES-dependent substrates. All three proteins bound nucleotide as effectively as did GroEL, but the binding of GroES in the presence of ATP or ADP was reduced significantly relative to the wild-type. These active single rings should provide a useful tool for studying the nature of the allosteric changes that occur in the GroEL reaction cycle.     

Type I chaperonins: not all are created equal

Type I chaperonins play an essential role in the folding of newly translated and stress-denatured proteins in eubacteria, mitochondria and chloroplasts. Since their discovery, the bacterial chaperonins have provided an excellent model system for investigating the mechanism by which chaperonins mediate protein folding. Due to the high conservation of the primary sequence among Type I chaperonins, it is generally accepted that organellar chaperonins function similar to the bacterial ones. However, recent studies indicate that the chloroplast and mitochondrial chaperonins possess unique structural and functional properties that distinguish them from their bacterial homologs. This review focuses on the unique properties of organellar chaperonins.     

In vitro reassembly of tobacco ribulose-1,5-bisphosphate carboxylase/oxygenase from fully denatured subunits

It has been generally proved impossible to reassemble ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from fully denatured subunits in vitro in higher plant,because large subunit of fullydenatured Rubisco is liable to precipitate when the denaturant is removed by common methods of directdilution and one-step dialysis.In our experiment,the problem of precipitation was resolved by an improvedgradual dialysis method,which gradually decreased the concentration of denaturant.However,fully denaturedRubisco subunits still could not be reassembled into holoenzyme using gradual dialysis unless chaperonin 60was added.The restored activity of reassembled Rubisco was approximately 8% of natural enzyme.Thequantity of reassembled Rubisco increased greatly when heat shock protein 70 was present in the reassemblyprocess.ATP and Mg~(2 ) were unnecessary for in vitro reassembly of Rubisco,and Mg~(2 ) inhibited the reassemblyprocess.The reassembly was weakened when ATP,Mg~(2 ) and K~ existed together in the reassembly process.     

Purification and Characterization of the GroESLx Chaperonins from the Symbiotic X-Bacteria in Amoeba proteus

GroELx and GroESx proteins of symbiotic X-bacteria from Amoeba proteus were overproduced in Escherichia coli transformed with pAJX91 and pUXGPRM, respectively, and their chaperonin functions were assayed. We utilized σ⁷⁰-dependent specific promoters of groEx in the expression vectors and grew recombinant cells at 37°C to minimize coexpression of host groE of E. coli. For purifying the proteins, we applied the principle of heat stability for GroELx and pI difference for GroESx to minimize copurification with the hosts GroEL and GroES, respectively. After ultracentrifugation in a sucrose density gradient, the yield and purity of GroELx were 56 and 89%, respectively. The yield and purity of GroESx after anion-exchange chromatography were 62 and 91%, respectively. Purified GroELx had an ATPase activity of 53.2 nmol Pi released/min/mg protein at 37°C. The GroESx protein inhibited ATPase activity of GroELx to 60% of the control at a ratio of 1 for GroESx-7mer/GroELx-14mer. GroESLx helped refolding of urea-unfolded rhodanese up to 80% of the native activity at 37°C. By chemical cross-linking analysis, oligomeric properties of GroESx and GroELx were confirmed as GroESx₇ and GroELx₁₄ in two stacks of GroELx₇. In this study, we developed a method for the purification of GroESLx and demonstrated that their chaperonin function is homologous to GroESL of E. coli.     

Protein folding rates and thermodynamic stability are key determinants for interaction with the Hsp70 chaperone system

The Hsp70 family of molecular chaperones participates in vital cellular processes including the heat shock response and protein homeostasis. E. coli''s Hsp70, known as DnaK, works in concert with the DnaJ and GrpE co-chaperones (K/J/E chaperone system), and mediates cotranslational and post-translational protein folding in the cytoplasm. While the role of the K/J/E chaperones is well understood in the presence of large substrates unable to fold independently, it is not known if and how K/J/E modulates the folding of smaller proteins able to fold even in the absence of chaperones. Here, we combine experiments and computation to evaluate the significance of kinetic partitioning as a model to describe the interplay between protein folding and binding to the K/J/E chaperone system. First, we target three nonobligatory substrates, that is, proteins that do not require chaperones to fold. The experimentally observed chaperone association of these client proteins during folding is entirely consistent with predictions from kinetic partitioning. Next, we develop and validate a computational model (CHAMP70) that assumes kinetic partitioning of substrates between folding and interaction with K/J/E. CHAMP70 quantitatively predicts the experimentally measured interaction of RNase H^D as it refolds in the presence of various chaperones. CHAMP70 shows that substrates are posed to interact with K/J/E only if they are slow-folding proteins with a folding rate constant k_f <50 s⁻¹, and/or thermodynamically unstable proteins with a folding free energy ΔG⁰_UN ≥−2 kcal mol⁻¹. Hence, the K/J/E system is tuned to use specific protein folding rates and thermodynamic stabilities as substrate selection criteria.     

The Hsp60 folding machinery is crucial for manganese superoxide dismutase folding and function

Abstract

Even though the deleterious effects of increased reactive oxygen species (ROS) levels have been implicated in a variety of neurodegenerative disorders, the triggering events that lead to the increased ROS and successive damages are still ill-defined. Mitochondria are the key organelles controlling the ROS balance, being their main source and also counteracting them by the action of the ROS scavenging system. Mitochondria, moreover, control the presence of ROS-damaged proteins by action of the protein quality control (PQC) system. One of its components is the mitochondrial chaperone Hsp60 assisting the folding of a subset of mitochondrial matrix proteins. Mutations in Hsp60 cause a late onset form of the neurodegenerative disease hereditary spastic paraplegia (SPG13). In this study, we aimed to address the molecular consequences of Hsp60 shortage. We here demonstrate that a heterozygous knockout Hsp60 model that recapitulates features of the human disease and exhibits increased oxidative stress in neuronal tissues. Moreover, we indicate that the increase of ROS is, at least in part, due to impaired folding of the manganese superoxide dismutase (MnSOD), a key antioxidant enzyme. We observed that the Hsp60 and MnSOD proteins interact. Based on these results, we propose that MnSOD is a substrate of the Hsp60 folding machinery and that under conditions of diminished availability of Hsp60, MnSOD is impaired in reaching the native state. This suggests a possible link between Hsp60-dependent PQC and the ROS scavenging systems that may have the function to increase ROS production under conditions of folding stress.     

Archaeal group II chaperonin mediates protein folding in the cis-cavity without a detachable GroES-like co-chaperonin.

Group II chaperonins of archaea and eukaryotes are distinct from group I chaperonins of bacteria. Whereas group I chaperonins require the co-chaperonin Cpn-10 or GroES for protein folding, no co-chaperonin has been known for group II. The protein folding mechanism of group II chaperonins is not yet clear. To understand this mechanism, we examined protein refolding by the recombinant alpha or beta-subunit chaperonin homo-oligomer (alpha16mer and beta16mer) from a hyperthermoplilic archaeum, Thermococcus strain KS-1, using a model substrate, green fluorescent protein (GFP). The alpha16mer and beta16mer captured the non-native GFP and promoted its refolding without any co-chaperonin in an ATP dependent manner. A non-hydrolyzable ATP analog, AMP-PNP, induced the GFP refolding mediated by beta16mer but not by the alpha16mer. A mutant alpha-subunit chaperonin homo-oligomer (trap-alpha) could capture the non-native protein but lacked the ability to refold it. Although trap-alpha suppressed ATP-dependent refolding of GFP mediated by alpha16mer or beta16mer, it did not affect the AMP-PNP-dependent refolding. This indicated that the GFP refolding mediated by beta16mer with AMP-PNP was not accessible to the trap-alpha. Gel filtration chromatography and a protease protection experiment revealed that this refolded GFP, in the presence of AMP-PNP, was associated with beta16mer. After the completion of GFP refolding mediated by beta16mer with AMP-PNP, addition of ATP induced an additional refolding of GFP. Furthermore, the beta16mer preincubated with AMP-PNP showed the ability to capture the non-native GFP. These suggest that AMP-PNP induced one of two chaperonin rings (cis-ring) to close and induced protein refolding in this ring, and that the other ring (trans-ring) could capture the unfolded GFP which was refolded by adding ATP. The present data indicate that, in the group II chaperonin of Thermococcus strain KS-1, the protein folding proceeds in its cis-ring in an ATP-dependent fashion without any co-chaperonin.     

Comparative analysis of the protein folding activities of two chaperonin subunits of Thermococcus strain KS-1: the effects of beryllium fluoride

We conducted a comparative analysis of the effects of beryllium fluoride (BeFx) on protein folding mediated by the α- and β-subunit homooligomers (α16mer or β16mer) from the hyperthermophilic archaeum Thermococcus strain KS-1. BeFx inhibited the ATPase activities of both α16mer and β16mer with equal efficiency. This indicated that BeFx replaces the γ-phosphate of chaperonin-bound ATP, thereby forming a stable chaperonin–ADP–BeFx complex. In the presence of ATP and BeFx, both of the two chaperonin subunits mediated green fluorescent protein (GFP) folding. Gel filtration chromatography revealed that the refolded GFP was retained by both chaperonins. Protease digestion and electron microscopic analyses showed that both chaperonin–ADP–BeFx complexes of the two subunits adopted a symmetric closed conformation with the built-in lids of both rings closed and that protein folding took place in their central cavities. These data indicated that basic protein folding mechanisms of α16mer and β16mer are likely similar although there were some apparent differences. While β16mer-mediated GFP refolding in the presence of ATP–BeFx that proceeded more rapidly than in the presence of ATP alone and reached a twofold higher plateau than that achieved with AMP–PNP, the folding mediated by the α16mer that proceeded with much lower yields. A mutant of α16mer, trapα, which traps the unfolded and partially folded substrate protein, did not affect the ATP–BeFx-dependent GFP folding by β16mer but it suppressed that mediated by α16mer to the level of spontaneous folding. These results suggested that β16mer differed from the α16mer in nucleotide binding affinity or the rate of nucleotide hydrolysis.     

Function of phosducin-like proteins in G protein signaling and chaperone-assisted protein folding

Members of the phosducin gene family were initially proposed to act as down-regulators of G protein signaling by binding G protein βγ dimers (Gβγ) and inhibiting their ability to interact with G protein subunits (G) and effectors. However, recent findings have over-turned this hypothesis by showing that most members of the phosducin family act as co-chaperones with the cytosolic chaperonin complex (CCT) to assist in the folding of a variety of proteins from their nascent polypeptides. In fact rather than inhibiting G protein pathways, phosducin-like protein 1 (PhLP1) has been shown to be essential for G protein signaling by catalyzing the folding and assembly of the Gβγ dimer. PhLP2 and PhLP3 have no role in G protein signaling, but they appear to assist in the folding of proteins essential in regulating cell cycle progression as well as actin and tubulin. Phosducin itself is the only family member that does not participate with CCT in protein folding, but it is believed to have a specific role in visual signal transduction to chaperone Gβγ subunits as they translocate to and from the outer and inner segments of photoreceptor cells during light-adaptation.     

Structural stability and solution structure of chaperonin GroES heptamer studied by synchrotron small-angle X-ray scattering

The GroES protein from Escherichia coli is a well-known member of the molecular chaperones. GroES consists of seven identical 10 kDa subunits, and forms a dome-like oligomeric structure. In order to obtain information on the structural stability and unfolding-refolding mechanism of GroES protein, especially at protein concentrations (0.4-1.2 mM GroES monomer) that would mimic heat stress conditions in vivo, we have performed synchrotron small-angle X-ray scattering (SAXS) experiments. Surprisingly, in spite of the high protein concentration, reversibility in the unfolding-refolding reaction was confirmed by SAXS experiments structurally. Although the unfolding-refolding reaction showed an apparent single transition with a Cm of 1.1 M guanidium hydrochloride, a more detailed analysis of this transition demonstrated that the unfolding mechanism could be best explained by a sequential three-state model, which consists of native heptamer, dissociated monomer, and unfolded monomer. Together with our previous result that GroES unfolded completely via a partially folded monomer according to a three-state model at low protein concentration (5 microM monomer), the unfolding-refolding mechanism of GroES protein could be explained uniformly by the three-state model from low to high protein concentrations. Furthermore, to clarify an ambiguity of the native GroES structure in solution, especially mobile loop structures, we have estimated a solution structure of GroES using SAXS profiles obtained from experiments and simulation analysis. The result suggested that the native structure of GroES in solution was very similar to that seen in GroES-GroEL complex determined by crystallography.     

Population analyses of kinetic partitioning in protein folding

A simple lattice model of protein folding is studied in order to analyze the kinetic partitioning phenomena in the energy landscape perspective. By restricting the area of conformational space, it becomes possible to follow many Monte Carlo trajectories until they reach equilibrium. Alteration of population of trajectories is monitored and the relations between the energy landscape and kinetics are examined. Kinetic partitioning phenomena are categorized into different types in terms of characteristic time constants and partitioning ratio. In a specific partitioning process, refolding proceeds along the parallel pathways; the time constants have a temperature dependence similar to that observed in hen lysozyme. High-energy conformations are classified into groups according to the probability that the trajectories starting from those conformations will reach each energy valley. The partitioning ratio is determined by the way in which the conformational space is organized into these groups.     

A cell-based screen for inhibitors of protein folding and degradation

Cancer cells are exposed to external and internal stresses by virtue of their unrestrained growth, hostile microenvironment, and increased mutation rate. These stresses impose a burden on protein folding and degradation pathways and suggest a route for therapeutic intervention in cancer. Proteasome and Hsp90 inhibitors are in clinical trials and a 20S proteasome inhibitor, Velcade, is an approved drug. Other points of intervention in the folding and degradation pathway may therefore be of interest. We describe a simple screen for inhibitors of protein synthesis, folding, and proteasomal degradation pathways in this paper. The molecular chaperone-dependent client v-Src was fused to firefly luciferase and expressed in HCT-116 colorectal tumor cells. Both luciferase and protein tyrosine kinase activity were preserved in cells expressing this fusion construct. Exposing these cells to the Hsp90 inhibitor geldanamycin caused a rapid reduction of luciferase and kinase activities and depletion of detergent-soluble v-Src::luciferase fusion protein. Hsp70 knockdown reduced v-Src::luciferase activity and, when combined with geldanamycin, caused a buildup of v-Src::luciferase and ubiquitinated proteins in a detergent-insoluble fraction. Proteasome inhibitors also decreased luciferase activity and caused a buildup of phosphotyrosine-containing proteins in a detergent-insoluble fraction. Protein synthesis inhibitors also reduced luciferase activity, but had less of an effect on phosphotyrosine levels. In contrast, certain histone deacetylase inhibitors increased luciferase and phosphotyrosine activity. A mass screen led to the identification of Hsp90 inhibitors, ubiquitin pathway inhibitors, inhibitors of Hsp70/Hsp40-mediated refolding, and protein synthesis inhibitors. The largest group of compounds identified in the screen increased luciferase activity, and some of these increase v-Src levels and activity. When used in conjunction with appropriate secondary assays, this screen is a powerful cell-based tool for studying compounds that affect protein synthesis, folding, and degradation.     

Heat shock proteins: Molecules with assorted functions

Heat shock proteins (Hsps) or molecular chaperones, are highly conserved protein families present in all studied organisms. Following cellular stress, the intracellular concentration of Hsps generally increases several folds. Hsps undergo ATP-driven conformational changes to stabilize unfolded proteins or unfold them for translocation across membranes or mark them for degradation. They are broadly classified in several families according to their molecular weights and functional properties. Extensive studies during the past few decades suggest that Hsps play a vital role in both normal cellular homeostasis and stress response. Hsps have been reported to interact with numerous substrates and are involved in many biological functions such as cellular communication, immune response, protein transport, apoptosis, cell cycle regulation, gametogenesis and aging. The present review attempts to provide a brief overview of various Hsps and summarizes their involvement in diverse biological activities.     

Monoclonal antibodies to heat shock protein 60 induce a protective immune response against experimental Paracoccidioides lutzii

Paracoccidioidomycosis (PCM) is an endemic mycosis in Latin America. PCM is primarily caused by Paracoccidioides brasiliensis and less frequently by the recently described, closely related species Paracoccidioides lutzii. Current treatment requires protracted administration of systemic antibiotics and relapses may frequently occur despite months of initial therapy. Hence, there is a need for innovative approaches to treatment. In the present study we analyzed the impact of two monoclonal antibodies (mAbs) generated against Heat Shock 60 (Hsp60) from Histoplasma capsulatum on the interactions of P. lutzii with macrophages and on the experimental P. lutzii infection. We demonstrated that the Hsp60-binding mAbs labeled P. lutzii yeast cells and enhanced their phagocytosis by macrophage cells. Treatment of mice with the mAbs to Hsp60 before infection reduced the pulmonary fungal burden as compared to mice treated with irrelevant mAb. Hence, mAbs raised to H. capsulatum Hsp60 are protective against P. lutzii, including mAb 7B6 which was non-protective against H. capsulatum, suggesting differences in their capacity to bind to these fungi and to be recognized by macrophages. These findings indicate that mAbs raised to one dimorphic fungus may be therapeutic against additional dimorphic fungi, but also suggests that biological differences in diseases may influence whether a mAb is beneficial or harmful.