Functional crosstalk between the TIM22 complex and YME1 machinery maintains mitochondrial proteostasis and integrity.

TIM22 pathway cargos are essential for sustaining mitochondrial homeostasis as an excess of these proteins leads to proteostatic stress and cell death. Yme1 is an inner membrane metalloprotease that regulates protein quality control with chaperone-like and proteolytic activities. Although the mitochondrial translocase and protease machinery are critical for organelle health, their functional association remains unexplored. The present study unravels a novel genetic connection between the TIM22 complex and YME1 machinery in Saccharomyces cerevisiae that is required for maintaining mitochondrial health. Our genetic analyses indicate that impairment in the TIM22 complex rescues the respiratory growth defects of cells without Yme1. Furthermore, Yme1 is essential for the stability of the TIM22 complex and regulates the proteostasis of TIM22 pathway substrates. Moreover, impairment in the TIM22 complex suppressed the mitochondrial structural and functional defects of Yme1-devoid cells. In summary, excessive levels of TIM22 pathway substrates could be one of the reasons for respiratory growth defects of cells lacking Yme1, and compromising the TIM22 complex can compensate for the imbalance in mitochondrial proteostasis caused by the loss of Yme1.

Multiple variants of the human presequence translocase motor subunit Magmas govern the mitochondrial import.

Mitochondrial protein translocation is an intricately regulated process that requires dedicated translocases at the outer and inner membranes. The presequence translocase complex, translocase of the inner membrane 23, facilitates most of the import of preproteins containing presequences into the mitochondria, and its primary structural organization is highly conserved. As part of the translocase motor, two J-proteins, DnaJC15 and DnaJC19, are recruited to form two independent translocation machineries (translocase A and translocase B, respectively). On the other hand, the J-like protein subunit of translocase of the inner membrane 23, Mitochondria-associated granulocyte-macrophage colony-stimulating factor signaling molecule (Magmas) (orthologous to the yeast subunit Pam16), can regulate human import-motor activity by forming a heterodimer with DnaJC19 and DnaJC15. However, the precise coordinated regulation of two human import motors by a single Magmas protein is poorly understood. Here, we report two additional Magmas variants (Magmas-1 and Magmas-2) constitutively expressed in the mammalian system. Both the Magmas variants are functional orthologs of Pam16 with an evolutionarily conserved J-like domain critical for cell survival. Moreover, the Magmas variants are peripherally associated with the inner membrane as part of the human import motor for translocation. Our results demonstrate that Magmas-1 is predominantly recruited to translocase B, whereas Magmas-2 is majorly associated with translocase A. Strikingly, both the variants exhibit differential J-protein inhibitory activity in modulating import motor, thereby regulating overall translocase function. Based on our findings, we hypothesize that additional Magmas variants are of evolutionary significance in humans to maximize protein import in familial-linked pathological conditions.

Conserved regions of budding yeast Tim22 have a role in structural organization of the carrier translocase.

Mitochondrial biogenesis requires efficient sorting of various proteins into different mitochondrial sub-compartments, mediated by dedicated protein machinery present in the outer and inner membrane. Among them, the TIM22 complex enables the integration of complex membrane proteins with internal targeting signals into the inner membrane. Although the Tim22 protein forms the core of the complex, the dynamic recruitment of subunits to the channel is still enigmatic. In this study, we highlight that the intermembrane space (IMS) and transmembrane 4 (TM4) regions of Tim22 are critically required for interactions with the membrane-embedded subunits, including Tim54, Tim18, and Sdh3, and thereby maintain the functional architecture of the TIM22 translocase. Furthermore, we find that the TM1 and TM2 regions of Tim22 are important for association with Tim18, whereas TM3 is exclusively required for the interaction with Sdh3. Moreover, impairment of TIM22 complex assembly influences its translocase activity, the mitochondrial network, and the viability of cells lacking mitochondrial DNA. Overall, our findings provide compelling evidence highlighting the significance of conserved regions of Tim22 that are important for the maintenance of the TIM22 complex and mitochondrial integrity.

Double DJ-1 domain containing Arabidopsis DJ-1D is a robust macromolecule deglycase.

Plants, being sessile, are prone to genotoxin-induced macromolecule damage. Among the inevitable damaging agents are reactive carbonyls that induce glycation of DNA, RNA and proteins to result in the build-up of advanced glycated end-products. However, it is unclear how plants repair glycated macromolecules. DJ-1/PARK7 members are a highly conserved family of moonlighting proteins having double domains in higher plants and single domains in other phyla. Here we show that Arabidopsis DJ-1D offers robust tolerance to endogenous and exogenous stresses through its ability to repair glycated DNA, RNA and proteins. DJ-1D also reduced the formation of reactive carbonyls through its efficient methylglyoxalase activity. Strikingly, full-length double domain-containing DJ-1D suppressed the formation of advanced glycated end-products in yeast and plants. DJ-1D also efficiently repaired glycated nucleic acids and nucleotides in vitro and mitochondrial DNA in vivo under stress, indicating the existence of a new DNA repair pathway in plants. We propose that multi-stress responding plant DJ-1 members, often present in multiple copies among plants, probably contributed to the adaptation to a variety of endogenous and exogenous stresses.

Marf-mediated mitochondrial fusion is imperative for the development and functioning of indirect flight muscles (IFMs) in drosophila.

Dynamic changes in mitochondrial shape and size are vital for mitochondrial health and for tissue development and function. Adult Drosophila indirect flight muscles contain densely packed mitochondria. We show here that mitochondrial fusion is critical during early muscle development (in pupa) and that silencing of the outer mitochondrial membrane fusion gene, Marf, in muscles results in smaller mitochondria that are functionally defective. This leads to abnormal muscle development resulting in muscle dysfunction in adult flies. However, post-developmental silencing of Marf has no obvious effects on mitochondrial and muscle phenotype in adult flies, indicating the importance of mitochondrial fusion during early muscle development.

Evolving paradigms on the interplay of mitochondrial Hsp70 chaperone system in cell survival and senescence.

The role of mitochondria within a cell has grown beyond being the prime source of cellular energy to one of the major signaling platforms. Recent evidence provides several insights into the crucial roles of mitochondrial chaperones in regulating the organellar response to external triggers. The mitochondrial Hsp70 (mtHsp70/Mortalin/Grp75) chaperone system plays a critical role in the maintenance of proteostasis balance in the organelle. Defects in mtHsp70 network result in attenuated protein transport and misfolding of polypeptides leading to mitochondrial dysfunction. The functions of Hsp70 are primarily governed by J-protein cochaperones. Although human mitochondria possess a single Hsp70, its multifunctionality is characterized by the presence of multiple specific J-proteins. Several studies have shown a potential association of Hsp70 and J-proteins with diverse pathological states that are not limited to their canonical role as chaperones. The role of mitochondrial Hsp70 and its co-chaperones in disease pathogenesis has not been critically reviewed in recent years. We evaluated some of the cellular interfaces where Hsp70 machinery associated with pathophysiological conditions, particularly in context of tumorigenesis and neurodegeneration. The mitochondrial Hsp70 machinery shows a variable localization and integrates multiple components of the cellular processes with varied phenotypic consequences. Although Hsp70 and J-proteins function synergistically in proteins folding, their precise involvement in pathological conditions is mainly idiosyncratic. This machinery is associated with a heterogeneous set of molecules during the progression of a disorder. However, the precise binding to the substrate for a specific physiological response under a disease subtype is still an undocumented area of analysis.

Iron-Sulfur Protein Assembly in Human Cells.

Iron-sulfur (Fe-S) clusters serve as a fundamental inorganic constituent of living cells ranging from bacteria to human. The importance of Fe-S clusters is underscored by their requirement as a co-factor for the functioning of different enzymes and proteins. The biogenesis of Fe-S cluster is a highly coordinated process which requires specialized cellular machinery. Presently, understanding of Fe-S cluster biogenesis in human draws meticulous attention since defects in the biogenesis process result in development of multiple diseases with unresolved solutions. Mitochondrion is the major cellular compartment of Fe-S cluster biogenesis, although cytosolic biogenesis machinery has been reported in eukaryotes, including in human. The core biogenesis pathway comprises two steps. The process initiates with the assembly of Fe-S cluster on a platform scaffold protein in the presence of iron and sulfur donor proteins. Subsequent process is the transfer and maturation of the cluster to a bonafide target protein. Human Fe-S cluster biogenesis machinery comprises the mitochondrial iron-sulfur cluster (ISC) assembly and export system along with the cytosolic Fe-S cluster assembly (CIA) machinery. Impairment in the Fe-S cluster machinery components results in cellular dysfunction leading to various mitochondrial pathophysiological consequences. The current review highlights recent developments and understanding in the domain of Fe-S cluster assembly biology in higher eukaryotes, particularly in human cells.

Regulation of mitochondrial protein import by the nucleotide exchange factors GrpEL1 and GrpEL2 in human cells.

Mitochondria are organelles indispensable for maintenance of cellular energy homeostasis. Most mitochondrial proteins are nuclearly encoded and are imported into the matrix compartment where they are properly folded. This process is facilitated by the mitochondrial heat shock protein 70 (mtHsp70), a chaperone contributing to mitochondrial protein quality control. The affinity of mtHsp70 for its protein clients and its chaperone function are regulated by binding of ATP/ADP to mtHsp70's nucleotide-binding domain. Nucleotide exchange factors (NEFs) play a crucial role in exchanging ADP for ATP at mtHsp70's nucleotide-binding domain, thereby modulating mtHsp70's chaperone activity. A single NEF, Mge1, regulates mtHsp70's chaperone activity in lower eukaryotes, but the mammalian orthologs are unknown. Here, we report that two putative NEF orthologs, GrpE-like 1 (GrpEL1) and GrpEL2, modulate mtHsp70's function in human cells. We found that both GrpEL1 and GrpEL2 associate with mtHsp70 as a hetero-oligomeric subcomplex and regulate mtHsp70 function. The formation of this subcomplex was critical for conferring stability to the NEFs, helped fine-tune mitochondrial protein quality control, and regulated crucial mtHsp70 functions, such as import of preproteins and biogenesis of Fe-S clusters. Our results also suggested that GrpEL2 has evolved as a possible stress resistance protein in higher vertebrates to maintain chaperone activity under stress conditions. In conclusion, our findings support the idea that GrpEL1 has a role as a stress modulator in mammalian cells and highlight that multiple NEFs are involved in controlling protein quality in mammalian mitochondria.

Functional Diversity of Human Mitochondrial J-proteins Is Independent of Their Association with the Inner Membrane Presequence Translocase.

Mitochondrial J-proteins play a critical role in governing Hsp70 activity and, hence, are essential for organellar protein translocation and folding. In contrast to yeast, which has a single J-protein Pam18, humans involve two J-proteins, DnaJC15 and DnaJC19, associated with contrasting cellular phenotype, to transport proteins into the mitochondria. Mutation in DnaJC19 results in dilated cardiomyopathy and ataxia syndrome, whereas expression of DnaJC15 regulates the response of cancer cells to chemotherapy. In the present study we have comparatively assessed the biochemical properties of the J-protein paralogs in relation to their association with the import channel. Both DnaJC15 and DnaJC19 formed two distinct subcomplexes with Magmas at the import channel. Knockdown analysis suggested an essential role for Magmas and DnaJC19 in organellar protein translocation and mitochondria biogenesis, whereas DnaJC15 had dispensable supportive function. The J-proteins were found to have equal affinity for Magmas and could stimulate mitochondrial Hsp70 ATPase activity by equivalent levels. Interestingly, we observed that DnaJC15 exhibits bifunctional properties. At the translocation channel, it involves conserved interactions and mechanism to translocate the precursors into mitochondria. In addition to protein transport, DnaJC15 also showed a dual role in yeast where its expression elicited enhanced sensitivity of cells to cisplatin that required the presence of a functional J-domain. The amount of DnaJC15 expressed in the cell was directly proportional to the sensitivity of cells. Our analysis indicates that the differential cellular phenotype displayed by human mitochondrial J-proteins is independent of their activity and association with Magmas at the translocation channel.

Methylglyoxal detoxification by a DJ-1 family protein provides dual abiotic and biotic stress tolerance in transgenic plants.

Methylglyoxal (MG) is a key signaling molecule resulting from glycolysis and other metabolic pathways. During abiotic stress, MG levels accumulate to toxic levels in affected cells. However, MG is routinely detoxified through the action of DJ1/PARK7/Hsp31 proteins that are highly conserved across kingdoms and mutations in such genes are associated with neurodegenerative diseases. Here, we report for the first time that, similar to abiotic stresses, MG levels increase during biotic stresses in plants, likely contributing to enhanced susceptibility to a wide range of stresses. We show that overexpression of yeast Heat shock protein 31 (Hsp31), a DJ-1 homolog with robust MG detoxifying capabilities, confers dual biotic and abiotic stress tolerance in model plant Nicotiana tabacum. Strikingly, overexpression of Hsp31 in tobacco imparts robust stress tolerance against diverse biotic stress inducers such as viruses, bacteria and fungi, in addition to tolerance against a range of abiotic stress inducers. During stress, Hsp31 was targeted to mitochondria and induced expression of key stress-related genes. These results indicate that Hsp31 is a novel attractive tool to engineer plants against both biotic and abiotic stresses.

A Redox Modulatory Mn3 O4 Nanozyme with Multi-Enzyme Activity Provides Efficient Cytoprotection to Human Cells in a Parkinson's Disease Model.

Nanomaterials with enzyme-like activities (nanozymes) attracts significant interest due to their therapeutic potential for the treatment of various diseases. Herein, we report that a Mn3 O4 nanozyme functionally mimics three major antioxidant enzymes, that is, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) and the multienzyme activity is size as well as morphology-dependent. The redox modulatory effect of Mn3 O4 plays a crucial role in protecting the cells from MPP+ induced cytotoxicity in a Parkinson disease (PD)-like cellular model, indicating that manganese-based nanomaterials having multi-enzyme activity can robustly rescue the cells from oxidative damage and thereby possess therapeutic potential to prevent ROS-mediated neurological disorders.

Mapping Key Residues of ISD11 Critical for NFS1-ISD11 Subcomplex Stability: IMPLICATIONS IN THE DEVELOPMENT OF MITOCHONDRIAL DISORDER, COXPD19.

Biogenesis of the iron-sulfur (Fe-S) cluster is an indispensable process in living cells. In mammalian mitochondria, the initial step of the Fe-S cluster assembly process is assisted by the NFS1-ISD11 complex, which delivers sulfur to scaffold protein ISCU during Fe-S cluster synthesis. Although ISD11 is an essential protein, its cellular role in Fe-S cluster biogenesis is still not defined. Our study maps the important ISD11 amino acid residues belonging to putative helix 1 (Phe-40), helix 3 (Leu-63, Arg-68, Gln-69, Ile-72, Tyr-76), and C-terminal segment (Leu-81, Glu-84) are critical for in vivo Fe-S cluster biogenesis. Importantly, mutation of these conserved ISD11 residues into alanine leads to its compromised interaction with NFS1, resulting in reduced stability and enhanced aggregation of NFS1 in the mitochondria. Due to altered interaction with ISD11 mutants, the levels of NFS1 and Isu1 were significantly depleted, which affects Fe-S cluster biosynthesis, leading to reduced electron transport chain complex (ETC) activity and mitochondrial respiration. In humans, a clinically relevant ISD11 mutation (R68L) has been associated in the development of a mitochondrial genetic disorder, COXPD19. Our findings highlight that the ISD11 R68A/R68L mutation display reduced affinity to form a stable subcomplex with NFS1, and thereby fails to prevent NFS1 aggregation resulting in impairment of the Fe-S cluster biogenesis. The prime affected machinery is the ETC complex, which showed compromised redox properties, causing diminished mitochondrial respiration. Furthermore, the R68L ISD11 mutant displayed accumulation of mitochondrial iron and reactive oxygen species, leading to mitochondrial dysfunction, which correlates with the phenotype observed in COXPD19 patients.

Robust glyoxalase activity of Hsp31, a ThiJ/DJ-1/PfpI family member protein, is critical for oxidative stress resistance in Saccharomyces cerevisiae.

Methylglyoxal (MG) is a reactive metabolic intermediate generated during various cellular biochemical reactions, including glycolysis. The accumulation of MG indiscriminately modifies proteins, including important cellular antioxidant machinery, leading to severe oxidative stress, which is implicated in multiple neurodegenerative disorders, aging, and cardiac disorders. Although cells possess efficient glyoxalase systems for detoxification, their functions are largely dependent on the glutathione cofactor, the availability of which is self-limiting under oxidative stress. Thus, higher organisms require alternate modes of reducing the MG-mediated toxicity and maintaining redox balance. In this report, we demonstrate that Hsp31 protein, a member of the ThiJ/DJ-1/PfpI family in Saccharomyces cerevisiae, plays an indispensable role in regulating redox homeostasis. Our results show that Hsp31 possesses robust glutathione-independent methylglyoxalase activity and suppresses MG-mediated toxicity and ROS levels as compared with another paralog, Hsp34. On the other hand, glyoxalase-defective mutants of Hsp31 were found highly compromised in regulating the ROS levels. Additionally, Hsp31 maintains cellular glutathione and NADPH levels, thus conferring protection against oxidative stress, and Hsp31 relocalizes to mitochondria to provide cytoprotection to the organelle under oxidative stress conditions. Importantly, human DJ-1, which is implicated in the familial form of Parkinson disease, complements the function of Hsp31 by suppressing methylglyoxal and oxidative stress, thus signifying the importance of these proteins in the maintenance of ROS homeostasis across phylogeny.

The presence of multiple cellular defects associated with a novel G50E iron-sulfur cluster scaffold protein (ISCU) mutation leads to development of mitochondrial myopathy.

Iron-sulfur (Fe-S) clusters are versatile cofactors involved in regulating multiple physiological activities, including energy generation through cellular respiration. Initially, the Fe-S clusters are assembled on a conserved scaffold protein, iron-sulfur cluster scaffold protein (ISCU), in coordination with iron and sulfur donor proteins in human mitochondria. Loss of ISCU function leads to myopathy, characterized by muscle wasting and cardiac hypertrophy. In addition to the homozygous ISCU mutation (g.7044G→C), compound heterozygous patients with severe myopathy have been identified to carry the c.149G→A missense mutation converting the glycine 50 residue to glutamate. However, the physiological defects and molecular mechanism associated with G50E mutation have not been elucidated. In this report, we uncover mechanistic insights concerning how the G50E ISCU mutation in humans leads to the development of severe ISCU myopathy, using a human cell line and yeast as the model systems. The biochemical results highlight that the G50E mutation results in compromised interaction with the sulfur donor NFS1 and the J-protein HSCB, thus impairing the rate of Fe-S cluster synthesis. As a result, electron transport chain complexes show significant reduction in their redox properties, leading to loss of cellular respiration. Furthermore, the G50E mutant mitochondria display enhancement in iron level and reactive oxygen species, thereby causing oxidative stress leading to impairment in the mitochondrial functions. Thus, our findings provide compelling evidence that the respiration defect due to impaired biogenesis of Fe-S clusters in myopathy patients leads to manifestation of complex clinical symptoms.

A Pd8 Tetrafacial Molecular Barrel as Carrier for Water Insoluble Fluorophore.

A new carbazole-based tetraimidazole ligand 1,3,6,8-tetra(1H-imidazol-1-yl)-9-methyl-9H-carbazole (L) has been synthesized. The unsymmetrical nature of L as well as the rotational freedom of imidazole donor moieties around C-N bond make it a special building unit, which upon treatment with cis-(tmeda)Pd(NO3)2 produced an unprecedented single linkage-isomeric Pd8 tetrafacial molecular nanobarrel (PSMBR-1) [tmeda = N,N,N',N'-tetramethylethane-1,2-diamine]. Unlike closed architectures, open barrel architecture of water-soluble PSMBR-1 makes it an ideal host for some water insoluble polyaromatic hydrocarbons in aqueous medium; one such inclusion complex coronene⊂PSMBR-1 was characterized by X-ray diffraction study. Moreover, the potential application of PSMBR-1 as carrier in aqueous medium for the transportation of water insoluble fluorophore (perylene) for live cell imaging is explored.

Highly Efficient Glutathione Peroxidase and Peroxiredoxin Mimetics Protect Mammalian Cells against Oxidative Damage.

Novel isoselenazoles with high glutathione peroxidase (GPx) and peroxiredoxin (Prx) activities provide remarkable cytoprotection to human cells, mainly by exhibiting antioxidant activities in the presence of cellular thiols. The cytotoxicity of the isoselenazoles is found to be significantly lower than that of ebselen, which is being clinically evaluated by several groups for the treatment of reperfusion injuries and stroke, hearing loss, and bipolar disorder. The compounds reported in this paper have the potential to be used as therapeutic agents for disorders mediated by reactive oxygen species.

Enhanced J-protein interaction and compromised protein stability of mtHsp70 variants lead to mitochondrial dysfunction in Parkinson's disease.

Parkinson's disease (PD) is the second most prevalent progressive neurological disorder commonly associated with impaired mitochondrial function in dopaminergic neurons. Although familial PD is multifactorial in nature, a recent genetic screen involving PD patients identified two mitochondrial Hsp70 variants (P509S and R126W) that are suggested in PD pathogenesis. However, molecular mechanisms underlying how mtHsp70 PD variants are centrally involved in PD progression is totally elusive. In this article, we provide mechanistic insights into the mitochondrial dysfunction associated with human mtHsp70 PD variants. Biochemically, the R126W variant showed severely compromised protein stability and was found highly susceptible to aggregation at physiological conditions. Strikingly, on the other hand, the P509S variant exhibits significantly enhanced interaction with J-protein cochaperones involved in folding and import machinery, thus altering the overall regulation of chaperone-mediated folding cycle and protein homeostasis. To assess the impact of mtHsp70 PD mutations at the cellular level, we developed yeast as a model system by making analogous mutations in Ssc1 ortholog. Interestingly, PD mutations in yeast (R103W and P486S) exhibit multiple in vivo phenotypes, which are associated with 'mitochondrial dysfunction', including compromised growth, impairment in protein translocation, reduced functional mitochondrial mass, mitochondrial DNA loss, respiratory incompetency and increased susceptibility to oxidative stress. In addition to that, R103W protein is prone to aggregate in vivo due to reduced stability, whereas P486S showed enhanced interaction with J-proteins, thus remarkably recapitulating the cellular defects that are observed in human PD variants. Taken together, our findings provide evidence in favor of direct involvement of mtHsp70 as a susceptibility factor in PD.

Structure and mechanistic insights into novel iron-mediated moonlighting functions of human J-protein cochaperone, Dph4.

J-proteins are obligate cochaperones of Hsp70s and stimulate their ATPase activity via the J-domain. Although the functions of J-proteins have been well understood in the context of Hsp70s, their additional co-evolved "physiological functions" are still elusive. We report here the solution structure and mechanism of novel iron-mediated functional roles of human Dph4, a type III J-protein playing a vital role in diphthamide biosynthesis and normal development. The NMR structure of Dph4 reveals two domains: a conserved J-domain and a CSL-domain connected via a flexible linker-helix. The linker-helix modulates the conformational flexibility between the two domains, regulating thereby the protein function. Dph4 exhibits a unique ability to bind iron in tetrahedral coordination geometry through cysteines of its CSL-domain. The oxidized Fe-Dph4 shows characteristic UV-visible and electron paramagnetic resonance spectral properties similar to rubredoxins. Iron-bound Dph4 (Fe-Dph4) also undergoes oligomerization, thus potentially functioning as a transient "iron storage protein," thereby regulating the intracellular iron homeostasis. Remarkably, Fe-Dph4 exhibits vital redox and electron carrier activity, which is critical for important metabolic reactions, including diphthamide biosynthesis. Further, we observed that Fe-Dph4 is conformationally better poised to perform Hsp70-dependent functions, thus underlining the significance of iron binding in Dph4. Yeast Jjj3, a functional ortholog of human Dph4 also shows a similar iron-binding property, indicating the conserved nature of iron sequestration across species. Taken together, our findings provide invaluable evidence in favor of additional co-evolved specialized functions of J-proteins, previously not well appreciated.

HSPIR: a manually annotated heat shock protein information resource.

SUMMARY: Heat shock protein information resource (HSPIR) is a concerted database of six major heat shock proteins (HSPs), namely, Hsp70, Hsp40, Hsp60, Hsp90, Hsp100 and small HSP. The HSPs are essential for the survival of all living organisms, as they protect the conformations of proteins on exposure to various stress conditions. They are a highly conserved group of proteins involved in diverse physiological functions, including de novo folding, disaggregation and protein trafficking. Moreover, their critical role in the control of disease progression made them a prime target of research. Presently, limited information is available on HSPs in reference to their identification and structural classification across genera. To that extent, HSPIR provides manually curated information on sequence, structure, classification, ontology, domain organization, localization and possible biological functions extracted from UniProt, GenBank, Protein Data Bank and the literature. The database offers interactive search with incorporated tools, which enhances the analysis. HSPIR is a reliable resource for researchers exploring structure, function and evolution of HSPs. AVAILABILITY: http://pdslab.biochem.iisc.ernet.in/hspir/

Rapid characterization of hydrogen exchange in proteins.

Kinetics and thermodynamics of amide hydrogen exchange in proteins can be investigated with two-dimensional (13)CO-(15)N NMR correlation experiments. The spectra are acquired with high resolution and sensitivity. A single type of experiment on one sample serves to characterize hydrogen-deuterium fractionation factors and hydrogen-exchange rates that span three orders of magnitude.