La RNase P mitochondriale chez Neurospora crassa

Résumé La Ribonucléase P (RNase P) est une enzyme principalement reconnue pour sa participation à la maturation en 5’des ARN de transfert (ARNt). Cependant, d’autres substrats sont reconnus par l’enzyme. En général, la RNase P est composée d’une sous-unité ARN (le P-ARN, codé par le gène rnpB) qui porte le centre actif de l’enzyme et d’une ou de plusieurs sous-unités protéiques (la P-protéine). Les P-ARN chez toutes les bactéries, la majorité des archéobactéries et dans le génome nucléaire de la plupart des eucaryotes, possèdent généralement une structure secondaire très conservée qui inclut le noyau (P1-P4); l’hélice P4 constitue le site catalytique de l’enzyme et l’hélice P1 apparie les extrémités du P-ARN en stabilisant sa structure globale. Les P-ARN mitochondriaux sont souvent moins conservés et difficiles à découvrir. Dans certains cas, les seules régions de structure primaire qui restent conservées sont celles qui définissent le P4 et le P1. Pour la détection des gènes rnpB, un outil de recherche bioinformatique, basé sur la séquence et le profil de structure secondaire, a été développé dans le laboratoire. Cet outil permet le dépistage de toutes les séquences eucaryotes (nucléaires et mitochondriales) du gène avec une très grande confiance (basée sur une valeur statistique, E-value). Chez les champignons, plusieurs ascomycètes encodent un gène rnpB dans leur génome mitochondrial y compris tous les membres du genre d’Aspergillus. Cependant, chez les espèces voisines, Neurospora crassa, Podospora anserina et Sordaria macrospora, une version mitochondriale de ce gène n’existe pas. Au lieu de cela, elles contiennent deux copies nucléaires du gène, légèrement différentes en taille et en contenu nucléotidique. Mon projet a été établi dans le but d’éclaircir l’évolution de la RNase P mitochondriale (mtRNase P) chez ces trois espèces voisines d’Aspergillus. En ce qui concerne les résultats, des modèles de structures secondaires pour les transcrits de ces gènes ont été construits en se basant sur la structure consensus universelle de la sous-unité ARN de la RNase P. Pour les trois espèces, par la comparaison de ces modèles, nous avons établi que les deux copies nucléaires du gène rnpB sont assez distinctes en séquence et en structure pour pouvoir y penser à une spécialisation de fonction de la RNase P. Chez N. crassa, les deux P-ARN sont modifiés probablement par une coiffe et les extrémités 5’, 3’ sont conformes à nos modèles, ayant un P1 allongé. Encore chez N. crassa, nous avons constaté que les deux copies sont transcrites au même niveau dans le cytoplasme et que la plus petite et la plus stable d’entre elles (Nc1) se retrouve dans l’extrait matriciel mitochondrial. Lors du suivi du P-ARN dans diverses sous-fractions provenant de la matrice mitochondriale soluble, Nc1 est associée avec l’activité de la RNase P. La caractérisation du complexe protéique, isolé à partir de la fraction active sur un gel non dénaturant, révèle qu’il contient au moins 87 protéines, 73 d’entre elles ayant déjà une localisation mitochondriale connue. Comme chez la levure, les protéines de ce complexe sont impliquées dans plusieurs fonctions cellulaires comme le processing de l’ADN/ARN, le métabolisme, dans la traduction et d’autres (par exemple : la protéolyse et le repliement des protéines, ainsi que la maintenance du génome mitochondrial). Pour trois protéines, leur fonction est non déterminée.

Characterization and Pathogenicity of Vibrio cholerae and Vibrio vulnificus from Marine environments

The genus Vibrioof the family Vibrionaceae are Gram negative, oxidasepositive, rod- or curved- rodshaped facultative anaerobes, widespread in marine and estuarine environments. Vibrio species are opportunistic human pathogens responsible for diarrhoeal disease, gastroenteritis, septicaemia and wound infections and are also pathogens of aquatic organisms, causing infections to crustaceans, bivalves and fishes. In the present study, marine environmental samples like seafood and water and sediment samples from aquafarms and mangroves were screened for the presence of Vibrio species. Of the134 isolates obtained from the various samples, 45 were segregated to the genus Vibrio on the basis of phenotypic characterization.like Gram staining, oxidase test, MoF test and salinity tolerance. Partial 16S rDNA sequence analysis was utilized for species level identification of the isolates and the strains were identified as V. cholerae(N=21), V. vulnificus(N=18), V. parahaemolyticus(N=3), V. alginolyticus (N=2) and V. azureus (N=1). The genetic relatedness and variations among the 45 Vibrio isolates were elucidated based on 16S rDNA sequences. Phenotypic characterization of the isolates was based on their response to 12 biochemical tests namely Voges-Proskauers’s (VP test), arginine dihydrolase , tolerance to 3% NaCl test, ONPG test that detects β-galactosidase activity, and tests for utilization of citrate, ornithine, mannitol, arabinose, sucrose, glucose, salicin and cellobiose. The isolates exhibited diverse biochemical patterns, some specific for the species and others indicative of their environmental source.Antibiogram for the isolates was determined subsequent to testing their susceptibility to 12 antibiotics by the disc diffusion method. Varying degrees of resistance to gentamycin (2.22%), ampicillin(62.22%), nalidixic acid (4.44%), vancomycin (86.66), cefixime (17.77%), rifampicin (20%), tetracycline (42.22%) and chloramphenicol (2.22%) was exhibited. All the isolates were susceptible to streptomycin, co-trimoxazole, trimethoprim and azithromycin. Isolates from all the three marine environments exhibited multiple antibiotic resistance, with high MAR index value. The molecular typing methods such as ERIC PCR and BOX PCR revealed intraspecies relatedness and genetic heterogeneity within the environmental isolatesof V. cholerae and V. vulnificus. The 21 strains of V. choleraewere serogroupedas non O1/ non O139 by screening for the presence O1rfb and O139 rfb marker genes by PCR. The virulence/virulence associated genes namely ctxA, ctxB, ace, VPI, hlyA, ompU, rtxA, toxR, zot, nagst, tcpA, nin and nanwere screened in V. cholerae and V. vulnificusstrains.The V. vulnificusstrains were also screened for three species specific genes viz., cps, vvhand viu. In V. cholerae strains, the virulence associated genes like VPI, hlyA, rtxA, ompU and toxR were confirmed by PCR. All the isolates, except for strain BTOS6, harbored at least one or a combination of the tested genes and V. choleraestrain BTPR5 isolated from prawn hosted the highest number of virulence associated genes. Among the V. vulnificusstrains, only 3 virulence genes, VPI, toxR and cps, were confirmed out of the 16 tested and only 7 of the isolates had these genes in one or more combinations. Strain BTPS6 from aquafarm and strain BTVE4 from mangrove samples yielded positive amplification for the three genes. The toxRgene from 9 strains of V. choleraeand 3 strains of V. vulnificus were cloned and sequenced for phylogenetic analysis based on nucleotide and the amino acid sequences. Multiple sequence alignment of the nucleotide sequences and amino acid sequences of the environmental strains of V. choleraerevealed that the toxRgene in the environmental strains are 100% homologous to themselves and to the V. choleraetoxR gene sequence available in the Genbank database. The 3 strains of V. vulnificus displayed high nucleotide and amino acid sequence similarity among themselves and to the sequences of V. cholerae and V. harveyi obtained from the GenBank database, but exhibited only 72% homology to the sequences of its close relative V. vulnificus. Structure prediction of the ToxR protein of Vibrio cholerae strain BTMA5 was by PHYRE2 software. The deduced amino acid sequence showed maximum resemblance with the structure of DNA-binding domain of response regulator2 from Escherichia coli k-12 Template based homology modelling in PHYRE2 successfully modelled the predicted protein and its secondary structure based on protein data bank (PDB) template c3zq7A. The pathogenicity studies were performed using the nematode Caenorhabditiselegansas a model system. The assessment of pathogenicity of environmental strain of V. choleraewas conducted with E. coli strain OP50 as the food source in control plates, environmental V. cholerae strain BTOS6, negative for all tested virulence genes, to check for the suitability of Vibrio sp. as a food source for the nematode;V. cholerae Co 366 ElTor, a clinical pathogenic strain and V. cholerae strain BTPR5 from seafood (Prawn) and positive for the tested virulence genes like VPI, hlyA, ompU,rtxA and toxR. It was found that V. cholerae strain BTOS6 could serve as a food source in place of E. coli strain OP50 but behavioral aberrations like sluggish movement and lawn avoidance and morphological abnormalities like pharyngeal and intestinal distensions and bagging were exhibited by the worms fed on V. cholerae Co 366 ElTor strain and environmental BTPR5 indicating their pathogenicity to the nematode. Assessment of pathogenicity of the environmental strains of V. vulnificus was performed with V. vulnificus strain BTPS6 which tested positive for 3 virulence genes, namely, cps, toxRand VPI, and V. vulnificus strain BTMM7 that did not possess any of the tested virulence genes. A reduction was observed in the life span of worms fed on environmental strain of V. vulnificusBTMM7 rather than on the ordinary laboratory food source, E. coli OP50. Behavioral abnormalities like sluggish movement, lawn avoidance and bagging were also observed in the worms fed with strain BTPS6, but the pharynx and the intestine were intact. The presence of multi drug resistant environmental Vibrio strainsthat constitute a major reservoir of diverse virulence genes are to be dealt with caution as they play a decisive role in pathogenicity and horizontal gene transfer in the marine environments.

Estabilitat conformacional de variants de la ribonucleasa A: pressió i temperatura com a inductors dels desplegament proteic

A fi d'analitzar la contribució de la regió C-terminal proposada com a iniciadora del plegament (CFIS 106-118) a l'estabilitat de l'RNasa A, els residus alifàtics d'aquesta regió es van substituir, mitjançant mutagènesi dirigida, per altres residus en els quals la cadena lateral alifàtica era rogressivament escurçada. La major part de les substitucions projectades suposaven delecions no disruptives de grups metil(è). A més, es va reemplaçar la Tyr115 per un Trp, de manera que, potencialment, s'introduïa una única sonda fluorescent, no desestabilitzant, per tal de seguir els canvis conformacionals que es poguessin generar en la regió durant el procés de legament/desplegament de la proteïna. Tant els paràmetres cinètics, com els espectres d'FTIR i CD, determinats per cadascuna de les ribonucleases variants, indiquen que els reemplaçaments aminoacídics efectuats presenten, en general, poc o cap efecte en l'estructura nativa i en l'activitat de l'enzim. Es va emprar l'espectroscòpia d'absorció a l'ultraviolat de quarta derivada, la fluorescència (per la variant amb Trp) i l'espectroscòpia d'infraroig per transformada de Fourier, per tal de seguir i caracteritzar, en condicions d'equilibri, les transicions conformacionals de cada variant en funció de la pressió i de la temperatura. Els resultats es van comparar amb els que es van obtenir per la proteïna salvatge. Per determinar més a fons les característiques del procés de desplegament de la variant Y115W, les transicions de desnaturalització induïdes per urea d'aquesta variant i de la proteïna salvatge, van ésser examinades per mitjà d'electroforesi en gradient d'urea i espectroscòpia de fluorescència. Curiosament, els canvis conformacionals que resulten de la desnaturalització per pressió són molt semblants als que s'obtenen per temperatura. Enfront d'un augment gradual tant de pressió com de temperatura, l'estructura terciària i els elements d'estructura secundària de les proteïnes estudiades es perden de manera conjunta i reversible. Aquestes variacions estructurals que es promouen descriuen un procés de desplegament molt cooperatiu i en dos estats. Atès que ambdues tècniques (UV i FTIR) utilitzen cadascuna un règim de concentració proteica molt diferent, els resultats indiquen que el procés de desplegament per pressió i per temperatura és intramolecular. Els resultats obtinguts suggereixen que la hidrofobicitat i el volum de les cadenes laterals del CFIS, juntament amb les interaccions de van der Waals entre elements d'estructura secundària intervenen de manera molt notable en l'estabilització de la proteïna. Entre els diferents aminoàcids alifàtics que pertanyen al CFIS C-terminal, la Val108 és el residu més important per tal de preservar la integritat estructural de l'estat natiu. Els reemplaçaments en aquesta posició causen petites alteracions conformacionals i una gran desestabilització de la proteïna (per exemple, el punt mig de la transició de desnaturalització per pressió i per temperatura de la variant V108G disminueix uns 592 MPa i 25ºC, respectivament, respecte a la proteïna salvatge). D'acord amb els resultats obtinguts, la variant Y115W ofereix una sonda útil per tal de seguir la cinètica de plegament/desplegament de l'RNasa A.

Paper del nucli hidrofòbic principal de la RNasa A en el plegament i desplegament induïts per pressió i temperatura

Utilitzant temperatura i pressió com a agents desnaturalitzants s'ha explorat la contribució a l'estabilitat de diferents residus del principal nucli hidrofòbic de la RNasa A. Aquests resutats suggereixen que el principal nucli hidrofòbic d'aquest enzim, està fortament empaquetat i ha revelat l'existència de reordenacions en l'interior de la proteïna. El mètode dels valors , han permès estudiar el paper de les interaccions hidrofòbiques establertes pels residus del principal nucli hidrofòbic de la RNasa A en el seu estat de transició induït per pressió. En conjunt, aquests resultats suggereixen que l'estat de transició de la RNasa A, s'assemblaria a un glòbul col·lapsat amb una cadena estructurada però amb un debilitat nucli hidrofòbic. S'ha explorat també, el paisatge energètic del plegament/desplegament proteic de la variant Y115W de la RNasa A. L'estat de transició sembla interaccionar fortament amb la capa d'hidratació d'aquest estat, tal i com indiquen els resultats en presència de glicerol.

Use of essential and molecular dynamics to study gamma B-crystallin unfolding after non-enzymic post-translational modifications

Essential and Molecular Dynamics (ED/MD) have been used to model the conformational changes of a protein implicated in a conformational disease-cataract, the largest cause of blindness in the world-after non-enzymic post-translational modification. Cyanate modification did not significantly alter flexibility, while the Schiff's base adduct produced a more flexible N-terminal domain, and intra-secondary structure regions, than either the cyanate adduct or the native structure. Glycation also increased linker flexibility and disrupted the charge network. A number of post-translational adducts showed structural disruption around Cys15 and increased linker flexibility; this may be important in subsequent protein aggregation. Our modelling results are in accord with experimental evidence, and show that ED/MD is a useful tool in modelling conformational changes in proteins implicated in disease processes. (C) 2003 Published by Elsevier Ltd.

Interactions of KLVFF-PEG peptide conjugate with fibrinogen in neutral aqueous solutions

In this work we report on the interaction of KLVFF-PEG with fibrinogen (Fbg) in neutral aqueous solutions at 20 degrees C, for particular ratios of KLVFF-PEG to Fbg concentration, Delta = CKLVFF-PEG/C-Fbg- Our results show the formation of Fbg/KLVFF-PEG complexes for Delta > 0, such that there is not an extended network of complexes throughout the solution. In addition, cleaved protein and Fbg dimers are identified in the solution for Delta >= 0. There is a dramatic change in the tertiary structure of the Fbg upon KLVFF-PEG binding, although the KLVFF-PEG binds to the Fbg without affecting the secondary structure elements of the glycoprotein.

Interactions of bovine serum albumin with ethylene oxide/butylene oxide copolymers in aqueous solution

The interactions of bovine serum albumin (BSA) with three ethylene oxide/butylene oxide (E/B) copolymers having different block lengths and varying molecular architectures is examined in this study in aqueous solutions. Dynamic light scattering (DLS) indicates the absence of BSA-polymer binding in micellar systems of copolymers with lengthy hydrophilic blocks. On the contrary, stable protein-polyrner aggregates were observed in the case of E18B10 block copolymer. Results from DLS and SAXS suggest the dissociation of E/B copolymer micelles in the presence of protein and the absorption of polymer chains to BSA surface. At high protein loadings, bound BSA adopts a more compact conformation in solution. The secondary structure of the protein remains essentially unaffected even at high polymer concentrations. Raman spectroscopy was used to give insight to the configurations of the bound molecules in concentrated solutions. In the vicinity of the critical gel concentration of E18B10 introduction of BSA can dramatically modify the phase diagram, inducing a gel-sol-gel transition. The overall picture of the interaction diagram of the E18B10-BSA reflects the shrinkage of the suspended particles due to destabilization of micelles induced by BSA and the gelator nature of the globular protein. SAXS and rheology were used to further characterize the structure and flow behavior of the polymer-protein hybrid gels and sols.

The adsorbed conformation of globular proteins at the air/water interface

External reflection FTIR spectroscopy and surface pressure measurements were used to compare conformational changes in the adsorbed structures of three globular proteins at the air/water interface. Of the three proteins studied, lysozyme, bovine serum albumin and P-lactoglobulin, lysozyme was unique in its behaviour. Lysozyme adsorption was slow, taking approximately 2.5 h to reach a surface pressure plateau (from a 0.07 mM solution), and led to significant structural change. The FTIR spectra revealed that lysozyme formed a highly networked adsorbed layer of unfolded protein with high antiparallel beta-sheet content and that these changes occurred rapidly (within 10 min). This non-native secondary structure is analogous to that of a 3D heat-set protein gel, suggesting that the adsorbed protein formed a highly networked interfacial layer. Albumin and P-lactoglobulin adsorbed rapidly (reaching a plateau within 10 min) and with little chance to their native secondary structure.

Hydrogelation and self-assembly of Fmoc-tripeptides: unexpected influence of sequence on self-assembled fibril structure, and hydrogel modulus and anisotropy

The self-assembly and hydrogelation properties of two Fmoc-tripeptides [Fmoc = N-(fluorenyl-9-methoxycarbonyl)] are investigated, in borate buffer and other basic solutions. A remarkable difference in self-assembly properties is observed comparing Fmoc-VLK(Boc) with Fmoc-K(Boc)LV, both containing K protected by N(epsilon)-tert-butyloxycarbonate (Boc). In borate buffer, the former peptide forms highly anisotropic fibrils which show local alignment, and the hydrogels show flow-aligning properties. In contrast, Fmoc-K(Boc)LV forms highly branched fibrils that produce isotropic hydrogels with a much higher modulus (G' > 10(4) Pa), and lower concentration for hydrogel formation. The distinct self-assembled structures are ascribed to conformational differences, as revealed by secondary structure probes (CD, FTIR, Raman spectroscopy) and X-ray diffraction. Fmoc-VLK(Boc) forms well-defined beta-sheets with a cross-beta X-ray diffraction pattern, whereas Fmoc-KLV(Boc) forms unoriented assemblies with multiple stacked sheets. Interchange of the K and V residues when inverting the tripeptide sequence thus leads to substantial differences in self-assembled structures, suggesting a promising approach to control hydrogel properties.

Prediction of novel and analogous folds using fragment assembly and fold recognition

A number of new and newly improved methods for predicting protein structure developed by the Jones–University College London group were used to make predictions for the CASP6 experiment. Structures were predicted with a combination of fold recognition methods (mGenTHREADER, nFOLD, and THREADER) and a substantially enhanced version of FRAGFOLD, our fragment assembly method. Attempts at automatic domain parsing were made using DomPred and DomSSEA, which are based on a secondary structure parsing algorithm and additionally for DomPred, a simple local sequence alignment scoring function. Disorder prediction was carried out using a new SVM-based version of DISOPRED. Attempts were also made at domain docking and “microdomain” folding in order to build complete chain models for some targets.

Assessing the quality of modelled 3D protein structures using the ModFOLD server

Model quality assessment programs (MQAPs) aim to assess the quality of modelled 3D protein structures. The provision of quality scores, describing both global and local (per-residue) accuracy are extremely important, as without quality scores we are unable to determine the usefulness of a 3D model for further computational and experimental wet lab studies.Here, we briefly discuss protein tertiary structure prediction, along with the biennial Critical Assessment of Techniques for Protein Structure Prediction (CASP) competition and their key role in driving the field of protein model quality assessment methods (MQAPs). We also briefly discuss the top MQAPs from the previous CASP competitions. Additionally, we describe our downloadable and webserver-based model quality assessment methods: ModFOLD3, ModFOLDclust, ModFOLDclustQ, ModFOLDclust2, and IntFOLD-QA. We provide a practical step-by-step guide on using our downloadable and webserver-based tools and include examples of their application for improving tertiary structure prediction, ligand binding site residue prediction, and oligomer predictions.

Genetic structure, phylogeny, and biogeography of Brazilian eyelid-less lizards of genera Calyptommatus and Nothobachia (Squamata, Gymnophthalmidae) as inferred from mitochondrial DNA sequences

Calyptommatus and Nothobachia genera of gymnophthalmid lizards are restricted to sandy open habitats on Sao Francisco River margins, northeastern Brazil. Phylogenetic relationships and geographic distribution of the four recognized species of Calyptommatus were analyzed from partial mitochondrial cyt b, 12S, and 16S rRNA genes sequencing, taking allopatric populations of the monotypic Nothobachia ablephara as the outgroup. In Calyptommatus a basal split separated C. sinebrachiatus, a species restricted to the eastern bank of the river, from the three other species. In this clade, C. confusionibus, found on western margin, was recovered as the sister group of the two other species, C. leiolepis and C. nicterus, from opposite margins. According to approximate date estimations, C. sinebrachiatus would have separated from the other congeneric species by 4.4-6.5 my, and C. nicterus, also from eastern bank, would be diverging by 1.8-2.6 my from C. leiolepis, the sister species on the opposite margin. C. confusionibus and C. leiolepis, both from western sandy areas, would be differentiating by 2.8-5.0 my. Divergence times of about 3.0-4.0 my were estimated for allopatric populations of Nothobachia restricted to western margin. Significant differences in 16S rRNA secondary structure relatively to other vertebrates are reported. Distinct evolutionary patterns are proposed for different taxa in those sandy areas, probably related to historical changes in the course of Sao Francisco River. (C) 2010 Elsevier Inc. All rights reserved.

The three-dimensional structure of bothropasin, the main hemorrhagic factor from Bothrops jararaca venom: Insights for a new classification of snake venom metalloprotease subgroups

Bothropasin is a 48 kDa hemorrhagic PIII snake venom metalloprotease (SVMP) isolated from Bothrops jararaca, containing disintegrin/cysteine-rich adhesive domains. Here we present the crystal structure of bothropasin complexed with the inhibitor POL647. The catalytic domain consists of a scaffold of two subdomains organized similarly to those described for other SVMPs, including the zinc and calcium-binding sites. The free cysteine residue Cys(189) is located within a hydrophobic core and it is not available for disulfide bonding or other interactions. There is no identifiable secondary structure for the disintegrin domain, but instead it is composed mostly of loops stabilized by seven disulfide bonds and by two calcium ions. The ECD region is in a loop and is structurally related to the RGD region of RGD disintegrins, which are derived from I`ll SVMPs. The ECD motif is stabilized by the Cys(117)_Cys(310) disulfide bond (between the disintegrin and cysteine-rich domains) and by one calcium ion. The side chain of Glu(276) of the ECD motif is exposed to solvent and free to make interactions. In bothropasin, the HVR (hyper-variable region) described for other Pill SVMPs in the cysteine-rich domain, presents a well-conserved sequence with respect to several other Pill members from different species. We propose that this subset be referred to as PIII-HCR (highly conserved region) SVMPs. The differences in the disintegrin-like, cysteine-rich or disintegrin-like cysteine-rich domains may be involved in selecting target binding, which in turn could generate substrate diversity or specificity for the catalytic domain. (C) 2008 Elsevier Ltd. All rights reserved.

Sugarcane Phosphoribosyl Pyrophosphate Synthetase: Molecular Characterization of a Phosphate-independent PRS

Phosphoribosyl pyrophosphate synthetase (PRS-EC:2.7.6.1) is an important enzyme present in several metabolic pathways, thus forming a complex family of isoenzymes. However, plant PRS enzymes have not been extensively investigated. In this study, a sugarcane prs gene has been characterized from the Sugar Cane Expressed Sequence Tag Genome Project. This gene contains a 984-bp open reading frame encoding a 328-amino acid protein. The predicted amino acid sequence has 77% and 78% amino acid sequence identity to Arabidopsis thaliana and Spinacia oleracea PRS4, respectively. The assignment of sugarcane PRS as a phosphate-independent PRS isoenzyme (Class II PRS) is verified following enzyme assay and phylogenetic reconstruction of PRS homologues. To gain further insight into the structural framework of the phosphate independence of sugarcane PRS, a molecular model is described. This model reveals the formation of two conserved domains elucidating the structural features involved in sugarcane PRS phosphate independence. The recombinant PRS retains secondary structure elements and a quaternary arrangement consistent with known PRS homologues, based on circular dichroism measurements.

On the Denaturation Mechanisms of the Ligand Binding Domain of Thyroid Hormone Receptors

The ligand binding domain (LBD) of nuclear hormone receptors adopts a very compact, mostly alpha-helical structure that binds specific ligands with very high affinity. We use circular dichroism spectroscopy and high-temperature molecular dynamics Simulations to investigate unfolding of the LBDs of thyroid hormone receptors (TRs). A molecular description of the denaturation mechanisms is obtained by molecular dynamics Simulations of the TR alpha and TR beta LBDs in the absence and in the presence of the natural ligand Triac. The Simulations Show that the thermal unfolding of the LBD starts with the loss of native contacts and secondary Structure elements, while the Structure remains essentially compact, resembling a molten globule state. This differs From most protein denaturation simulations reported to date and suggests that the folding mechanism may start with the hydrophobic collapse of the TR LBDs. Our results reveal that the stabilities of the LBDs of the TR alpha and TR beta Subtypes are affected to different degrees by the binding of the isoform selective ligand Triac and that ligand binding confers protection against thermal denaturation and unfolding in a subtype specific manner. Our Simulations indicate two mechanisms by which the ligand stabilizes the LBD: (1) by enhancing the interactions between H8 and H 11, and the interaction of the region between H I and the Omega-loop with the core of the LBD, and (2) by shielding the hydrophobic H6 from hydration.