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Fator de Transcrição Ikaros/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Doença Aguda , Sequência de Bases , Haploinsuficiência , Humanos , Fator de Transcrição Ikaros/biossíntese , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Deleção de SequênciaRESUMO
INTRODUCTION: Toll-like receptors play crucial roles in the sepsis-induced systemic inflammatory response. Septic shock mortality correlates with overexpression of neutrophilic TLR2 and TLR9, while the role of TLR4 overexpression remains a debate. In addition, TLRs are involved in the pathogenesis of viral infections such as COVID-19, where the single-stranded RNA of SARS-CoV-2 is recognized by TLR7 and TLR8, and the spike protein activates TLR4. METHODS: In this study, we conducted a comprehensive analysis of TLRs 1-10 expressions in white blood cells from 71 patients with bacterial and viral infections. Patients were divided into 4 groups based on disease type and severity (sepsis, septic shock, moderate, and severe COVID-19) and compared to 7 healthy volunteers. RESULTS: We observed a significant reduction in the expression of TLR4 and its co-receptor CD14 in septic shock neutrophils compared to the control group (p < 0.001). Severe COVID-19 patients exhibited a significant increase in TLR3 and TLR7 levels in neutrophils compared to controls (p < 0.05). Septic shock patients also showed a similar increase in TLR7 in neutrophils along with elevated intermediate monocytes (CD14+CD16+) compared to the control group (p < 0.005 and p < 0.001, respectively). However, TLR expression remained unchanged in lymphocytes. CONCLUSION: This study provides further insights into the mechanisms of TLR activation in various infectious conditions. Additional analysis is needed to assess their correlation with patient outcome and to evaluate the impact of TLR-pathway modulation during septic shock and severe COVID-19.
Assuntos
COVID-19 , SARS-CoV-2 , Receptor 10 Toll-Like , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Infecções Bacterianas/imunologia , COVID-19/imunologia , COVID-19/sangue , Leucócitos/imunologia , Leucócitos/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Neutrófilos/imunologia , SARS-CoV-2/imunologia , Sepse/imunologia , Choque Séptico/imunologia , Choque Séptico/sangue , Receptor 1 Toll-Like/metabolismo , Receptor 1 Toll-Like/genética , Receptor 7 Toll-Like/metabolismo , Receptor 7 Toll-Like/genética , Receptores Toll-Like/metabolismo , Idoso de 80 Anos ou maisRESUMO
Acetylation of the ε-amino group of lysine (Lys) is a reversible posttranslational modification recently discovered to be widespread, occurring on proteins outside the nucleus, in most subcellular locations in mammalian cells. Almost nothing is known about this modification in plants beyond the well-studied acetylation of histone proteins in the nucleus. Here, we report that Lys acetylation in plants also occurs on organellar and cytosolic proteins. We identified 91 Lys-acetylated sites on 74 proteins of diverse functional classes. Furthermore, our study suggests that Lys acetylation may be an important posttranslational modification in the chloroplast, since four Calvin cycle enzymes were acetylated. The plastid-encoded large subunit of Rubisco stands out because of the large number of acetylated sites occurring at important Lys residues that are involved in Rubisco tertiary structure formation and catalytic function. Using the human recombinant deacetylase sirtuin 3, it was demonstrated that Lys deacetylation significantly affects Rubisco activity as well as the activities of other central metabolic enzymes, such as the Calvin cycle enzyme phosphoglycerate kinase, the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase, and the tricarboxylic acid cycle enzyme malate dehydrogenase. Our results demonstrate that Lys acetylation also occurs on proteins outside the nucleus in Arabidopsis (Arabidopsis thaliana) and that Lys acetylation could be important in the regulation of key metabolic enzymes.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Acetilação , Núcleo Celular/metabolismo , Cloroplastos/metabolismo , Cromatografia Líquida , Mitocôndrias/metabolismo , Fotossíntese , Estrutura Terciária de Proteína , Proteoma/análise , Ribulose-Bifosfato Carboxilase/metabolismo , Espectrometria de Massas em TandemRESUMO
The AGC protein kinase OXI1 is a key protein in plant responses to oxidative signals, and is important for two oxidative burst-mediated processes: basal resistance to microbial pathogens and root hair growth. To identify possible components of the OXI1 signalling pathway, phosphoproteomic techniques were used to detect alterations in the abundance of phosphorylated proteins and peptides in an oxi1 null mutant of Arabidopsis thaliana. The relative abundance of phosphorylated proteins was assessed either using two-dimensional gel electrophoresis and staining with the phosphoprotein stain Pro-Q Diamond or by the identification and quantification, by mass spectrometry, of stable-isotope labelled phosphopeptides. A number of proteins show altered phosphorylation in the oxi1 mutant. Five proteins, including a putative F-box and 3-phosphoinositide-dependent kinase 1, show reduced phosphorylation in the oxi1 mutant, and may be direct or indirect targets of OXI1. Four proteins, including ethylene insensitive 2 and phospholipase d-gamma, show increased phosphorylation in the oxi1 mutant. This study has identified a range of candidate proteins from the OXI1 signalling pathway. The diverse activities of these proteins, including protein degradation and hormone signalling, may suggest crosstalk between OXI1 and other signal transduction cascades.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteoma/metabolismo , Sequência de Aminoácidos , Proteínas de Arabidopsis/química , Cromatografia Líquida , Eletroforese em Gel Bidimensional , Marcação por Isótopo , Espectrometria de Massas , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação/genética , Fosfopeptídeos/química , Fosfopeptídeos/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/química , Proteoma/químicaRESUMO
B-cell receptor (BCR) signaling is crucial for the pathophysiology of most mature B-cell lymphomas/leukemias and has emerged as a therapeutic target whose effectiveness remains limited by the occurrence of mutations. Therefore, deciphering the cellular program activated downstream this pathway has become of paramount importance for the development of innovative therapies. Using an original ex vivo model of BCR-induced proliferation of chronic lymphocytic leukemia cells, we generated 108 temporal transcriptional and proteomic profiles from 1 h up to 4 days after BCR activation. This dataset revealed a structured temporal response composed of 13,065 transcripts and 4027 proteins, comprising a leukemic proliferative signature consisting of 430 genes and 374 proteins. Mathematical modeling of this complex cellular response further highlighted a transcriptional network driven by 14 early genes linked to proteins involved in cell proliferation. This group includes expected genes (EGR1/2, NF-kB) and genes involved in NF-kB signaling modulation (TANK, ROHF) and immune evasion (KMO, IL4I1) that have not yet been associated with leukemic cells proliferation. Our study unveils the BCR-activated proliferative genetic program in primary leukemic cells. This approach combining temporal measurements with modeling allows identifying new putative targets for innovative therapy of lymphoid malignancies and also cancers dependent on ligand-receptor interactions.
Assuntos
Linfócitos B/metabolismo , Proliferação de Células/genética , Leucemia Linfocítica Crônica de Células B/genética , Receptores de Antígenos de Linfócitos B/genética , Idoso , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Ativação Linfocitária/genética , Masculino , Pessoa de Meia-Idade , Proteoma/genética , Proteômica/métodos , Transdução de Sinais/genética , Transcrição Gênica/genéticaRESUMO
BACKGROUND: The difficulty in interpreting somatic alterations is correlated with the increase in sequencing panel size. To correctly guide the clinical management of patients with cancer, there needs to be accurate classification of pathogenicity followed by actionability assessment. Here, we describe a specific detailed workflow for the classification of the pathogenicity of somatic variants in cancer into five categories: benign, likely benign, unknown significance, likely pathogenic and pathogenic. METHODS: Classification is obtained by combining a set of eight relevant criteria in favour of either a pathogenic or a benign effect (pathogenic stand-alone, pathogenic very strong, pathogenic strong, pathogenic moderate, pathogenic supporting, benign supporting, benign strong and benign stand-alone). RESULTS: Our guide is concordant with the ACMG/AMP 2015 guidelines for germline variants. Interpretation of somatic variants requires considering specific criteria, such as the disease and therapeutic context, co-occurring genomic events in the tumour when available and the use of cancer-specific variant databases. In addition, the gene role in tumorigenesis (oncogene or tumour suppressor gene) also needs to be taken into consideration. CONCLUSION: Our classification could contribute to homogenize best practices on somatic variant pathogenicity interpretation and improve interpretation consistency both within and between laboratories.
Assuntos
Neoplasias/genética , Patologia Molecular/métodos , Patologia Molecular/normas , Humanos , Fluxo de TrabalhoRESUMO
We describe the construction and analysis of a genome-scale metabolic model of Arabidopsis (Arabidopsis thaliana) primarily derived from the annotations in the Aracyc database. We used techniques based on linear programming to demonstrate the following: (1) that the model is capable of producing biomass components (amino acids, nucleotides, lipid, starch, and cellulose) in the proportions observed experimentally in a heterotrophic suspension culture; (2) that approximately only 15% of the available reactions are needed for this purpose and that the size of this network is comparable to estimates of minimal network size for other organisms; (3) that reactions may be grouped according to the changes in flux resulting from a hypothetical stimulus (in this case demand for ATP) and that this allows the identification of potential metabolic modules; and (4) that total ATP demand for growth and maintenance can be inferred and that this is consistent with previous estimates in prokaryotes and yeast.
Assuntos
Arabidopsis/metabolismo , Metaboloma , Modelos Biológicos , Trifosfato de Adenosina/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Biomassa , Células Cultivadas , Genoma de Planta , Redes e Vias MetabólicasRESUMO
A chronic antigenic stimulation is believed to sustain the leukemogenic development of chronic lymphocytic leukemia (CLL) and most of lymphoproliferative malignancies developed from mature B cells. Reproducing a proliferative stimulation ex vivo is critical to decipher the mechanisms of leukemogenesis in these malignancies. However, functional studies of CLL cells remains limited since current ex vivo B cell receptor (BCR) stimulation protocols are not sufficient to induce the proliferation of these cells, pointing out the need of mandatory BCR co-factors in this process. Here, we investigated benefits of several BCR co-stimulatory molecules (IL-2, IL-4, IL-15, IL-21 and CD40 ligand) in multiple culture conditions. Our results demonstrated that BCR engagement (anti-IgM ligation) concomitant to CD40 ligand, IL-4 and IL-21 stimulation allowed CLL cells proliferation ex vivo. In addition, we established a proliferative advantage for ZAP70 positive CLL cells, associated to an increased phosphorylation of ZAP70/SYK and STAT6. Moreover, the use of a tri-dimensional matrix of methylcellulose and the addition of TLR9 agonists further increased this proliferative response. This ex vivo model of BCR stimulation with T-derived cytokines is a relevant and efficient model for functional studies of CLL as well as lymphoproliferative malignancies.
Assuntos
Linfócitos B/patologia , Proliferação de Células , Leucemia Linfocítica Crônica de Células B/patologia , Receptores de Antígenos de Linfócitos B/metabolismo , Fator de Transcrição STAT6/metabolismo , Quinase Syk/metabolismo , Proteína-Tirosina Quinase ZAP-70/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Linfócitos B/metabolismo , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Masculino , Pessoa de Meia-Idade , Fosforilação , Células Tumorais CultivadasRESUMO
CD180, a related member of the Toll-like receptor family, is lost or underexpressed at the plasma membrane in circulating cells of various B-cell lymphomas except marginal zone lymphomas (MZL). In order to confirm its clinical relevance in routine analysis, we evaluated prospectively the expression of CD180 in 236 patients from 5 French University Hospital laboratories on behalf of the GEIL. Highly comparable results were obtained in all centers using the EuroFlow standardization protocol. We observed that CD180 median fluorescence (MdFI) was significantly higher in MZL and hairy cell leukaemia (HCL) compared to all other B-cell proliferations (P < 0.05). CD180 intensity could distinguish lymphomas with numerous villous lymphocytes from other MZL. ROC curve analysis identified a CD180 MdFI threshold for which the diagnosis of MZL could be assessed with 77% sensitivity and 92% specificity. This study showed that CD180 can be considered as a single positive robust marker of MZL and should be therefore included in flow cytometry panels for the diagnosis of mature B-cell neoplasms. Harmonization process is of great interest in order to evaluate new markers in multicentric studies and to set up decisional thresholds. © 2015 International Clinical Cytometry Society.
Assuntos
Antígenos CD/metabolismo , Linfócitos B/metabolismo , Citometria de Fluxo , Ativação Linfocitária/imunologia , Linfoma de Células B/metabolismo , Linfócitos B/imunologia , Proliferação de Células/fisiologia , Citometria de Fluxo/métodos , Humanos , Linfoma de Células B/diagnóstico , Linfoma de Células B/patologiaRESUMO
The difficulty and expense of preparing protein samples highly enriched in stable isotopes is a bottleneck for structural studies by nuclear magnetic resonance (NMR) spectroscopy. We have developed a new regulatable expression/labeling vector system in the cyanobacterium Anabaena sp. PCC 7120 using the endogenous promoter of the nitrate assimilation nir operon. Standard proteins were overexpressed upon induction with NaNO3, yielding up to 250 mg/L of culture. When the cyanobacteria were grown in the presence of inexpensive 15N-, 13C-labeled mineral salts and 2H2O, the expressed polypeptides were highly (>90%) enriched in stable isotopes. Furthermore, the tight repression of the nir promoter upon induction allowed the production of the toxic oncoprotein E6. In addition, under these conditions, the malE31 protein, while insoluble in Escherichia coli, was found to be soluble in Anabaena. Together, these properties render the described system especially suitable for the production and/or triple labeling of recombinant protein samples. It represents an interesting alternative to conventional protein expression systems used in structural genomics.
Assuntos
Anabaena/genética , Anabaena/metabolismo , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Espectroscopia de Ressonância Magnética/métodos , Engenharia de Proteínas/métodos , Proteínas Recombinantes/biossíntese , Proteínas de Bactérias/química , Marcação por Isótopo/métodosRESUMO
Nitrilase enzymes catalyse the hydrolysis of nitrile compounds to the corresponding carboxylic acid and ammonia, and have been identified in plants, bacteria and fungi. There is mounting evidence to support a role for nitrilases in plant-microbe interactions, but the activity of these enzymes in plant pathogenic bacteria remains unexplored. The genomes of the plant pathogenic bacteria Pseudomonas syringae pv. syringae B728a and Pseudomonas syringae pv. tomato DC3000 contain nitrilase genes with high similarity to characterized bacterial arylacetonitrilases. In this study, we show that the nitrilase of P. syringae pv. syringae B728a is an arylacetonitrilase, which is capable of hydrolysing indole-3-acetonitrile to the plant hormone indole-3-acetic acid, and allows P. syringae pv. syringae B728a to use indole-3-acetonitrile as a nitrogen source. This enzyme may represent an additional mechanism for indole-3-acetic acid biosynthesis by P. syringae pv. syringae B728a, or may be used to degrade and assimilate aldoximes and nitriles produced during plant secondary metabolism. Nitrilase activity was not detected in P. syringae pv. tomato DC3000, despite the presence of a homologous nitrilase gene. This raises the interesting question of why nitrilase activity has been retained in P. syringae pv. syringae B728a and not in P. syringae pv. tomato DC3000.
Assuntos
Ácidos Indolacéticos/metabolismo , Indóis/metabolismo , Pseudomonas syringae/metabolismo , Aminoidrolases/classificação , Aminoidrolases/genética , Filogenia , Pseudomonas syringae/classificação , Pseudomonas syringae/genéticaRESUMO
The diagnosis of mature B-cell neoplasms (MBCN) remains difficult in a number of cases, especially leukemic phases of non-Hodgkin lymphoma, for which discriminating criteria or biomarker are often lacking. To identify new surface biomarkers, we developed an original proteomic approach based on mass spectrometry analysis of plasma membrane microparticles derived from chronic B-cell lymphoproliferations of single patients: chronic lymphocytic leukemia (CLL), small cell lymphoma (SLL) and mantle cell lymphoma (MCL). A straightforward selection process for proteomic-based candidate biomarker identification was further constructed in order to propose potentially useful and relevant biomarkers. Comparison of the lists of the proteins identified in each pathology combined to highly stringent MS validation criteria for protein identification allowed to propose CD148, a membrane receptor with phosphatase activity, as a discriminating biomarker candidate. Flow cytometry analyses, performed on 158 patients and 30 controls, showed that an anti-CD148 antibody stained significantly higher MCL than CLL and SLL circulating cells (p<0.0001), which validates CD148 overexpression in MCL. Our results indicate that a medium or high CD148 expression level may exclude the diagnosis of CLL and high CD148 expression levels (CD148 MFI equal or superior to 2 times the value obtained with CLL/SLL) allows MCL diagnosis to be suspected with 91% specificity (versus CLL and SLL) and 78% sensitivity. This study is one of the first where proteomic strategies allowed to identify a potentially useful biomarker.
Assuntos
Linfócitos B/citologia , Biomarcadores Tumorais/metabolismo , Linfoma de Células B/diagnóstico , Linfoma de Células B/patologia , Linfoma de Célula do Manto/diagnóstico , Linfoma de Célula do Manto/patologia , Proteômica/métodos , Sequência de Aminoácidos , Membrana Celular/metabolismo , Micropartículas Derivadas de Células/metabolismo , Citometria de Fluxo/métodos , Técnica Indireta de Fluorescência para Anticorpo/métodos , Humanos , Imunofenotipagem , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/biossínteseRESUMO
Steady-state labeling experiments with [1-(13)C]Glc were used to measure multiple metabolic fluxes through the pathways of central metabolism in a heterotrophic cell suspension culture of Arabidopsis (Arabidopsis thaliana). The protocol was based on in silico modeling to establish the optimal labeled precursor, validation of the isotopic and metabolic steady state, extensive nuclear magnetic resonance analysis of the redistribution of label into soluble metabolites, starch, and protein, and a comprehensive set of biomass measurements. Following a simple modification of the cell culture procedure, cells were grown at two oxygen concentrations, and flux maps of central metabolism were constructed on the basis of replicated experiments and rigorous statistical analysis. Increased growth rate at the higher O(2) concentration was associated with an increase in fluxes throughout the network, and this was achieved without any significant change in relative fluxes despite differences in the metabolite profile of organic acids, amino acids, and carbohydrates. The balance between biosynthesis and respiration within the tricarboxylic acid cycle was unchanged, with 38% +/- 5% of carbon entering used for biosynthesis under standard O(2) conditions and 33% +/- 2% under elevated O(2). These results add to the emerging picture of the stability of the central metabolic network and its capacity to respond to physiological perturbations with the minimum of rearrangement. The lack of correlation between the change in metabolite profile, which implied significant disruption of the metabolic network following the alteration in the oxygen supply, and the unchanging flux distribution highlights a potential difficulty in the interpretation of metabolomic data.
Assuntos
Arabidopsis/metabolismo , Ciclo do Ácido Cítrico , Consumo de Oxigênio , Aerobiose , Biomassa , Isótopos de Carbono/metabolismo , Respiração Celular , Células Cultivadas , Meios de Cultura , Espectroscopia de Ressonância Magnética , Modelos BiológicosRESUMO
The accurate diagnosis of the different forms of chronic mature B-cell lymphocytic malignancies is of primary importance to determine an appropriate and efficient treatment. Usually, the diagnosis is achieved by morphology and immunophenotyping. Nevertheless, the diagnostic tools available are not able to discriminate pathologies with variable evolution, or to classify some of them. To discover new biomarkers, we used peptide and protein profiling SELDI-TOF-MS, to analyze 39 chronic B-cell malignancies and 20 control serum samples. Markers of interest were subsequently identified and characterized. In the obtained SELDI-MS profiles, most of the differences were observed in three mass ranges (m/z = 13 000; m/z = 9000; m/z < 2000). Identification of these biomarkers was achieved either by direct enrichment on the ProteinChip arrays followed by on-chip-MS/MS or by chromatographic fractionation, 1D-gel followed by nanoLC-MS/MS analysis. An increase of a sulfite form of transthyretin (13,841 Da) was observed in the patient group. A second set of markers at 8.6 and 8.9 kDa was identified as complement related fragment proteins, the C3a and C4a anaphylatoxins. In the low mass range, several peptides originating from N-terminal and C-terminal processing of the C3 alpha and C4 alpha chains were specifically observed in 38% of the patient sera, but in none of the control sera. This study emphasizes the usefulness of mass spectrometry studies in such malignancies.
Assuntos
Biomarcadores/análise , Proteínas Sanguíneas/análise , Linfoma de Células B/sangue , Linfoma de Células B/diagnóstico , Sequência de Aminoácidos , Proteínas Sanguíneas/genética , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Espectrometria de Massas , Dados de Sequência Molecular , Análise Serial de Proteínas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
Shed membrane microparticles (MPs) are microvesicles generated from the plasma membrane when cells are submitted to stress conditions. Although MPs reflect the cell state (at least in vitro), little is known on their protein composition. We describe the first set of experiments aiming to characterize the MP proteome. Two ways of triggering MP formation from a T-lymphocytic cell line were analyzed using a 1-D gel approach coupled with LC-MS/MS and the results were compared with those obtained from a classic membrane preparation. In total, 390 proteins were identified in MPs, among which 34% were localized to the plasma membrane. The MPs revealed a broad representation of plasma membrane proteins including 17 hematopoietic clusters of differentiation. This approach was successfully applied to one human chronic B-cell lymphoid malignancy. In all, 413 proteins were identified, including 117 membrane proteins, many of them being pathology associated. The sequence coverage in identified proteins was improved combining both nano-LC-MS/MS and MALDI-MS data. The suppression effect, observed on very complex peptide mixtures, was remediated by chromatographic fractionation. MPs may represent a new tool for studying plasma membrane proteins, displaying the advantages of reproducibility, minimal organelle contamination, and being potentially applicable to most cell types.
Assuntos
Linfócitos/metabolismo , Linfoma de Células B/metabolismo , Proteoma , Membrana Celular/metabolismo , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Humanos , Linfoma de Células B/patologia , Nanotecnologia , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Células Tumorais CultivadasRESUMO
E6 is a viral oncoprotein implicated in cervical cancers, produced by human papillomaviruses (HPVs). E6 contains two putative zinc-binding domains of about 75 residues each. The difficulty in producing recombinant E6 has long hindered the obtention of structural data. Recently, we described the expression and purification of E6-C 4C/4S, a stable, folded mutant of the C-terminal domain of HPV16 E6. Here, we have produced 15N-labelled samples of E6-C 4C/4S for structural studies by NMR. We have assigned most 1H and 15N resonances and identified the elements of secondary structure of the domain. The domain displays an original alpha/beta topology with roughly equal proportions of alpha-helix and beta-sheet. The PDZ-binding region of E6, located at the extreme C-terminus of the domain, is in a random conformation. Mass spectrometry demonstrated the presence of one zinc ion per protein molecule. Kinetics of replacement of zinc by cadmium followed by 1H,15N-HSQC experiments revealed specific frequency changes for the zinc-binding cysteines and their immediate neighbours. NMR spectra were affected by severe line-broadening effects which seriously hindered the assignment work. Investigation of these effects by 15N relaxation experiments showed that they are due to heterogeneous dynamic behaviour with mus-ms time scale motions occurring in localised regions of the monomeric domain.