C T Craescu

Université Paris-Sud 11, Orsay, Île-de-France, France

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Publications (113)384.93 Total impact

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    ABSTRACT: Apo-calmodulin, a small soluble mainly α protein is a calcium-dependent protein activator. Calcium binding affects the calmodulin conformation but also its stability. Calcium free form unfolds between 40 and 80°C, whereas the calcium-saturated form is stable up to temperatures as high as 100°C, forbidding comparison of the thermal unfolding pathways of the two forms. Thus, this paper focuses especially on the conformation of pressure-induced unfolding states of both forms of calmodulin, by combining small-angle neutron scattering (SANS) with biophysical techniques such as tyrosines and ANS fluorescence. In contrast to heat denaturation (Gibrat et al, BBA, 2012), the pressure denaturation of calmodulin is reversible up to pressures of 3000bar (300MPa). A pressure-induced compact intermediate state has been found for the two calmodulin forms, but their unfolding pathways are different. A domain compaction and an increase of the ANS fluorescence of holo form has been evidenced. On the contrary, a domain dilatation and an ANS fluorescence decrease has been found for the apo form. The pressure induced an increase of the interdomain distance for both calmodulin forms, suggesting that the central linker of calmodulin is flexible in solution.
    Biochimica et biophysica acta. 05/2014;
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    ABSTRACT: Apo-calmodulin, a small soluble mainly α protein is a calcium-dependent protein activator. Calcium binding affects the calmodulin conformation but also its stability. Calcium free form unfolds between 40 and 80 °C, whereas the calcium-saturated form is stable up to temperatures as high as 100 °C, forbidding comparison of the thermal unfolding pathways of the two forms. Thus, this paper focuses especially on the conformation of pressure-induced unfolding states of both forms of calmodulin, by combining small-angle neutron scattering (SANS) with biophysical techniques such as tyrosines and ANS fluorescence. In contrast to heat denaturation (Gibrat et al, BBA, 2012), the pressure denaturation of calmodulin is reversible up to pressures of 3000 bar (300 MPa). A pressure-induced compact intermediate state has been found for the two calmodulin forms, but their unfolding pathways are different. A domain compaction and an increase of the ANS fluorescence of holo form has been evidenced. On the contrary, a domain dilatation and an ANS fluorescence decrease has been found for the apo form. The pressure induced an increase of the interdomain distance for both calmodulin forms, suggesting that the central linker of calmodulin is flexible in solution.
    Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics 01/2014; · 3.73 Impact Factor
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    ABSTRACT: Apo-calmodulin, a small, mainly α, soluble protein is a calcium-dependent protein activator. It is made of two N- and C-terminal domains having a sequence homology of 70%, an identical folding but different stabilities, and is thus an interesting system for unfolding studies. The use of small angle neutron scattering (SANS) and other biophysical techniques has permitted to reveal conformational difference between native and thermal denatured states of apo-calmodulin. The results show that secondary and tertiary structures of apo-calmodulin evolve in a synchronous way, indicating the absence in the unfolding pathway of molten-globule state sufficiently stable to affect transition curves. From SANS experiments, at 85 °C, apo-calmodulin adopts a polymer chain conformation with some residual local structures. After cooling down, apo-calmodulin recovers a compact state, with a secondary structure close to the native one but with a higher radius of gyration and a different tyrosine environment. In fact on a timescale of few minutes, heat denaturation of apo-calmodulin is partially reversible, but on a time scale of hours (for SANS experiments), the long exposure to heat may lead to a non-reversibility due to some chemical perturbation of the protein. In fact, from Mass Spectrometry measurements, we got evidence of dehydration and deamidation of heated apo-calmodulin.
    Biochimica et Biophysica Acta 06/2012; 1824(10):1097-106. · 4.66 Impact Factor
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    ABSTRACT: We have examined the influence of centrin 2 (Cen2) on the interaction of nucleotide excision repair factors (XPC-HR23b, RPA, and XPA) with 48-mer DNA duplexes bearing the dUMP derivative 5-{3-[6-(carboxyamidofluoresceinyl)amidocapromoyl]allyl}-2'-deoxyuridine-5'-monophosphate. The fluorescein residue linked to the nucleotide base imitates a bulky lesion of DNA. Cen2 stimulated the binding and increased the yield of DNA adducts with XPC-HR23b, a protein recognizing bulky damages in DNA. Stimulation of the binding was most pronounced in the presence of Mg(2+) and demonstrated a bell-shaped dependence on Cen2 concentration. The addition of Cen2 changed the stoichiometry of RPA-DNA complexes and diminished the yield of RPA-DNA covalent crosslinks. We have shown that Cen2 influences the binding of RPA and XPA with DNA, which results in formation of additional DNA-protein complexes possibly including Cen2. We have also found some evidence of direct contacts between Cen2 and DNA. These results in concert with the literature data suggest that Cen2 can be a regulatory element in the nucleotide excision repair system.
    Biochemistry (Moscow) 04/2012; 77(4):346-53. · 1.15 Impact Factor
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    ABSTRACT: Lamins A and C are nuclear intermediate filament proteins expressed in most differentiated somatic cells. Previous data suggested that prelamin A, the lamin A precursor, accumulates in some lipodystrophy syndromes caused by mutations in the lamin A/C gene, and binds and inactivates the sterol regulatory element binding protein 1 (SREBP1). Here we show that, in vitro, the tail regions of prelamin A, lamin A and lamin C bind a polypeptide of SREBP1. Such interactions also occur in HeLa cells, since expression of lamin tail regions impedes nucleolar accumulation of the SREBP1 polypeptide fused to a nucleolar localization signal sequence. In addition, the tail regions of A-type lamin variants that occur in Dunnigan-type familial partial lipodystrophy of (R482W) and Hutchison Gilford progeria syndrome (∆607-656) bind to the SREBP1 polypeptide in vitro, and the corresponding FLAG-tagged full-length lamin variants co-immunoprecipitate the SREBP1 polypeptide in cells. Overexpression of wild-type A-type lamins and variants favors SREBP1 polypeptide localization at the intranuclear periphery, suggesting its sequestration. Our data support the hypothesis that variation of A-type lamin protein level and spatial organization, in particular due to disease-linked mutations, influences the sequestration of SREBP1 at the nuclear envelope and thus contributes to the regulation of SREBP1 function.
    Experimental Cell Research 12/2011; 317(20):2800-13. · 3.56 Impact Factor
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    ABSTRACT: Cdc31, the Saccharomyces cerevisiae centrin, is an EF-hand calcium-binding protein essential for the cell division and mRNA nuclear export. We used biophysical techniques to investigate its calcium, magnesium, and protein target binding properties as well as their conformations in solution. We show here that Cdc31 displays one Ca(2+)/Mg(2+) mixed site in the N-terminal domain and two low-affinity Ca(2+) sites in the C-terminal domain. The affinity of Cdc31 for different natural target peptides (from Kar1, Sfi1, Sac3) that we obtained by isothermal titration calorimetry shows weakly Ca(2+), but also Mg(2+) dependence. The characteristics of target surface binding were shown to be similar; we highlight that the 1-4 hydrophobic amino acid motif, in a stable amphipathic α-helix, is critical for binding. Ca(2+) and Mg(2+) binding increase the α-helix content and stabilize the structure. Analysis of small-angle X-ray scattering experiments revealed that N- and C-terminal domains are not individualized in apo-Cdc31; in contrast, they are separated in the Mg(2+) state, creating a groove in the middle of the molecule that is occupied by the target peptide in the liganded form. Consequently, Mg(2+) seems to have consequences on Cdc31's function and could be important to stimulate interactions in resting cells.
    Biochemistry 06/2011; 50(29):6409-22. · 3.38 Impact Factor
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    ABSTRACT: Several genes in human cells are activated by physical genotoxic agents in order to regenerate cell homeostasis. Among the pathways contributing to this response, nucleotide excision repair (NER) is unique in restoring the nucleotide sequence of the DNA molecule without generating mutations. The first step of NER is mediated by a protein complex composed of XPC, RAD23B, an ubiquitin receptor and CENTRIN 2, an EF-hand calcium binding protein. These three proteins are multifunctional and participate in other important biochemical pathways. We silenced the XPC, RAD23A or RAD23B genes in HeLa cells for a long period of time by using Epstein Barr Virus-derived plasmids carrying sequences coding for small interfering RNA. XPC silencing confirms an essential role for XPC in DNA repair and cell survival after ultraviolet light irradiation. RAD23A and RAD23B participate in DNA repair and cell survival with diverging functions. Our data also indicate that CENTRIN 2 is recruited onto nuclear damaged areas quickly after irradiation and that XPC plays an important role during its internalization into the nucleus of human cells. Furthermore, the inhibition of XPC expression correlates with a decreased amount of CENTRIN 2 transcript and protein, indicating that XPC is required for the fine tuning of CENTRIN 2 gene expression. Moreover, XPC-silenced cells present a reduced concentration of CENTRIN 2 that affects both its centrosomal and nuclear localization suggesting that XPC deficiency may indirectly slow down cell division.
    DNA repair 06/2011; 10(8):835-47. · 4.20 Impact Factor
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    ABSTRACT: Disrupting protein-protein interactions by small organic molecules is nowadays a promising strategy employed to block protein targets involved in different pathologies. However, structural changes occurring at the binding interfaces make difficult drug discovery processes using structure-based drug design/virtual screening approaches. Here we focused on two homologous calcium binding proteins, calmodulin and human centrin 2, involved in different cellular functions via protein-protein interactions, and known to undergo important conformational changes upon ligand binding. In order to find suitable protein conformations of calmodulin and centrin for further structure-based drug design/virtual screening, we performed in silico structural/energetic analysis and molecular docking of terphenyl (a mimicking alpha-helical molecule known to inhibit protein-protein interactions of calmodulin) into X-ray and NMR ensembles of calmodulin and centrin. We employed several scoring methods in order to find the best protein conformations. Our results show that docking on NMR structures of calmodulin and centrin can be very helpful to take into account conformational changes occurring at protein-protein interfaces. NMR structures of protein-protein complexes nowadays available could efficiently be exploited for further structure-based drug design/virtual screening processes employed to design small molecule inhibitors of protein-protein interactions.
    BMC Structural Biology 01/2011; 11:24. · 2.10 Impact Factor
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    ABSTRACT: Centrins are calcium binding proteins that belong to the EF-hand (or calmodulin) superfamily, which are highly conserved among eukaryotes. Herein, we report the molecular features and binding properties of the green alga Scherffelia dubia centrin (SdCen), a member of the Chlamydomonas reinhardtii centrin (CrCen) subfamily. The Ca(2+) binding capacity of SdCen and its isolated N- and C-terminal domains (N-SdCen and C-SdCen, respectively) was investigated using flow dialysis and isothermal titration calorimetry. In contrast with human centrin 1 and 2 (from the same subfamily), but like CrCen, SdCen exhibits three physiologically significant Ca(2+) binding sites, two in the N-terminal domain and one in the C-terminal domain. Mg(2+) ions could compete with Ca(2+) in one of the N-terminal sites. When Ca(2+) binds, the N-terminal domain becomes more stable and exposes a significant hydrophobic surface that binds hydrophobic fluorescent probes. The Ca(2+) binding properties and the metal ion-induced structural changes in the C-terminal domain are comparable to those of human centrins. We used isothermal titration calorimetry to quantify the binding of SdCen, N-SdCen, and C-SdCen to three types of natural target peptides, derived from the human XPC protein (P17-XPC), the human Sfi1 protein (R17-hSfi1), and the yeast Kar1 protein (P19-Kar1). The three peptides possess the complete (P17-XPC and R17-hSfi1) or partial (P19-Kar1) centrin binding motif (W(1)L(4)L(8)). The integral SdCen exhibits two binding sites for each target peptide, with distinct affinities for each site and each peptide. The high-affinity peptide binding site corresponds to the C-terminal domain of SdCen and displays binding constants and the poor Ca(2+) sensitivities similar to those observed for human centrins. The low-affinity site constituted by the N-terminal domain is active only in the presence of Ca(2+). The thermodynamic binding parameters suggest that the C-terminal domain of SdCen may be constitutively bound to a target, while the N-terminal domain could bind a target only after a Ca(2+) signal. SdCen is also able to interact with calmodulin binding peptides (W(1)F(5)V(8)F(14) motif) with a 1:1 stoichiometry, whereas the isolated N- and C-terminal domains have a much lower affinity. These data suggest particular molecular mechanisms used by SdCen (and probably by other algal centrins) to respond to cellular Ca(2+) signals.
    Biochemistry 05/2010; 49(20):4383-94. · 3.38 Impact Factor
  • Fabiana Tirone, Laura Radu, Constantin T Craescu, Jos A Cox
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    ABSTRACT: NADPH oxidases (NOX) are important superoxide producing enzymes that regulate a variety of physiological and pathological processes such as bacteria killing, angiogenesis, sperm-oocyte fusion, and oxygen sensing. NOX5 is a member of the NOX family but distinct from the others by the fact that it contains a long N-terminus with four EF-hand Ca(2+)-binding sites (NOX5-EF). NOX5 generates superoxide in response to intracellular Ca(2+) elevation in vivo and in a cell-free system. Previously, we have shown that the regulatory N-terminal EF-hand domain interacts directly and in a Ca(2+)-dependent manner with the catalytic C-terminal catalytic dehydrogenase domain (CDHD) of the enzyme, leading to its activation. Here we have characterized the interaction site for the regulatory NOX5-EF in the catalytic CDHD of NOX5 using cloned fragments and synthetic peptides of the CDHD. The interaction was monitored with pull-down techniques, cross-linking experiments, tryptophan fluorescence, hydrophobic exposure, isothermal titration calorimetry, and cell-free system enzymatic assays. This site is composed of two short segments: the 637-660 segment, referred to as the regulatory EF-hand-binding domain (REFBD), and the 489-505 segment, previously identified as the phosphorylation region (PhosR). NOX5-EF binds to these two segments in a Ca(2+)-dependent way, and the superoxide generation by NOX5 depends on this interaction. Controlled proteolysis suggests that the REFBD is autoinhibitory and inhibition is relieved by NOX5-EF.
    Biochemistry 12/2009; 49(4):761-71. · 3.38 Impact Factor
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    ABSTRACT: Polyoxometalates (POMs) show promising antibacterial, antiviral (particularly anti-HIV), antitumor, and anticancer activities, but the mechanism of these potential therapeutic effects remains to be elucidated at the molecular level. The interaction between the Gd-containing tungstosilicate [Gd(β2-SiW11O39)2]13– and human serum albumin (HSA) was studied by several techniques. Fluorescence spectroscopy showed an energy transfer between the single tryptophan residue of HSA and the POM. Circular dichroism led to the conclusion that the POM significantly altered the secondary structure of HSA. Isothermal titration calorimetry revealed an enthalpy-driven binding reaction between HSA and the POM, resulting in the formation of a 1:1 complex.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)
    Berichte der deutschen chemischen Gesellschaft 10/2009; 2009(34):5189 - 5193. · 2.94 Impact Factor
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    ABSTRACT: Centrin, an EF-hand calcium-binding protein, has been shown to be involved in the duplication of centrosomes, and Sfi1 (Suppressor of fermentation-induced loss of stress resistance protein 1) is one of its centrosomal targets. There are three isoforms of human centrin, but here we only considered centrin 2 (HsCen2). This protein has the ability to bind to any of the approximately 25 repeats of human Sfi1 (hSfi1) with more or less affinity. In this study, we mainly focused on the 17th repeat (R17-hSfi1-20), which presents the highest level of similarity with a well-studied 17-residue peptide (P17-XPC) from human xeroderma pigmentosum complementation group C protein, another centrin target for DNA repair. The only known structure of HsCen2 was resolved in complex with P17-XPC. The 20-residue peptide R17-hSfi1-20 exhibits the motif L8L4W1, which is the reverse of the XPC motif, W1L4L8. Consequently, the dipole of the helix formed by this motif has a reverse orientation. We wished to ascertain the impact of this reversal on the structure, dynamics and affinity of centrin. To address this question, we determined the structure of C-HsCen2 [the C-terminal domain of HsCen2 (T94-Y172)] in complex with R17-hSfi1-20 and monitored its dynamics by NMR, after having verified that the N-terminal domain of HsCen2 does not interact with the peptide. The structure shows that the binding mode is similar to that of P17-XPC. However, we observed a 2 -A translation of the R17-hSfi1-20 helix along its axis, inducing less anchorage in the protein and the disruption of a hydrogen bond between a tryptophan residue in the peptide and a well-conserved nearby glutamate in C-HsCen2. NMR dynamic studies of the complex strongly suggested the existence of an unusual calcium secondary binding mode in calcium-binding loop III, made possible by the uncommon residue composition of this loop. The secondary metal site is only populated at high calcium concentration and depends on the type of bound ligand.
    Journal of Molecular Biology 10/2009; 395(1):191-204. · 3.91 Impact Factor
  • Liliane Mouawad, Adriana Isvoran, Eric Quiniou, Constantin T Craescu
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    ABSTRACT: The EF-hand calcium-binding proteins may exist either in an extended or a compact conformation. This conformation is sometimes correlated with the function of the calcium-binding protein. For those proteins whose structure and function are known, calcium sensors are usually extended and calcium buffers compact; hence, there is interest in predicting the form of the protein starting from its sequence. In the present study, we used two different procedures: one that already exists in the literature, the sosuidumbbell algorithm, mainly based on the charges of the two EF-hand domains, and the other comprising a novel procedure that is based on linker average hydrophilicity. The linker consists of the residues that connect the domains. The two procedures were tested on 17 known-structure calcium-binding proteins and then applied to 59 unknown-structure centrins. The sosuidumbbell algorithm yielded the correct conformations for only 15 of the known-structure proteins and predicted that all centrins should be in a closed form. The linker average hydrophilicity procedure discriminated well between all the extended and non-extended forms of the known-structure calcium-binding proteins, and its prediction concerning centrins reflected well their phylogenetic classification. The linker average hydrophilicity criterion is a simple and powerful means to discriminate between extended and non-extended forms of calcium-binding proteins. What is remarkable is that only a few residues that constitute the linker (between 2 and 20 in our tested sample of proteins) are responsible for the form of the calcium-binding protein, showing that this form is mainly governed by short-range interactions.
    FEBS Journal 02/2009; 276(4):1082-93. · 4.25 Impact Factor
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    ABSTRACT: The activity of ERK2, an essential component of MAP-kinase pathway, is under the strict control of various effector proteins. Despite numerous efforts, no crystal structure of ERK2 complexed with such partners has been obtained so far. PTP-SL is a major regulator of ERK2 activity. To investigate the ERK2-PTP-SL complex we used a combined method based on cross-linking, MALDI-TOF analysis, isothermal titration calorimetry, molecular modeling and docking. Hence, new insights into the stoichiometry, thermodynamics and interacting regions of the complex are obtained and a structural model of ERK2-PTP-SL complex in a state consistent with PTP-SL phosphatase activity is developed incorporating all the experimental constraints available at hand to date. According to this model, part of the N-terminal region of PTP-SL has propensity for intrinsic disorder and becomes structured within the complex with ERK2. The proposed model accounts for the structural basis of several experimental findings such as the complex-dissociating effect of ATP, or PTP-SL blocking effect on the ERK2 export to the nucleus. A general observation emerging from this model is that regions involved in substrate binding in PTP-SL and ERK2, respectively are interacting within the interface of the complex.
    PLoS ONE 02/2009; 4(5):e5432. · 3.53 Impact Factor
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    L. Pitulice, A. Isvoran, C. T. Craescu, A. Chiriac
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    ABSTRACT: In this paper, we analyze the scaling properties of both the radius of gyration and the surface area for EF-hand calcium binding proteins. These properties are different for two conformational subfamilies: proteins with extended and compact structures, respectively. The radius of gyration is a measure of the shape of protein, whereas its surface fractal dimension is a measure of its interatomic packing. Different scaling properties for the radius of gyration underline that these two subfamilies present different shapes whilst different scaling properties for the surface area reveal different strengths of their intermolecular forces. All these data suggest different mechanisms responsible for the global folding of proteins belonging to these two subfamilies.
    Chaos Solitons & Fractals 01/2009; 40(2):684-690. · 1.25 Impact Factor
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    ABSTRACT: Binding human serum albumin (HSA) of three polyoxometalates (POMs) with the Wells-Dawson structure, alpha(2)-[P2W17O61]10- (abbreviated as alpha(2)-P2W17) and two of its metal-substituted derivatives, alpha(2)-[NiP2W17O61]8- and alpha(2)-[CuP2W17O61]8- (alpha(2)-P2W17Ni and alpha(2)-P2W17Cu, respectively) was studied in an aqueous medium at pH 7.5. Fluorescence quenching, circular dichroism (CD), thermal denaturation, and isothermal titration calorimetry (ITC) were used for this purpose. The results were compared with those obtained previously with the Keggin structure POM, [H2W12O40]6- (H2W12), and the wheel-shaped structure, [NaP5W30O110]14- (P5W30). All these POMs bind HSA mainly by electrostatic interactions. Comparison of the physical characteristics and HSA interaction parameters for the POMs of the present work and those studied previously showed that the overall charge of the clusters is not the single parameter governing the binding process and its consequences. In contrast, besides the influences of the structure, the dimension and/or weight of the POMs, the results have permitted highlighting of the importance of each POM atomic composition for its binding behavior.
    Biomacromolecules 04/2008; 9(3):812-7. · 5.37 Impact Factor
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    ABSTRACT: Xeroderma pigmentousum group C protein (XPC) is involved in the first step of nucleotide excision repair, with multiple functional roles including DNA damage recognition and recruitment of the repair machinery. This human protein of 940 residues forms a strong heterotrimeric complex with Rad23B and centrin 2. The structure of XPC is actually not known, and lack of significant sequence homology with proteins from structural data bases precludes any relevant prediction. Here, we present the molecular and structural characterization of a C-terminal fragment of XPC (C-XPC: 126 residues, 815-940), which was shown to be involved in centrin 2 and TFIIH binding. C-XPC may be highly expressed in E. coli, but because of its limited solubility it was purified under 6 M urea. Using bioinformatics tools, and a combination of several experimental methods (circular dichroism, fluorescence, nuclear magnetic resonance, and small-angle X-ray scattering), we show that C-XPC has a highly flexible structure under native physiological conditions, with a propensity to form helical secondary structures. Isothermal titration calorimetry experiments show that the C-XPC fragment binds human centrin 2 with high affinity and a 1:1 stoichiometry. NMR analysis indicates that the physical interaction between C-XPC and centrin 2 induces only minor conformational changes into XPC, localized around the 17-mer segment (847-863), showed to be critically involved in the centrin binding.
    Biochemistry 03/2008; 47(5):1403-13. · 3.38 Impact Factor
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    ABSTRACT: Apo-calmodulin, a small, mainly alpha, soluble protein is a calcium-dependent protein activator. This article presents a study of internal dynamics of native and thermal unfolded apo-calmodulin, using quasi-elastic neutron scattering. This technique can probe protein internal dynamics in the picosecond timescale and in the nanometer length-scale. It appears that a dynamical transition is associated with thermal denaturation of apo-calmodulin. This dynamical transition goes together with a decrease of the confinement of hydrogen atoms, a decrease of immobile protons proportion and an increase of dynamical heterogeneity. The comparison of native and unfolded states dynamics suggests that the dynamics of protein atoms is more influenced by their distance to the backbone than by their solvent exposure.
    Biophysical Journal 02/2008; 95(11):5247-56. · 3.67 Impact Factor
  • Adriana Isvoran, Laura Pitulice, Constantin T. Craescu, Adrian Chiriac
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    ABSTRACT: The structures of EF-hand calcium binding proteins may be classified into two distinct groups: extended and compact structures. In this paper we studied 20 different structures of calcium binding proteins using the fractal analysis. Nine structures show extended shapes, one is semi-compact and the other 10 have compact shapes. Our study reveals different fractal characteristics for protein backbones belonging to different structural classes and these observations may be correlated to the physicochemical forces governing the protein folding.
    Chaos Solitons & Fractals 01/2008; 35(5):960-966. · 1.25 Impact Factor
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    ABSTRACT: Human centrin 2 (HsCen2), an EF-hand calcium binding protein, plays a regulatory role in the DNA damage recognition during the first steps of the nucleotide excision repair. This biological action is mediated by the binding to a short fragment (N847-R863) from the C-terminal region of xeroderma pigmentosum group C (XPC) protein. This work presents a detailed structural and energetic characterization of the HsCen2/XPC interaction. Using a truncated form of HsCen2 we obtained a high resolution (1.8 A) X-ray structure of the complex with the peptide N847-R863 from XPC. Structural and thermodynamic analysis of the interface revealed the existence of both electrostatic and apolar inter-molecular interactions, but the binding energy is mainly determined by the burial of apolar bulky side-chains into the hydrophobic pocket of the HsCen2 C-terminal domain. Binding studies with various peptide variants showed that XPC residues W848 and L851 constitute the critical anchoring side-chains. This enabled us to define a minimal centrin binding peptide variant of five residues, which accounts for about 75% of the total free energy of interaction between the two proteins. Immunofluorescence imaging in HeLa cells demonstrated that HsCen2 binding to the integral XPC protein may be observed in living cells, and is determined by the same interface residues identified in the X-ray structure of the complex. Overexpression of XPC perturbs the cellular distribution of HsCen2, by inducing a translocation of centrin molecules from the cytoplasm to the nucleus. The present data confirm that the in vitro structural features of the centrin/XPC peptide complex are highly relevant to the cellular context.
    Journal of Molecular Biology 12/2007; 373(4):1032-46. · 3.91 Impact Factor

Publication Stats

925 Citations
384.93 Total Impact Points

Institutions

  • 2009–2011
    • Université Paris-Sud 11
      Orsay, Île-de-France, France
    • Christian-Albrechts-Universität zu Kiel
      • Zoological Institute and Museum
      Kiel, Schleswig-Holstein, Germany
  • 2008
    • French National Centre for Scientific Research
      Lutetia Parisorum, Île-de-France, France
  • 2007
    • Atomic Energy and Alternative Energies Commission
      • Laboratoire de Biologie Structurale et Radiobiologie (LBSR)
      Gif-sur-Yvette, Ile-de-France, France
  • 1998–2006
    • Institut Curie
      Lutetia Parisorum, Île-de-France, France
  • 2005
    • University of Geneva
      • Department of Biochemistry
      Genève, GE, Switzerland
  • 1989–2005
    • French Institute of Health and Medical Research
      Lutetia Parisorum, Île-de-France, France
  • 1999
    • Institut Universitaire de France
      Lutetia Parisorum, Île-de-France, France
  • 1989–1991
    • Hôpital Henri Mondor (Hôpitaux Universitaires Henri Mondor)
      Créteil, Île-de-France, France
  • 1985
    • Unité Inserm U1077
      Caen, Lower Normandy, France