Functional and cellular analysis of autoimmune regulator (AIRE) protein

Autoimmune diseases are a major health problem. Usually autoimmune disorders are multifactorial and their pathogenesis involves a combination of predisposing variations in the genome and other factors such as environmental triggers. APECED (autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy) is a rare, recessively inherited, autoimmune disease caused by mutations in a single gene. Patients with APECED suffer from several organ-specific autoimmune disorders, often affecting the endocrine glands. The defective gene, AIRE, codes for a transcriptional regulator. The AIRE (autoimmune regulator) protein controls the expression of hundreds of genes, representing a substantial subset of tissue-specific antigens which are presented to developing T cells in the thymus and has proven to be a key molecule in the establishment of immunological tolerance. However, the molecular mechanisms by which AIRE mediates its functions are still largely obscure. The aim of this thesis has been to elucidate the functions of AIRE by studying the molecular interactions it is involved in by utilizing different cultured cell models. A potential molecular mechanism for exceptional, dominant, inheritance of APECED in one family, carrying a glycine 228 to tryptophan (G228W) mutation, was described in this thesis. It was shown that the AIRE polypeptide with G228W mutation has a dominant negative effect by binding the wild type AIRE and inhibiting its transactivation capacity in vitro. The data also emphasizes the importance of homomultimerization of AIRE in vivo. Furthermore, two novel protein families interacting with AIRE were identified. The importin alpha molecules regulate the nuclear import of AIRE by binding to the nuclear localization signal of AIRE, delineated as a classical monopartite signal sequence. The interaction of AIRE with PIAS E3 SUMO ligases, indicates a link to the sumoylation pathway, which plays an important role in the regulation of nuclear architecture. It was shown that AIRE is not a target for SUMO modification but enhances the localization of SUMO1 and PIAS1 proteins to nuclear bodies. Additional support for the suggestion that AIRE would preferably up-regulate genes with tissue-specific expression pattern and down-regulate housekeeping genes was obtained from transactivation studies performed with two models: human insulin and cystatin B promoters. Furthermore, AIRE and PIAS activate the insulin promoter concurrently in a transactivation assay, indicating that their interaction is biologically relevant. Identification of novel interaction partners for AIRE provides us information about the molecular pathways involved in the establishment of immunological tolerance and deepens our understanding of the role played by AIRE not only in APECED but possibly also in several other autoimmune diseases.

Stress and developmental responses in Arabidopsis thaliana: regulation through the transcription factor-interacting protein RCD1

Plants constantly face adverse environmental conditions, such as drought or extreme temperatures that threaten their survival. They demonstrate astonishing metabolic flexibility in overcoming these challenges and one of the key responses to stresses is changes in gene expression leading to alterations in cellular functions. This is brought about by an intricate network of transcription factors and associated regulatory proteins. Protein-protein interactions and post-translational modifications are important steps in this control system along with carefully regulated degradation of signaling proteins. This work concentrates on the RADICAL-INDUCED CELL DEATH1 (RCD1) protein which is an important regulator of abiotic stress-related and developmental responses in Arabidopsis thaliana. Plants lacking this protein function display pleiotropic phenotypes including sensitivity to apoplastic reactive oxygen species (ROS) and salt, ultraviolet B (UV-B) and paraquat tolerance, early flowering and senescence. Additionally, the mutant plants overproduce nitric oxide, have alterations in their responses to several plant hormones and perturbations in gene expression profiles. The RCD1 gene is transcriptionally unresponsive to environmental signals and the regulation of the protein function is likely to happen post-translationally. RCD1 belongs to a small protein family and, together with its closest homolog SRO1, contains three distinguishable domains: In the N-terminus, there is a WWE domain followed by a poly(ADP-ribose) polymerase-like domain which, despite sequence conservation, does not seem to be functional. The C-terminus of RCD1 contains a novel domain called RST. It is present in RCD1-like proteins throughout the plant kingdom and is able to mediate physical interactions with multiple transcription factors. In conclusion, RCD1 is a key point of signal integration that links ROS-mediated cues to transcriptional regulation by yet unidentified means, which are likely to include post-translational mechanisms. The identification of RCD1-interacting transcription factors, most of whose functions are still unknown, opens new avenues for studies on plant stress as well as developmental responses.

Comparative and functional genome analysis of fungi for development of the protein production host Trichoderma reesei

Filamentous fungi of the subphylum Pezizomycotina are well known as protein and secondary metabolite producers. Various industries take advantage of these capabilities. However, the molecular biology of yeasts, i.e. Saccharomycotina and especially that of Saccharomyces cerevisiae, the baker's yeast, is much better known. In an effort to explain fungal phenotypes through their genotypes we have compared protein coding gene contents of Pezizomycotina and Saccharomycotina. Only biomass degradation and secondary metabolism related protein families seem to have expanded recently in Pezizomycotina. Of the protein families clearly diverged between Pezizomycotina and Saccharomycotina, those related to mitochondrial functions emerge as the most prominent. However, the primary metabolism as described in S. cerevisiae is largely conserved in all fungi. Apart from the known secondary metabolism, Pezizomycotina have pathways that could link secondary metabolism to primary metabolism and a wealth of undescribed enzymes. Previous studies of individual Pezizomycotina genomes have shown that regardless of the difference in production efficiency and diversity of secreted proteins, the content of the known secretion machinery genes in Pezizomycotina and Saccharomycotina appears very similar. Genome wide analysis of gene products is therefore needed to better understand the efficient secretion of Pezizomycotina. We have developed methods applicable to transcriptome analysis of non-sequenced organisms. TRAC (Transcriptional profiling with the aid of affinity capture) has been previously developed at VTT for fast, focused transcription analysis. We introduce a version of TRAC that allows more powerful signal amplification and multiplexing. We also present computational optimisations of transcriptome analysis of non-sequenced organism and TRAC analysis in general. Trichoderma reesei is one of the most commonly used Pezizomycotina in the protein production industry. In order to understand its secretion system better and find clues for improvement of its industrial performance, we have analysed its transcriptomic response to protein secretion stress conditions. In comparison to S. cerevisiae, the response of T. reesei appears different, but still impacts on the same cellular functions. We also discovered in T. reesei interesting similarities to mammalian protein secretion stress response. Together these findings highlight targets for more detailed studies.

Hyperexpression of chicken riboflavin carrier protein: Antibodies to the recombinant protein curtail pregnancy in rodents

Chicken riboflavin carrier protein (RCP) is a phosphoglycoprotein present in the egg white and yolk of egg-laying animals and in the sera of laying hens and of estrogenized chicks. The RCP cDNA, encoding a protein of predictedMr27,000, has been cloned into a T7 polymerase-driven vector, and high-level expression was observed on induction with IPTG inEscherichia coli.The protein was largely localized in inclusion bodies when expressed at 37°C but was present in the cytosolic fraction when induced at 22°C. At 37°C, two major bands were detected in whole-cell lysates of the strain expressing the protein. N-terminal sequence analysis indicated that the two proteins represented translated products with and without the pelB leader sequence encoded in the pET20b vector, but both included an additional 10 amino acids generated during cloning procedures. The inclusion body obtained at 37°C, on extraction with detergent, led to preferential solubilization of the protein without the pelB signal sequence. The solubilized recombinant RCP was recognized by polyclonal antisera to native RCP but radioimmunoassay revealed quantitative differences in the epitopes exhibited by the recombinant protein. Thus, sequence-specific monoclonal antibodies to chicken RCP also cross-reacted with the recombinant protein with almost equal efficiency, but antibodies which recognize conformation-dependent epitopes showed relatively reduced cross-reactivity with the recombinant protein. Polyclonal antibodies to recombinant RCP were able to recognize both the native and the denatured RCP. Administration of recombinant RCP antisera to pregnant mice led to embryonic resorption leading to early pregnancy termination. These findings reveal that the recombinant protein will be useful for investigations related to the mechanism of pregnancy termination on immunoneutralization of RCP in mammals, as well as in unraveling folding properties of RCP in terms of its ligand binding and antigenetic determinants exposed at its surface.

Functional diversity of human protein kinase splice variants marks significant expansion of human kinome

Background: Protein kinases are involved in diverse spectrum of cellular processes. Availability of draft version of the human genomic data in the year 2001 enabled recognition of repertoire of protein kinases. However, over the years the human genomic data is being refined and the current release of human genomic data has helped us to recognize a larger repertoire of over 900 human protein kinases represented mainly by splice variants. Results: Many of these identified protein kinases are alternatively spliced products. Interestingly, some of the human kinase splice variants appear to be significantly diverged in terms of their functional properties as represented by incorporation or absence of one or more domains. Many sets of protein kinase splice variants have substantially different domain organization and in a few sets of splice variants kinase domains belong to different subfamilies of kinases suggesting potential participation in different signal transduction pathways. Conclusions: Addition or deletion of a domain between splice variants of multi-domain kinases appears to be a means of generating differences in the functional features of otherwise similar kinases. It is intriguing that marked sequence diversity within the catalytic regions of some of the splice variant kinases result in kinases belonging to different subfamilies. These human kinase splice variants with different functions might contribute to diversity of eukaryotic cellular signaling.

How Effective Is The Data On Co-Occurrence Of Domains In Multi-Domain Proteins In Prediction Of Protein-Protein Interactions?

We explore the fuse of information on co-occurrence of domains in multi-domain proteins in predicting protein-protein interactions. The basic premise of our work is the assumption that domains co-occurring in a polypeptide chain undergo either structural or functional interactions among themselves. In this study we use a template dataset of domains in multidomain proteins and predict protein-protein interactions in a target organism. We note that maximum number of correct predictions of interacting protein domain families (158) is made in S. cerevisiae when the dataset of closely related organisms is used as the template followed by the more diverse dataset of bacterial proteins (48) and a dataset of randomly chosen proteins (23). We conclude that use of multi-domain information from organisms closely-related to the target can aid prediction of interacting protein families.

Classification of Protein Kinases on the Basis of Both Kinase and Non-Kinase Regions

Background: Protein phosphorylation is a generic way to regulate signal transduction pathways in all kingdoms of life. In many organisms, it is achieved by the large family of Ser/Thr/Tyr protein kinases which are traditionally classified into groups and subfamilies on the basis of the amino acid sequence of their catalytic domains. Many protein kinases are multidomain in nature but the diversity of the accessory domains and their organization are usually not taken into account while classifying kinases into groups or subfamilies. Methodology: Here, we present an approach which considers amino acid sequences of complete gene products, in order to suggest refinements in sets of pre-classified sequences. The strategy is based on alignment-free similarity scores and iterative Area Under the Curve (AUC) computation. Similarity scores are computed by detecting common patterns between two sequences and scoring them using a substitution matrix, with a consistent normalization scheme. This allows us to handle full-length sequences, and implicitly takes into account domain diversity and domain shuffling. We quantitatively validate our approach on a subset of 212 human protein kinases. We then employ it on the complete repertoire of human protein kinases and suggest few qualitative refinements in the subfamily assignment stored in the KinG database, which is based on catalytic domains only. Based on our new measure, we delineate 37 cases of potential hybrid kinases: sequences for which classical classification based entirely on catalytic domains is inconsistent with the full-length similarity scores computed here, which implicitly consider multi-domain nature and regions outside the catalytic kinase domain. We also provide some examples of hybrid kinases of the protozoan parasite Entamoeba histolytica. Conclusions: The implicit consideration of multi-domain architectures is a valuable inclusion to complement other classification schemes. The proposed algorithm may also be employed to classify other families of enzymes with multidomain architecture.

Protein Kinase C and Anaplastic Lymphoma Kinase Targeted Compounds

The protein kinases (PKs) belong to the largest single family of enzymes, phosphotransferases, which catalyze the phosphorylation of other enzymes and proteins and function primarily in signal transduction. Consequently, PKs regulate cell mechanisms such as growth, differentiation, and proliferation. Dysfunction of these cellular mechanisms may lead to cancer, a major predicament in health care. Even though there is a range of clinically available cancer-fighting drugs, increasing number of cancer cases and setbacks such as drug resistance, constantly keep cancer research active. At the commencement of this study an isophthalic acid derivative had been suggested to bind to the regulatory domain of protein kinase C (PKC). In order to investigate the biological effects and structure-activity relationships (SARs) of this new chemical entity, a library of compounds was synthesized. The best compounds induced apoptosis in human leukemia HL-60 cells and were not cytotoxic in Swiss 3T3 fibroblasts. In addition, the best apoptosis inducers were neither cytotoxic nor mutagenic. Furthermore, results from binding affinity assays of PKC isoforms revealed the pharmacophores of these isophthalic acid derivatives. The best inhibition constants of the tested compounds were measured to 210 nM for PKCα and to 530 nM for PKCδ. Among natural compounds targeting the regulatory domain of PKC, the target of bistramide A has been a matter of debate. It was initially found to activate PKCδ; however, actin was recently reported as the main target. In order to clarify and to further study the biological effects of bistramide A, the total syntheses of the natural compound and two isomers were performed. Biological assays of the compounds revealed accumulation of 4n polyploid cells as the primary mode of action and the compounds showed similar overall antiproliferative activities. However, each compound showed a distinct distribution of antimitotic effect presumably via actin binding, proapoptotic effect presumably via PKCδ, and pro-differentiation effect as evidenced by CD11b expression. Furthermore, it was shown that the antimitotic and proapoptotic effects of bistramide A were not secondary effects of actin binding but independent effects. The third aim in this study was to synthesize a library of a new class of urea-based type II inhibitors targeted at the kinase domain of anaplastic lymphoma kinase (ALK). The best compounds in this library showed IC50 values as low as 390 nM for ALK while the initial low cellular activities were successfully increased even by more than 70 times for NPM-ALK- positive BaF3 cells. More importantly, selective antiproliferative activity on ALK-positive cell lines was achieved; while the best compound affected the BaF3 and SU-DHL-1 cells with IC50 values of 0.5 and 0.8 μM, respectively, they were less toxic to the NPM-ALK-negative human leukemic cells U937 (IC50 = 3.2 μM) and BaF3 parental cells (IC50 = 5.4 μM). Furthermore, SAR studies of the synthesized compounds revealed functional groups and positions of the scaffold, which enhanced the enzymatic and cellular activities.

PeptideMine - A webserver for the design of peptides for protein-peptide binding studies derived from protein-protein interactomes

Background: Signal transduction events often involve transient, yet specific, interactions between structurally conserved protein domains and polypeptide sequences in target proteins. The identification and validation of these associating domains is crucial to understand signal transduction pathways that modulate different cellular or developmental processes. Bioinformatics strategies to extract and integrate information from diverse sources have been shown to facilitate the experimental design to understand complex biological events. These methods, primarily based on information from high-throughput experiments, have also led to the identification of new connections thus providing hypothetical models for cellular events. Such models, in turn, provide a framework for directing experimental efforts for validating the predicted molecular rationale for complex cellular processes. In this context, it is envisaged that the rational design of peptides for protein-peptide binding studies could substantially facilitate the experimental strategies to evaluate a predicted interaction. This rational design procedure involves the integration of protein-protein interaction data, gene ontology, physico-chemical calculations, domain-domain interaction data and information on functional sites or critical residues. Results: Here we describe an integrated approach called ``PeptideMine'' for the identification of peptides based on specific functional patterns present in the sequence of an interacting protein. This approach based on sequence searches in the interacting sequence space has been developed into a webserver, which can be used for the identification and analysis of peptides, peptide homologues or functional patterns from the interacting sequence space of a protein. To further facilitate experimental validation, the PeptideMine webserver also provides a list of physico-chemical parameters corresponding to the peptide to determine the feasibility of using the peptide for in vitro biochemical or biophysical studies. Conclusions: The strategy described here involves the integration of data and tools to identify potential interacting partners for a protein and design criteria for peptides based on desired biochemical properties. Alongside the search for interacting protein sequences using three different search programs, the server also provides the biochemical characteristics of candidate peptides to prune peptide sequences based on features that are most suited for a given experiment. The PeptideMine server is available at the URL: http://caps.ncbs.res.in/peptidemine

Molecular studies on LIM-domain protein CRP1

Various intrinsic and external factors are constantly attacking the cells causing damage to DNA and to other cellular structures. Cells in turn have evolved with different kinds of mechanisms to protect against the attacks and to repair the damage. Ultraviolet radiation (UVR) is one of the major environmental genotoxic carcinogens that causes inflammation, mutations, immunosuppression, accelerated aging of the skin and skin cancers. Epidermis is the outermost layer of the skin consisting mostly of keratinocytes, whose primary function is to protect the skin against e.g. UV radiation. LIM domain proteins are a group of proteins involved in regulation of cell growth, damage signalling, cell fate determination and signal transduction. Despite their two zinc fingers, LIM domains do not bind to DNA, but rather mediate protein-protein interactions and function as modular protein binding interfaces. We initially identified CSRP1 as UVR-regulated transcript by using expression profiling. Here we have further studied the regulation and function of CRP1, a representative of cysteine rich protein- family consisting of two LIM domains. We find that CRP1 is increased by UVR in primary human keratinocytes and in normal human skin fibroblasts. Ectopic expression of CRP1 protected the cells against UVR and provided a survival advantage, whereas silencing of CRP1 rendered the cells more photosensitive. Actinic keratosis is a premalignant lesion of skin caused by excess exposure to sunlight and sunburn, which may lead to formation of squamous cell carcinoma. The expression of CRP1 was increased in basal keratinocytes of Actinic keratosis patient specimens suggesting that CRP1 may be increased by constant exposure to UVR and may provide survival advantage for the cells also in vivo. In squamous cell carcinoma, CRP1 was only expressed in the fibroblasts surrounding the tumour. Moreover, we found that ectopic expression of CRP1 suppresses cell proliferation. Transforming growth factor beta (TGFbeta) is a multifunctional cytokine that regulates several functions in cell including growth, apoptosis and differentiation, and plays important roles in pathological disorders like cancer and fibrosis. We found that TGFbeta-signalling pathway regulates CRP1 at protein, but not at transcriptional level. The increase was mediated both through Smad and non-Smad signalling pathways involving MAPK/p38. Furthermore, we found that TGFbeta-mediated increase in CRP1 was associated with myofibroblast differentiation, and that CRP1 was significantly more expressed in idiopathic pulmonary fibrosis as compared to normal lung specimens. Since cell contractility is a distinct feature of myofibroblasts, and CRP1 is associated with actin cytoskeleton, we studied the role of CRP1 in cell contractility. CRP1 was found to localize to stress fibres that mediate contractility and to mediate myofibroblast contraction. These studies identify CRP1 as a stress responsive and cytokine regulated cytoskeletal protein that participates in pathological processes involved in fibrotic diseases and cancer.

Post-transcriptional Repair of a Split Heat Shock Protein 90 Gene by mRNA trans-Splicing

Heat shock protein 90 participates in diverse biological processes ranging from protein folding, cell cycle, signal transduction and development to evolution in all eukaryotes. It is also critically involved in regulating growth of protozoa such as Dictyostelium discoideum, Leishmania donovani, Plasmodium falciparum, Trypanosoma cruzi, and Trypanosoma evansi. Selective inhibition of Hsp90 has also been explored as an intervention strategy against important human diseases such as cancer, malaria, or trypanosomiasis. Giardia lamblia, a simple protozoan parasite of humans and animals, is an important cause of diarrheal disease with significant morbidity and some mortality in tropical countries. Here we show that the G. lamblia cytosolic hsp90 ( glhsp90) is split in two similar sized fragments located 777 kb apart on the same scaffold. Intrigued by this unique arrangement, which appears to be specific for the Giardiinae, we have investigated the biosynthesis of GlHsp90. We used genome sequencing to confirm the split nature of the giardial hsp90. However, a specific antibody raised against the peptide detected a product with a mass of about 80 kDa, suggesting a post-transcriptional rescue of the genomic defect. We show evidence for the joining of the two independent Hsp90 transcripts in-trans to one long mature mRNA presumably by RNA splicing. The splicing junction carries hallmarks of classical cis-spliced introns, suggesting that the regular cis-splicing machinery may be sufficient for repair of the open reading frame. A complementary 26-nt sequence in the ``intron'' regions adjacent to the splice sites may assist in positioning the two pre-mRNAs for processing. This is the first example of post-transcriptional rescue of a split gene by trans-splicing.

Inhibition of nuclear protein import by a monoclonal antibody against a novel class of nuclear pore proteins

Nuclear import of proteins is mediated by the nuclear pore complexes in the nuclear envelope and requires the presence of a nuclear localization signal (NLS) on the karyophilic protein. In this paper, we describe studies with a monoclonal antibody, Mab E2, which recognizes a class of nuclear pore proteins of 60-76 kDa with a common phosphorylated epitope on rat nuclear envelopes. The Mab Ea-reactive proteins fractionated with the relatively insoluble pore complex-containing component of the envelope and gave a finely punctate pattern of nuclear staining in immunofluorescence assays. The antibody did not bind to any cytosolic proteins. Mab E2 inhibited the interaction of a simian virus 40 large T antigen NLS peptide with a specific 60-kDa NLS-binding protein from rat nuclear envelopes in photoaffinity labeling experiments. The antibody blocked the nuclear import of NLS-albumin conjugates in an in vitro nuclear transport assay with digitonin-permeabilized cells, but did not affect passive diffusion of a small nonnuclear protein, lysozyme, across the pore. Mab E2 may inhibit protein transport by directly interacting with the 60-kDa NLS-binding protein, thereby blocking signal-mediated nuclear import across the nuclear pore complex. (C) 1994 Academic Press, Inc.

The repertoire of protein kinases encoded in the draft version of the human genome: atypical variations and uncommon domain combinations

Background: Phosphorylation by protein kinases is central to cellular signal transduction. Abnormal functioning of kinases has been implicated in developmental disorders and malignancies. Their activity is regulated by second messengers and by the binding of associated domains, which are also influential in translocating the catalytic component to their substrate sites, in mediating interaction with other proteins and carrying out their biological roles. Results: Using sensitive profile-search methods and manual analysis, the human genome has been surveyed for protein kinases. A set of 448 sequences, which show significant similarity to protein kinases and contain the critical residues essential for kinase function, have been selected for an analysis of domain combinations after classifying the kinase domains into subfamilies. The unusual domain combinations in particular kinases suggest their involvement in ubiquitination pathways and alternative modes of regulation for mitogen-activated protein kinase kinases (MAPKKs) and cyclin-dependent kinase (CDK)-like kinases. Previously unexplored kinases have been implicated in osteoblast differentiation and embryonic development on the basis of homology with kinases of known functions from other organisms. Kinases potentially unique to vertebrates are involved in highly evolved processes such as apoptosis, protein translation and tyrosine kinase signaling. In addition to coevolution with the kinase domain, duplication and recruitment of non-catalytic domains is apparent in signaling domains such as the PH, DAG-PE, SH2 and SH3 domains. Conclusions: Expansion of the functional repertoire and possible existence of alternative modes of regulation of certain kinases is suggested by their uncommon domain combinations. Experimental verification of the predicted implications of these kinases could enhance our understanding of their biological roles.

The requirements for Ca2+, protein phosphorylation and concurrent protein synthesis for zeatin signaling of acidic chitinase transcript accumulation in Cucumis sativus L

We have previously reported that both Ca2+ and staurosporine-sensitive protein kinase(s) are involved in the cytokinin zeatin induction of cucumber chitinase activity and its protein content (Barwe et al. 2001). To further characterize signal transduction events involved in this cytokinin induction of chitinase gene expression, Northern hybridizations of total RNAs prepared from excised, dark-grown cucumber cotyledons treated with cytokinins and/or various agonists and antagonists of signal transduction components, were carried out using a cucumber acidic chitinase (CACHT) cDNA probe (Metraux et al. 1989). CACHT mRNA increased by approximately 5- to 6-fold in response to exogenous zeatin (Z), zeatin riboside (ZR), and benzyladenine (BA) treatment, but failed to accumulate in response to kinetin (K). Among the cytokinins tested, Z was most effective. The Z-induced accumulation of CACHT mRNA was inhibited by a plasma membrane Ca2+ channel blocker verapamil. Treatment of cotyledons with exogenous CaCl2 and calcium ionophore A23187 in the presence and absence of cytokinin enhanced CACHT mRNA accumulation. These two observations suggest the participation of extracellular calcium in signaling Z-induction. Furthermore, the presence of staurosporine (an inhibitor of protein kinase) in Z treatment reduced CACHT mRNA, suggesting the involvement of phosphorylation of one or more cellular proteins. In addition, we provide evidence that the Z-induction of CACHT mRNA is blocked by protein synthesis inhibitor cycloheximide treatment. Taken together, these results suggest that Ca2+ influx from extracellular space, protein phosphorylation, and concurrent protein synthesis events participate in cytokinin signaling during Z-induced CACHT transcript accumulation.

Denaturant mediated unfolding of both native and molten globule states of maltose binding protein are accompanied by large [Delta]Cp's

Maltose binding protein (MBP) is a large, monomeric two domain protein containing 370 amino acids. In the absence of denaturant at neutral pH, the protein is in the native state, while at pH 3.0 it forms a molten globule. The molten globule lacks a tertiary circular dichroism signal but has secondary structure similar to that of the native state. The molten globule binds 8-anilino-1-naphthalene sulfonate (ANS). The unfolding thermodynamics of MBP at both pHs were measured by carrying out a series of isothermal urea melts at temperatures ranging from 274–329 K. At 298 K, values of [Delta]G°, [Delta]Cp, and Cm were 3.1 ± 0.2 kcal mol−1, 5.9 ± 0.8 kcal mol−1 K−1 (15.9 cal (mol-residue)−1 K−1), and 0.8 M, respectively, at pH 3.0 and 14.5 ± 0.4 kcal mol−1, 8.3 ± 0.7 kcal mol−1 K−1 (22.4 kcal (mol-residue)−1 K−1), and 3.3 M, respectively, at pH 7.1. Guanidine hydrochloride denaturation at pH 7.1 gave values of [Delta]G° and [Delta]Cp similar to those obtained with urea. The m values for denaturation are strongly temperature dependent, in contrast to what has been previously observed for small globular proteins. The value of [Delta]Cp per mol-residue for the molten globule is comparable to corresponding values of [Delta]Cp for the unfolding of typical globular proteins and suggests that it is a highly ordered structure, unlike molten globules of many small proteins. The value of [Delta]Cp per mol-residue for the unfolding of the native state is among the highest currently known for any protein.