Folding Activity Research Articles

A New Approach for the Identification of Allosteric Binding Sites in Proteins

Recent results indicate that small‐molecule‐protein interactions modulate the function of a large fraction of the enzyme complement in eukaryotic cells. In particular, modulation of enzyme functions by small metabolites that are not their substrates or products is increasingly recognized as a mechanism by which cells can adapt to changes in environmental conditions. While the effect of metabolite‐binding on conformational transitions and thus protein folding, stability, and catalytic activity is clearly of considerable importance, studying metabolite‐protein interactions is in most instances impeded by the low‐affinity nature of the interactions, their moderate effect on enzyme activity, and the complex nature of their interference with substrate turnover. Here, we describe an approach for the efficient mapping of the binding sites of nutrient polyphenols and small dye molecules to the 80 kDa myosin motor domain. We speculate that their binding occurs preferentially with sites that are important for protein‐metabolite interactions and can also be exploited as binding sites for therapeutic drugs. To determine the affinity of the prolific binders for the myosin‐2 motor domain and to select the most suitable compounds, we performed microscale thermophoresis experiments. Subsequently, we determined the structure of the binding pockets for the most promising compounds by X‐ray crystallography.

The Lid Domain of Caenorhabditis elegans Hsc70 Influences ATP Turnover, Cofactor Binding and Protein Folding Activity

Hsc70 is a conserved ATP-dependent molecular chaperone, which utilizes the energy of ATP hydrolysis to alter the folding state of its client proteins. In contrast to the Hsc70 systems of bacteria, yeast and humans, the Hsc70 system of C. elegans (CeHsc70) has not been studied to date.We find that CeHsc70 is characterized by a high ATP turnover rate and limited by post-hydrolysis nucleotide exchange. This rate-limiting step is defined by the helical lid domain at the C-terminus. A certain truncation in this domain (CeHsc70-Δ545) reduces the turnover rate and renders the hydrolysis step rate-limiting. The helical lid domain also affects cofactor affinities as the lidless mutant CeHsc70-Δ512 binds more strongly to DNJ-13, forming large protein complexes in the presence of ATP. Despite preserving the ability to hydrolyze ATP and interact with its cofactors DNJ-13 and BAG-1, the truncation of the helical lid domain leads to the loss of all protein folding activity, highlighting the requirement of this domain for the functionality of the nematode's Hsc70 protein.

Magnitude and Molecular Origin of Water Slowdown Next to a Protein

Hydration shell dynamics plays a critical role in protein folding and biochemical activity and has thus been actively studied through a broad range of techniques. While all observations concur with a slowdown of water dynamics relative to the bulk, the magnitude and molecular origin of this retardation remain unclear. Via numerical simulations and theoretical modeling, we establish a molecular description of protein hydration dynamics and identify the key protein features that govern it. Through detailed microscopic mapping of the water reorientation and hydrogen-bond (HB) dynamics around lysozyme, we first determine that 80% of the hydration layer waters experience a moderate slowdown factor of ~2-3, while the slower residual population is distributed along a power-law tail, in quantitative agreement with recent NMR results. We then establish that the water reorientation mechanism at the protein interface is dominated by large angular jumps similar to the bulk situation. A theoretical extended jump model is shown to provide the first rigorous determination of the two key contributions to the observed slowdown: a topological excluded-volume factor resulting from the local protein geometry, which governs the dynamics of the fastest 80% of the waters, and a free energetic factor arising from the water-protein HB strength, which is especially important for the remaining waters in confined sites at the protein interface. These simple local factors are shown to provide a nearly quantitative description of the hydration shell dynamics.

Multiple Biological Activities of the Mammalian Protein Hap46/BAG-1

One of the most prominent features of the mammalian protein Hap46/BAG-1M and its isoforms is the ability to form complexes with an enormous variety of unrelated proteins. In the majority of these interactions, however, molecular chaperones of the hsp70 heat shock protein family are the primary interaction partners which themselves are able to bind very different protein structures by making use of their respective substrate-binding domains. The carboxy terminal portion of Hap46/BAG-1M and its isoforms, called the BAG domain, interacts with the adenosine triphosphate (ATP)-binding domain of hsp70 chaperones and thereby affects their protein folding activities. On the other hand, the amino terminal region of Hap46/BAG-1M, rich in basic amino acid residues, mediates the binding to DNA which, however, has no nucleotide sequence specificity. Hap46/BAG-1M can simultaneously interact with DNA and a chaperone of the hsp70 family. Importantly, Hap46/BAG-1M and the larger isoform Hap50/BAG-1L are able to stimulate transcription in vitro and upon overexpression in intact cells, and both the DNA binding region as well as the BAG domain participate in this effect. A model discusses how such stimulation of transcription may occur molecularly. Increased transcriptional activity in cells expressing high levels of Hap46/BAG-1M and the large isoform Hap50/BAG-1L is related to positive effects on proliferation and survival of such cells. Thus, Hap46/BAG-1M and Hap50/BAG-1L now gain significance as tumor markers.

Proteopedia entry: Bovine pancreatic ribonuclease a

This Proteopedia article presents a structural analysis of the well-studied, model enzyme, bovine pancreatic ribonuclease A (RNase A; Fig. 1). RNase A serves as a common discussion point in introductory biochemistry courses, where RNase A is used to illustrate diverse topics ranging from protein structure, function, folding, and enzymatic activity to total protein synthesis. RNase A is standardly depicted in its simple, monomeric state oriented with the active site cleft of its kidney bean structure in the center of the picture (Figs. 1 a and 1 c). This simplistic depiction of the structure and biology of RNase A, based solely on its secondary structure of a four-strand antiparallel β-sheet and three α-helices, undervalues the wealth of additional structural, biological, and chemical advances that RNase A has facilitated. In an interconnected series of five Proteopedia pages, undergraduate students from a second semester biochemistry course used interactive structural applets and integrated descriptions of RNase A to illustrate its historical significance, enzymology, folding, synthesis, solution structure, oligomeric assemblies, and therapeutic applications. Specific focal points from the pages include illustrating the difference between NMR and X-ray crystallography structures with comparisons between flexible and rigid portions of the RNase A structure (Fig. 1 a). RNase A also forms domain-swapped multimers in solution, and experimenting with the oligomerization state of RNase A has served as a model for understanding oligomerization diseases like Alzheimer's disease (Fig. 1 b). Total chemical synthesis of proteins was also developed using RNase A, and newer semisynthetic methods of protein synthesis continue to be refined using RNase A (Fig. 1 c). The five Proteopedia pages are presented together as a resource for biochemistry courses where students can explore protein structure through the easily accessible package of RNase A and as an illustration of hands-on experiences in protein structure, protein visualization, and multimedia design using Proteopedia. Unusual structural features of RNase A. (a) NMR structure of RNase A (PDB code: 2AAS). Top 32 solution structures shown to highlight the flexibility in the loops and termini of RNase A. (b) Domain-swapped dimer of RNase A (PDB code: 1A2W). Dimer formed by swapping of the N-terminal helix between two RNase A monomers (blue and green). (c) Semisynthetic RNase A (PDB code: 1SRN). A noncovalent complex between residues 1 and 118 of RNase A (blue) and a synthetic peptide comprising residues 111–124 (yellow). The synthetic peptide is colored to indicate the hydrophobicity of the peptide: yellow = hydrophobic; red = acidic; brown = basic.

Caught in the Act: Covalent Cross-Linking Captures Activator–Coactivator Interactions in Vivo

Currently there are few methods suitable for the discovery and characterization of transient, moderate affinity protein-protein interactions in their native environment, despite their prominent role in a host of cellular functions including protein folding, signal transduction, and transcriptional activation. Here we demonstrate that a genetically encoded photoactivatable amino acid, p-benzoyl-l-phenylalanine, can be used to capture transient and/or low affinity binding partners in an in vivo setting. In this study, we focused on ensnaring the coactivator binding partners of the transcriptional activator VP16 in S. cerevisiae. The interactions between transcriptional activators and coactivators in eukaryotes are moderate in affinity and short-lived, and due in part to these characteristics, identification of the direct binding partners of activators in vivo has met with only limited success. We find through in vivo photo-cross-linking that VP16 contacts the Swi/Snf chromatin-remodeling complex through the ATPase Snf2(BRG1/BRM) and the subunit Snf5 with two distinct regions of the activation domain. An analogous experiment with Gal4 reveals that Snf2 is also a target of this activator. These results suggest that Snf2 may be a valuable target for small molecule probe discovery given the prominent role the Swi/Snf complex family plays in development and in disease. More significantly, the successful implementation of the in vivo cross-linking methodology in this setting demonstrates that it can be applied to the discovery and characterization of a broad range of transient and/or modest affinity protein-protein interactions.

Heat shock protein 70 purification and characterization from Cyprinion macrastomus macrastomus and Garra rufa obtusa

Organisms resist stress factors by common mechanisms at cellular and physiological levels. Heat shock proteins (Hsps) play an essential role as a protective mechanism against stressors, and Hsp70 has a central role in the heat shock response. Although several investigations have been made on the cellular functions of Hsps, very little is known about extreme temperature effects on organisms like fish. To address this point, we have studied two fish species, Cyprinion macrostomus macrostomus and Garra rufa obtuse, whose habitat is the Kangal hot spring in Turkey. The hot spring has multiple stress factors; extremely hot temperature, food deprivation, infectious medium and low levels of oxygen. Like other Hsp70s, two Cyprinidae species Hsp70 showed similar ATPase and substrate protein folding activities in vitro. These proteins also showed relatively higher thermal stability. Biochemical characterization of two Hsp70, suggested a Hsp mechanism capable of protecting the cell against stressors even under adverse conditions and in the presence of multiple stress factors.

The various facets of the protein‐folding activity of the ribosome

In addition to its involvement in protein synthesis, the ribosome is implicated in protein folding. Some co-translational events, such as the rhythm of protein synthesis, the passage through the exit tunnel of the ribosome, or the interaction with ribosome-associated chaperones may help protein folding. Ribosomes from prokaryotes, eukaryotes, and mitochondria have also been shown to assist the folding of denatured proteins in vitro in a translation-independent way. This intriguing protein-folding activity of the ribosome (PFAR, also termed RPFA) has been mapped to the domain V of the large rRNA of the large subunit of the ribosome. Unfolded, newly synthesized proteins catalyze the dissociation of the two ribosomal subunits in vitro, thereby promoting ribosome recycling and facilitating accessibility of domain V to these proteins, which in turn may help their folding by PFAR. The recent identification of 6AP and GA - the two first drugs that specifically inhibit PFAR without affecting protein translation - will help decipher the biological significance of PFAR in vivo. Of note, 6AP and GA were initially isolated on the basis of their activity against prion-based diseases. Recently, 6AP and GA were also shown to be active in vivo in a drosophila model for oculopharyngeal muscular dystrophy, which is another amyloid-based disease. This effect is mimicked by large deletions in the ribosomal DNA (rDNA) locus. In addition, small deletions in the rDNA locus show a synergistic effect with low doses of 6AP and GA. Hence, PFAR may be involved in various amyloid-based diseases.

Mode of action of the antiprion drugs 6AP and GA on ribosome assisted protein folding

The ribosome, the protein synthesis machinery of the cell, has also been implicated in protein folding. This activity resides within the domain V of the main RNA component of the large subunit of the ribosome. It has been shown that two antiprion drugs 6-aminophenanthridine (6AP) and Guanabenz (GA) bind to the ribosomal RNA and inhibit specifically the protein folding activity of the ribosome. Here, we have characterized with biochemical experiments, the mode of inhibition of these two drugs using ribosomes or ribosomal components active in protein folding (referred to as ’ribosomal folding modulators’ or RFMs) from both bacteria Escherichia coli and yeast Saccharomyces cerevisiae, and human carbonic anhydrase (HCA) as a sample protein. Our results indicate that 6AP and GA inhibit the protein folding activity of the ribosome by competition with the unfolded protein for binding to the ribosome. As a result, the yield of the refolded protein decreases, but the rate of its refolding remains unaffected. Further, 6AP- and GA mediated inhibition of RFM mediated refolding can be reversed by the addition of RFMs in excess. We also demonstrate with delayed addition of the ribosome and the antiprion drugs that there is a short time-span in the range of seconds within which the ribosome interacts with the unfolded protein. Thus we conclude that the protein folding activity of the ribosome is conserved from bacteria to eukaryotes and most likely the substrate for RFMs is an early refolding state of the target protein.

Optimal nitrogen supply as a key to increased and sustained production of a monoclonal full‐size antibody in BY‐2 suspension culture

Plant cell cultures have been used as expression hosts for recombinant proteins for over two decades. The quality of plant cell culture-produced proteins such as full-size monoclonal antibodies has been shown to be excellent in terms of protein folding and binding activity, but the productivity and yield fell short of what was achieved using mammalian cell culture, in which the key to gram-per-liter expression levels was strain selection and medium/process optimization. We carried out an extensive media analysis and optimization for the production of the full-size human anti-HIV antibody 2G12 in N. tabacum cv. BY-2. Nitrogen source and availability was found to be one key factor for the volumetric productivity of plant cell cultures. Increased amounts of nitrate in the culture medium had a dramatic impact on protein yields, resulting in a 10-20-fold increase in product accumulation through a combination of enhanced secretion and higher stability. The results were scalable from shake flasks to stirred-tank bioreactors, where the maximum yield per cultivation volume was 8 mg L(-1) over 7 days. During the stationary phase, antibody levels were 150-fold higher in nitrogen-enriched medium compared to standard medium. The enhanced medium appeared not to affect antibody quality and activity, as determined by Western blots, surface plasmon resonance binding assays and N-glycan analysis.

Mutagenesis Reveals the Complex Relationships between ATPase Rate and the Chaperone Activities of Escherichia coli Heat Shock Protein 70 (Hsp70/DnaK)

The Escherichia coli 70-kDa heat shock protein, DnaK, is a molecular chaperone that engages in a variety of cellular activities, including the folding of proteins. During this process, DnaK binds its substrates in coordination with a catalytic ATPase cycle. Both the ATPase and protein folding activities of DnaK are stimulated by its co-chaperones, DnaJ and GrpE. However, it is not yet clear how changes in the stimulated ATPase rate of DnaK impact the folding process. In this study, we performed mutagenesis throughout the nucleotide-binding domain of DnaK to generate a collection of mutants in which the stimulated ATPase rates varied from 0.7 to 13.6 pmol/microg/min(-1). We found that this range was largely established by differences in the ability of the mutants to be stimulated by one or both of the co-chaperones. Next, we explored how changes in ATPase rate might impact refolding of denatured luciferase in vitro and found that the two activities were poorly correlated. Unexpectedly, we found several mutants that refold luciferase normally in the absence of significant ATP turnover, presumably by increasing the flexibility of DnaK. Finally, we tested whether DnaK mutants could complement growth of DeltadnaK E. coli cells under heat shock and found that the ability to refold luciferase was more predictive of in vivo activity than ATPase rate. This study provides insights into how flexibility and co-chaperone interactions affect DnaK-mediated ATP turnover and protein folding.

Binding of a small molecule at a protein-protein interface regulates the chaperone activity of hsp70-hsp40.

Heat shock protein 70 (Hsp70) is a highly conserved molecular chaperone that plays multiple roles in protein homeostasis. In these various tasks, the activity of Hsp70 is shaped by interactions with co-chaperones, such as Hsp40. The Hsp40 family of co-chaperones binds to Hsp70 through a conserved J-domain, and these factors stimulate ATPase and protein-folding activity. Using chemical screens, we identified a compound, 115-7c, which acts as an artificial co-chaperone for Hsp70. Specifically, the activities of 115-7c mirrored those of a Hsp40; the compound stimulated the ATPase and protein-folding activities of a prokaryotic Hsp70 (DnaK) and partially compensated for a Hsp40 loss-of-function mutation in yeast. Consistent with these observations, NMR and mutagenesis studies indicate that the binding site for 115-7c is adjacent to a region on DnaK that is required for J-domain-mediated stimulation. Interestingly, we found that 115-7c and the Hsp40 do not compete for binding but act in concert. Using this information, we introduced additional steric bulk to 115-7c and converted it into an inhibitor. Thus, these chemical probes either promote or inhibit chaperone functions by regulating Hsp70-Hsp40 complex assembly at a native protein-protein interface. This unexpected mechanism may provide new avenues for exploring how chaperones and co-chaperones cooperate to shape protein homeostasis.

Proteomics of life at low temperatures: trigger factor is the primary chaperone in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125

The proteomes expressed at 4 degrees C and 18 degrees C by the psychrophilic Antarctic bacterium Pseudoalteromonas haloplanktis have been compared using two-dimensional differential in-gel electrophoresis, showing that translation, protein folding, membrane integrity and anti-oxidant activities are upregulated at 4 degrees C. This proteomic analysis revealed that the trigger factor is the main upregulated protein at low temperature. The trigger factor is the first molecular chaperone interacting with virtually all newly synthesized polypeptides on the ribosome and also possesses a peptidyl-prolyl cis-trans isomerase activity. This suggests that protein folding at low temperatures is a rate-limiting step for bacterial growth in cold environments. It is proposed that the psychrophilic trigger factor rescues the chaperone function as both DnaK and GroEL (the major bacterial chaperones but also heat-shock proteins) are downregulated at 4 degrees C. The recombinant psychrophilic trigger factor is a monomer that displays unusually low conformational stability with a Tm value of 33 degrees C, suggesting that the essential chaperone function requires considerable flexibility and dynamics to compensate for the reduction of molecular motions at freezing temperatures. Its chaperone activity is strongly temperature-dependent and requires near-zero temperature to stably bind a model-unfolded polypeptide.

Single-Molecule Kinetics Under Force: Probing Protein Folding and Enzymatic Activity with Optical Tweezers

Weak non-covalent bonds between and within single molecules govern many aspects of biological structure and function (e.g. receptor-ligand binding, protein folding) In living systems, these interactions are often subject to mechanical forces, which can greatly alter their kinetics and activity. My group develops and applies single-molecule manipulation techniques to explore and quantify these force-dependent kinetics. We have developed a variety of optical tweezers techniques, such as high-resolution 3D position tracking using interference imaging (0.2 nm resolution in z, 1 nm in x-y) [1,2], active feedback for longterm stability in trap height and focus (1-2 nm stability) [2], and intensity modulation imaging for quantifying high-frequency fluctuation above the acquistion rate of a detector (power spectrum measurements above 100 kHz can be made with a slow camera) [3]. We have used these methods to quantify the force-dependent unfolding and refolding kinetics of proteins, including the cytoskeletal protein spectrin in collaboration with E. Evans [4], and the A2 domain of the von Willebrand factor blood clotting protein in collaboration with T. Springer [5]. Furthermore, we have studied the kinetics of the ADAMTS13 enzyme acting on a single A2 domain, and have shown that physiolgical forces in the circulation can act as a cofactor for enzymatic cleavage, regulating hemostatic activity [5].

Using multi-objective computational design to extend protein promiscuity

Many enzymes possess, besides their native function, additional promiscuous activities. Proteins with several activities (multipurpose catalysts) may have a wide range of biotechnological and biomedical applications. Natural promiscuity, however, appears to be of limited scope in this context, because the latent (promiscuous) function is often related to the evolved one (sharing the active site and even the chemical mechanism) and its enhancement upon suitable mutations usually brings about a decrease in the native activity. Here we explore the use of computational protein design to overcome these limitations. The high-plasticity positions close to the original (“native”) active-site are the most promising candidates for mutations that create a second active-site associated to a new function. To avoid compromising protein folding and native activity, we propose a minimal-perturbation approach based on the combinatorial optimization of, both the de novo catalytic activity and the folding free-energy: essentially, we construct the Pareto Set of optimal stability/promiscuous-function solutions. We validate our approach by introducing a promiscuous esterase activity in E. coli thioredoxin on the basis of mutations at positions close to the native-active-site disulfide-bridge. Native oxidoreductase activity is not compromised and it is, in fact, found to be 1.5-fold enhanced, as determined by an insulin-reduction assay. This work provides general guidelines as to how computational design can be used to expand the scope and applications of protein promiscuity. From a more general viewpoint, it illustrates the potential of multi-objective optimization as the computational analogue of multi-feature natural selection.

Association of TXNDC5 gene polymorphisms and susceptibility to nonsegmental vitiligo in the Korean population

Vitiligo is a pigmentary skin disorder characterized by a chronic and progressive loss of melanocytes. Although the aetiology of vitiligo is currently unknown, several theories have been proposed to explain the pathogenesis of this disease, including autoimmune, neural, self-destruction, oxidative stress, and genetic theories. Thioredoxin domain containing 5 (TXNDC5) is a newly identified member of the thioredoxin family. TXNDC5 has a protein disulphide isomerase-like domain which plays an important role in protein folding and chaperone activity, against endoplasmic reticulum (ER) stress induced by oxidative stress within the ER. To determine whether variation in the TXNDC5 gene contributes to the risk of developing nonsegmental vitiligo (NSV) in the Korean population. We conducted a case-control association study of 230 patients with NSV and 417 matched, unaffected controls. Seven single nucleotide polymorphisms (SNPs) in the TXNDC5 gene were selected for study. Of the selected SNPs, three exonic SNPs (rs1043784, rs7764128 and rs8643) were statistically associated with NSV. Among them, rs1043784 remained a statistically significant association following Bonferroni correction. These three SNPs were located within a block of linkage disequilibrium; the haplotypes AGG and GAA, consisting of rs1043784, rs7764128 and rs8643, demonstrated a significant association with NSV. These results suggest that TXNDC5 gene polymorphisms are associated with the development of NSV in the Korean population.

Alternate Strategies of Hsp90 Modulation for the Treatment of Cancer and Other Diseases

The 90 kDa heat shock protein (Hsp90) has become a validated target for the development of anti-cancer agents. Several Hsp90 inhibitors are currently under clinical trial investigation for the treatment of cancer. All of these agents inhibit Hsp90's protein folding activity by binding to the N-terminal ATP binding site of the Hsp90 molecular chaperone. Administration of these investigational drugs elicits induction of the heat shock response, or the overexpression of several Hsps, which exhibit antiapoptotic and pro-survival effects that may complicate the application of these inhibitors. To circumvent this issue, alternate mechanisms for Hsp90 inhibition that do not elicit the heat shock response have been identified and pursued. After providing background on the structure, function, and mechanism of the Hsp90 protein folding machinery, this review describes several mechanisms of Hsp90 modulation via small molecules that do not induce the heat shock response.

Exploring the midgut proteome of partially fed female cattle tick (Rhipicephalus (Boophilus) microplus)

The continued development of effective anti-tick vaccines remains the most promising prospect for the control of the cattle tick, Rhipicephalus (Boophilus) microplus. A vaccine based on midgut proteins could interfere with successful tick feeding and additionally interfere with midgut developmental stages of Babesia parasites, providing opportunities for the control of both the tick and the pathogens it transmits. Midgut proteins from partially fed adult female cattle ticks were analysed using a combination of 2-DE and gel-free LC-MS/MS. Analysis of the urea-soluble protein fraction resulted in the confident identification of 105 gut proteins, while the PBS-soluble fraction yielded an additional 37 R. microplus proteins. The results show an abundance of proteins involved in mitochondrial ATP synthesis, electron transport chain, protein synthesis, chaperone, antioxidant and protein folding and transport activities in midgut tissues of adult female ticks. Among the novel products identified were clathrin-adaptor protein, which is involved in the assembly of clathrin-coated vesicles, and membrane-associated trafficking proteins such as syntaxin 6 and surfeit 4. The observations allow the formulation of hypotheses regarding midgut physiology and will serve as a basis for future vaccine development and tick-host interaction research.

Multifunctional host defense peptides: intracellular‐targeting antimicrobial peptides

There is widespread acceptance that cationic antimicrobial peptides, apart from their membrane-permeabilizing/disrupting properties, also operate through interactions with intracellular targets, or disruption of key cellular processes. Examples of intracellular activity include inhibition of DNA and protein synthesis, inhibition of chaperone-assisted protein folding and enzymatic activity, and inhibition of cytoplasmic membrane septum formation and cell wall synthesis. The purpose of this minireview is to question some widely held views about intracellular-targeting antimicrobial peptides. In particular, I focus on the relative contributions of intracellular targeting and membrane disruption to the overall killing strategy of antimicrobial peptides, as well as on mechanisms whereby some peptides are able to translocate spontaneously across the plasma membrane. Currently, there are no more than three peptides that have been convincingly demonstrated to enter microbial cells without the involvement of stereospecific interactions with a receptor/docking molecule and, once in the cell, to interfere with cellular functions. From the limited data currently available, it seems unlikely that this property, which is isolated in particular peptide families, is also shared by the hundreds of naturally occurring antimicrobial peptides that differ in length, amino acid composition, sequence, hydrophobicity, amphipathicity, and membrane-bound conformation. Microbial cell entry and/or membrane damage associated with membrane phase/transient pore or long-lived transitions could be a feature common to intracellular-targeting antimicrobial peptides and mammalian cell-penetrating peptides that have an overrepresentation of one or two amino acids, i.e. Trp and Pro, His, or Arg. Differences in membrane lipid composition, as well as differential lipid recruitment by peptides, may provide a basis for microbial cell killing on one hand, and mammalian cell passage on the other.

The MitCHAP-60 Disease Is Due to Entropic Destabilization of the Human Mitochondrial Hsp60 Oligomer

The 60-kDa heat shock protein (mHsp60) is a vital cellular complex that mediates the folding of many of the mitochondrial proteins. Its function is executed in cooperation with the co-chaperonin, mHsp10, and requires ATP. Recently, the discovery of a new mHsp60-associated neurodegenerative disorder, MitCHAP-60 disease, has been reported. The disease is caused by a point mutation at position 3 (D3G) of the mature mitochondrial Hsp60 protein, which renders it unable to complement the deletion of the homologous bacterial protein in Escherichia coli (Magen, D., Georgopoulos, C., Bross, P., Ang, D., Segev, Y., Goldsher, D., Nemirovski, A., Shahar, E., Ravid, S., Luder, A., Heno, B., Gershoni-Baruch, R., Skorecki, K., and Mandel, H. (2008) Am. J. Hum. Genet. 83, 30-42). The molecular basis of the MitCHAP-60 disease is still unknown. In this study, we present an in vitro structural and functional analysis of the purified wild-type human mHsp60 and the MitCHAP-60 mutant. We show that the D3G mutation leads to destabilization of the mHsp60 oligomer and causes its disassembly at low protein concentrations. We also show that the mutant protein has impaired protein folding and ATPase activities. An additional mutant that lacks the first three amino acids (N-del), including Asp-3, is similarly impaired in refolding activity. Surprisingly, however, this mutant exhibits profound stabilization of its oligomeric structure. These results suggest that the D3G mutation leads to entropic destabilization of the mHsp60 oligomer, which severely impairs its chaperone function, thereby causing the disease.