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The folding of staphylococcal nuclease (SNase) is known to proceed via a major intermediate in which the central OB subdomain is folded and the C-terminal helical subdomain is disordered. To identify the structural and energetic determinants of this folding free energy landscape, we have examined in detail, using high-pressure NMR, the consequences of cavity creating mutations in each of the two subdomains of an ultrastable SNase, Δ+PHS. The stabilizing mutations of Δ+PHS enhanced the population of the major folding intermediate. Cavity creation in two different regions of the Δ+PHS reference protein, despite equivalent effects on global stability, had very distinct consequences on the complexity of the folding free energy landscape. The L125A substitution in the C-terminal helix of Δ+PHS slightly suppressed the major intermediate and promoted an additional excited state involving disorder in the N-terminus, but otherwise decreased landscape heterogeneity with respect to the Δ+PHS background protein. The I92A substitution, located in the hydrophobic OB-fold core, had a much more profound effect, resulting in a significant increase in the number of intermediate states and implicating the entire protein structure. Denaturant (GuHCl) had very subtle and specific effects on the landscape, suppressing some states and favoring others, depending upon the mutational context. These results demonstrate that disrupting interactions in a region of the protein with highly cooperative, unfrustrated folding has very profound effects on the roughness of the folding landscape, whereas the effects are less pronounced for an energetically equivalent substitution in an already frustrated region.

Provided that care is taken in adjusting the WATERGATE element of a (1)H-(15)N TROSY-HSQC experiment, such that neither the water magnetization nor the (1)H(α) protons are inverted by its final 180° pulse, (3)JHNHα couplings can be measured directly from splittings in the (1)H dimension of the spectrum. With band-selective (1)H decoupling, very high (15)N resolution can be achieved. A complete set of (3)JHNHα values, ranging from 3.4 to 10.1 Hz was measured for the 56-residue third domain of IgG-binding protein G (GB3). Using the H-N-C(α)-H(α) dihedral angles extracted from a RDC-refined structure of GB3, (3)JHNHα values predicted by a previously parameterized Karplus equation agree to within a root-mean-square deviation (rmsd) of 0.37 Hz with the experimental data. Values measured for the Alzheimer's implicated Aβ(1-40) peptide fit to within an rmsd of 0.45 Hz to random coil (3)JHNHα values.

Lilliad eclaire lors de son inauguration le 17 novembre 2016 © Atmosphere Photo Lilliad a pour noyau l’ancienne bibliotheque de l’Universite des sciences et technologies de Lille (USTL). Au sein du campus de la Cite scientifique, le bâtiment a des l’origine constitue le symbole de l’universite, son cœur. Bâtiment de type « Pailleron », rond, ouvert et sans murs porteurs, mais reposant sur une foret de piliers metalliques, la bibliotheque universitaire se composait de trois niveaux – sous-sol...

Inquisition registers which are the main available sources for the knowledge of heresy in the South of France are particularly poor concerning Catharism at Caunes-Minervois. The only somewhat detailed mentions concern the last third of the XIIIth century. Other sources allow to rectify this deceptive impression. The royal inquiries concerning faidiments in Languedoc and certain documents copied in the Doat collection of the National French Library give valuable information as to the place of heresy at Caunes and in its region. Caunes played a particular role in the fight against heresy. By 1226, the Cathar Bishop of Carcassés Pierre Isarn is burnt there. Ten years later, inquisitor Ferrier is installed at Caunes, in the abbey probably, a place from which he launches the inquiries of the court against people suspected of heresy. At the end of the century, the heresy at Caunes will again be brought to light through the supposed links of abbot Pierre de Pelages with Catharism., FRE: Catharisme, Hérésie, Inquisition, Pierre Isarn, Ferrier, Caunes-Minervois., Roche Julien. Caunes-Minervois et l’hérésie cathare. In: Archéologie du Midi médiéval n°6, 2010. L’abbaye et le village de Caunes-Minervois (Aude). Archéologie et Histoire. pp. 105-113.

We investigate the pressure-induced structural changes in the mature human immunodeficiency virus type 1 protease dimer (HIV-1 PR), using residual dipolar coupling (RDC) measurements in a weakly oriented solution. 1DNH RDCs were measured under high-pressure conditions for an inhibitor-free PR and an inhibitor-bound complex, as well as for an inhibitor-free multidrug resistant protease bearing 20 mutations (PR20). While PR20 and the inhibitor-bound PR were little affected by pressure, inhibitor-free PR showed significant differences in the RDCs measured at 600 bar compared to 1 bar. The structural basis of such changes was investigated by MD simulations using the experimental RDC restraints, revealing substantial conformational perturbations, specifically a partial opening of the flaps and the penetration of water molecules into the hydrophobic core of the subunits at high-pressure. This study highlights the exquisite sensitivity of RDCs to pressure-induced conformational changes and illustrates how RDCs combined with MD simulations can be used to determine the structural properties of metastable intermediate states on the folding energy landscape.

Defining the physical-chemical determinants of protein folding and stability, under normal and pathological conditions has constituted a major subfield in biophysical chemistry for over 50 years. Although a great deal of progress has been made in recent years towards this goal, a number of important questions remain. These include characterizing the structural, thermodynamic and dynamic properties of the barriers between conformational states on the protein energy landscape, understanding the sequence dependence of folding cooperativity, defining more clearly the role of solvation in controlling protein stability and dynamics and probing the high energy thermodynamic states in the native state basin and their role in misfolding and aggregation. Fundamental to the elucidation of these questions is a complete thermodynamic parameterization of protein folding determinants. In this chapter, we describe the use of high-pressure coupled to Nuclear Magnetic Resonance (NMR) spectroscopy to reveal unprecedented details on the folding energy landscape of proteins.

The way in which the network of intramolecular interactions determines the cooperative folding and conformational dynamics of a protein remains poorly understood. High-pressure NMR spectroscopy is uniquely suited to examine this problem because it combines the site-specific resolution of the NMR experiments with the local character of pressure perturbations. Here we report on the temperature dependence of the site-specific volumetric properties of various forms of staphylococcal nuclease (SNase), including three variants with engineered internal cavities, as measured with high-pressure NMR spectroscopy. The strong temperature dependence of pressure-induced unfolding arises from poorly understood differences in thermal expansion between the folded and unfolded states. A significant inverse correlation was observed between the global thermal expansion of the folded proteins and the number of strong intramolecular hydrogen bonds, as determined by the temperature coefficient of the backbone amide chemical shifts. Comparison of the identity of these strong H-bonds with the co-evolution of pairs of residues in the SNase protein family suggests that the architecture of the interactions detected in the NMR experiments could be linked to a functional aspect of the protein. Moreover, the temperature dependence of the residue-specific volume changes of unfolding yielded residue-specific differences in expansivity and revealed how mutations impact intramolecular interaction patterns. These results show that intramolecular interactions in the folded states of proteins impose constraints against thermal expansion and that, hence, knowledge of site-specific thermal expansivity offers insight into the patterns of strong intramolecular interactions and other local determinants of protein stability, cooperativity, and potentially also of function.

The flexibility of the glycine-rich flaps are known to be essential for the catalytic activity of the HIV-1 Protease but their exact conformations at the different stages of the enzymatic pathway remain yet to be determined. While hundreds of crystal structures of protease-inhibitor complexes have been solved, only a dozen of unbound protease structures have been reported to date. These structures reveal a large heterogeneity of flap conformations, ranging from closed, to semi-open and wide-open conformations. We used here NMR spectroscopy to determine the flap orientation of the unbound protease in solution. The conformation of the flaps was determined by comparing the measured residual dipolar couplings to reference crystal structures representing the closed, semi-open and wide-open states. The data clearly indicate that the apo protease adopts a closed conformation in solution, similar to an inhibitor-bound state.We also compared the effect of high-pressure on the flap conformations of an apo and inhibitor-bound protease by measuring backbone residual dipolar couplings up to 1 kbar. A rigid-body motion model was derived to characterize the pressure-induced conformational changes of the flaps. While a minimal rearrangement of the flaps was observed in the case of the inhibitor-protease complex, we observed a significant pressure-induced rotation of the flaps in the case of the apo protease. These results highlight the intrinsic conformational flexibility of the unbound protease and offer a new perspective to understand the mechanism of ligand and inhibitor binding.

Defining the physical-chemical determinants of protein folding and stability, under normal and pathological conditions has constituted a major subfield in biophysical chemistry for over 50 years. Although a great deal of progress has been made in recent years towards this goal, a number of important questions remain. These include characterizing the structural, thermodynamic and dynamic properties of the barriers between conformational states on the protein energy landscape, understanding the sequence dependence of folding cooperativity, defining more clearly the role of solvation in controlling protein stability and dynamics and probing the high energy thermodynamic states in the native state basin and their role in misfolding and aggregation. Fundamental to the elucidation of these questions is a complete thermodynamic parameterization of protein folding determinants. In this chapter, we describe the use of high-pressure coupled to Nuclear Magnetic Resonance (NMR) spectroscopy to reveal unprecedented details on the folding energy landscape of proteins.

Flexibility of the glycine-rich flaps is known to be essential for catalytic activity of the HIV-1 protease, but their exact conformations at the different stages of the enzymatic pathway remain subject to much debate. Although hundreds of crystal structures of protease-inhibitor complexes have been solved, only about a dozen inhibitor-free protease structures have been reported. These latter structures reveal a large diversity of flap conformations, ranging from closed to semi-open to wide open. To evaluate the average structure in solution, we measured residual dipolar couplings (RDCs) and compared these to values calculated for crystal structures representative of the closed, semi-open, and wide-open states. The RDC data clearly indicate that the inhibitor-free protease, on average, adopts a closed conformation in solution that is very similar to the inhibitor-bound state. By contrast, a highly drug-resistant protease mutant, PR20, adopts the wide-open flap conformation.

The HIV-1 Protease (PR) mediates its own release and promotes the proteolysis of the Gag-Pol polyprotein into mature structural and functional forms indispensable for viral maturation. Due to its central role in the virus life cycle, the HIV-1 PR has been a prominent target for clinical protease inhibitors for almost 25 years. However the short replication cycle of the virus together with the error-prone reverse transcriptase lead to the rapidly evolving selection of inhibitor-resistant mutations in HIV-1 PR. We use here high-pressure NMR spectroscopy to characterize the cooperativity of unfolding, in the absence and in the presence of a symmetric inhibitor, of the active mature PR, the inactive variant PRD25N and of the clinical multi-drug resistant PR20. We found that binding of the inhibitor drastically decreases the cooperativity of unfolding by trapping the closed flap conformation in a deep free-energy minimum. We show that in order to avoid this conformational trap, PR20 has evolved to exhibit a nearly ideal two-state unfolding transition. Our study highlights the malleability of the HIV-1 PR folding pathways and illustrates how selection for drug resistant mutations can lead to a major remodeling of the free-energy landscape.

The envelope glycoprotein gp41 mediates the process of membrane fusion that enables entry of the HIV-1 virus into the host cell. Strong lipid affinity of the ectodomain suggests that its heptad repeat regions play an active role in destabilizing membranes by directly binding to the lipid bilayers and thereby lowering the free-energy barrier for membrane fusion. In such a model, immediately following the shedding of gp120, the N-heptad and C-heptad helices dissociate and melt into the host cell and viral membranes, respectively, pulling the destabilized membranes into juxtaposition, ready for fusion. Post-fusion, reaching the final 6-helix bundle (6 HB) conformation then involves competition between intermolecular interactions needed for formation of the symmetric 6 HB trimer and the membrane affinity of gp41's ectodomain, including its membrane-proximal regions. Our solution NMR study of the structural and dynamic properties of three constructs containing the ectodomain of gp41 with and without its membrane-proximal regions suggests that these segments do not form inter-helical interactions until the very late steps of the fusion process. Interactions between the polar termini of the heptad regions, which are not associating with the lipid surface, therefore may constitute the main driving force initiating formation of the final post-fusion states. The absence of significant intermolecular ectodomain interactions in the presence of dodecyl phosphocholine highlights the importance of trimerization of gp41's transmembrane helix to prevent complete dissociation of the trimer during the course of fusion.

We previously reported a series of antibodies, in fragment antigen binding domain (Fab) formats, selected from a human non-immune phage library, directed against the internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41. Broadly neutralizing antibodies from that series bind to both the fully exposed N-HR trimer, representing the pre-hairpin intermediate state of gp41, and to partially-exposed N-HR helices within the context of the gp41 six-helix bundle. While the affinities of the Fabs for pre-hairpin intermediate mimetics vary by only 2 to 20-fold between neutralizing and non-neutralizing antibodies, differences in inhibition of viral entry exceed three orders of magnitude. Here we compare the binding of neutralizing (8066) and non-neutralizing (8062) antibodies, differing in only four positions within the CDR-H2 binding loop, in Fab and single chain variable fragment (ScFv) formats, to several pre-hairpin intermediate and six-helix bundle constructs of gp41. Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity. There is a large differential (≥ 150-fold) in binding affinity between neutralizing and non-neutralizing antibodies to the six-helix bundle of gp41 and binding to the six-helix bundle does not involve displacement of the outer C-terminal helices of the bundle. The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs. We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.

The time required to fold proteins usually increases significantly under conditions of high pressure. Taking advantage of this general property of proteins, we combined P-jump experiments with NMR spectroscopy to examine in detail the folding reaction of staphylococcal nuclease (SNase) and of some of its cavity-containing variants. The nearly 100 observables that could be measured simultaneously collectively describe the kinetics of folding as a function of pressure and denaturant concentration with exquisite site-specific resolution. SNase variants with cavities in the central core of the protein exhibit a highly heterogeneous transition-state ensemble (TSE) with a smaller solvent-excluded void volume than the TSE of the parent SNase. This heterogeneous TSE experiences Hammond behavior, becoming more native-like (higher molar volume) with increasing denaturant concentration. In contrast, the TSE of the L125A variant, which has a cavity at the secondary core, is only slightly different from that of the parent SNase. Because pressure acts mainly to eliminate solvent-excluded voids, which are heterogeneously distributed throughout structures, it perturbs the protein more selectively than chemical denaturants, thereby facilitating the characterization of intermediates and the consequences of packing on folding mechanisms. Besides demonstrating how internal cavities can affect the routes and rates of folding of a protein, this study illustrates how the combination of P-jump and NMR spectroscopy can yield detailed mechanistic insight into protein folding reactions with exquisite site-specific temporal information.

The magnitude and sign of the volume change upon protein unfolding are strongly dependent on temperature. This temperature dependence reflects differences in the thermal expansivity of the folded and unfolded states. The factors that determine protein molar expansivities and the large differences in thermal expansivity for proteins of similar molar volume are not well understood. Model compound studies have suggested that a major contribution is made by differences in the molar volume of water molecules as they transfer from the protein surface to the bulk upon heating. The expansion of internal solvent-excluded voids upon heating is another possible contributing factor. Here, the contribution from hydration density to the molar thermal expansivity of a protein was examined by comparing bovine pancreatic trypsin inhibitor and variants with alanine substitutions at or near the protein-water interface. Variants of two of these proteins with an additional mutation that unfolded them under native conditions were also examined. A modest decrease in thermal expansivity was observed in both the folded and unfolded states for the alanine variants compared with the parent protein, revealing that large changes can be made to the external polarity of a protein without causing large ensuing changes in thermal expansivity. This modest effect is not surprising, given the small molar volume of the alanine residue. Contributions of the expansion of the internal void volume were probed by measuring the thermal expansion for cavity-containing variants of a highly stable form of staphylococcal nuclease. Significantly larger (2-3-fold) molar expansivities were found for these cavity-containing proteins relative to the reference protein. Taken together, these results suggest that a key determinant of the thermal expansivities of folded proteins lies in the expansion of internal solvent-excluded voids.

The impact of pressure on the backbone (15) N, (1) H and (13) C chemical shifts in N-terminally acetylated α-synuclein has been evaluated over a pressure range 1-2500 bar. Even while the chemical shifts fall very close to random coil values, as expected for an intrinsically disordered protein, substantial deviations in the pressure dependence of the chemical shifts are seen relative to those in short model peptides. In particular, the nonlinear pressure response of the (1) H(N) chemical shifts, which commonly is associated with the presence of low-lying "excited states", is much larger in α-synuclein than in model peptides. The linear pressure response of (1) H(N) chemical shift, commonly linked to H-bond length change, correlates well with those in short model peptides, and is found to be anticorrelated with its temperature dependence. The pressure dependence of (13) C chemical shifts shows remarkably large variations, even when accounting for residue type, and do not point to a clear shift in population between different regions of the Ramachandran map. However, a nearly universal decrease in (3) JHN-Hα by 0.22 ± 0.05 Hz suggests a slight increase in population of the polyproline II region at 2500 bar. The first six residues of N-terminally acetylated synuclein show a transient of approximately 15% population of α-helix, which slightly diminishes at 2500 bar. The backbone dynamics of the protein is not visibly affected beyond the effect of slight increase in water viscosity at 2500 bar.

Staphylococcal nuclease (SNase) protein is a model protein folding system. We recently reported that the magnitude of internal void volumes primarily determines proteins sensitivity to pressure (1) and that the tridimensional position of cavities can modulate the folding free-energy landscape (2). Here we characterize the effect of temperature on pressure denaturation of these previously studied cavity containing variants of SNase (L125A and I92A) to address the determinants of thermal expansivity using high-pressure NMR. The experiments show that thermal expansivity is sensitive to changes in the degree of internal packing. We corroborate these observations by measuring the coefficient of thermal expansion using pressure perturbation calorimetry. We also analyze the effect of temperature on the activation volume using pressure-jump kinetics NMR. We find that the transition state ensemble presents structurally heterogeneous expansion with significant differences among the mutants tested. We take advantage of the single residue resolution analysis to map the differences of expansivity on the tridimensional structure of proteins tested. We propose that the network of interactions in the protein interior is responsible for the degree of thermal expansion. Assessing this unexplored thermodynamic parameter will provide unique insight into protein behavior in the pressure-Temperature plane.References:1) Roche, J., Caro, J. A., Norberto, D. R., Barthe, P., Roumestand, C., Schlessman, J. L., Garcia, A. E., Garcia-Moreno, E. B., and Royer, C. A. (2012) Cavities determine the pressure unfolding of proteins, PNAS.2) Roche, J., Dellarole, M., Caro, J. A., Guca, E., Norberto, D. R., Yang, Y., Garcia A.E., Roumestand, C, Garcia-Moreno E. B., and Royer, C.A. (2012) Remodeling of the folding free-energy landscape of staphylococcal nuclease by cavity-creating mutations. Biochemistry, in press.

The transitioning of the ectodomain of gp41 from a pre-hairpin to a six-helix bundle conformation is a crucial aspect of virus-cell fusion. To gain insight into the intermediary steps of the fusion process we have studied the pH and dodecyl phosphocholine (DPC) micelle dependent trimer association of gp41 by systematic deletion analysis of an optimized construct termed 17-172 (residues 528 to 683 of Env) that spans the fusion peptide proximal region (FPPR) to the membrane proximal external region (MPER) of gp41, by sedimentation velocity and double electron-electron resonance (DEER) EPR spectroscopy. Trimerization at pH 7 requires the presence of both the FPPR and MPER regions. However, at pH 4, the protein completely dissociates to monomers. DEER measurements reveal a partial fraying of the C-terminal MPER residues in the 17-172 trimer while the other regions, including the FPPR, remain compact. In accordance, truncating nine C-terminal MPER residues (675-683) in the 17-172 construct does not shift the trimer-monomer equilibrium significantly. Thus, in the context of the gp41 ectodomain spanning residues 17-172, trimerization is clearly dependent on FPPR and MPER regions even when the terminal residues of MPER unravel. The antibody Z13e1, which spans both the 2F5 and 4E10 epitopes in MPER, binds to 17-172 with a Kd of 1 ± 0.12 μM. Accordingly, individual antibodies 2F5 and 4E10 also recognize the 17-172 trimer/DPC complex. We propose that binding of the C-terminal residues of MPER to the surface of the DPC micelles models a correct positioning of the trimeric transmembrane domain anchored in the viral membrane.

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It has been known for nearly 100 years that pressure unfolds proteins, yet the physical basis of this effect is not understood. Unfolding by pressure implies that the molar volume of the unfolded state of a protein is smaller than that of the folded state. This decrease in volume has been proposed to arise from differences between the density of bulk water and water associated with the protein, from pressure-dependent changes in the structure of bulk water, from the loss of internal cavities in the folded states of proteins, or from some combination of these three factors. Here, using 10 cavity-containing variants of staphylococcal nuclease, we demonstrate that pressure unfolds proteins primarily as a result of cavities that are present in the folded state and absent in the unfolded one. High-pressure NMR spectroscopy and simulations constrained by the NMR data were used to describe structural and energetic details of the folding landscape of staphylococcal nuclease that are usually inaccessible with existing experimental approaches using harsher denaturants. Besides solving a 100-year-old conundrum concerning the detailed structural origins of pressure unfolding of proteins, these studies illustrate the promise of pressure perturbation as a unique tool for examining the roles of packing, conformational fluctuations, and water penetration as determinants of solution properties of proteins, and for detecting folding intermediates and other structural details of protein-folding landscapes that are invisible to standard experimental approaches.

The application of hydrostatic pressure generally leads to protein unfolding, implying, in accordance with Le Chatelier's principle, that the unfolded state has a smaller molar volume than the folded state. However, the origin of the volume change upon unfolding, ΔV(u), has yet to be determined. We have examined systematically the effects of protein size and sequence on the value of ΔV(u) using as a model system a series of deletion variants of the ankyrin repeat domain of the Notch receptor. The results provide strong evidence in support of the notion that the major contributing factor to pressure effects on proteins is their imperfect internal packing in the folded state. These packing defects appear to be specifically localized in the 3D structure, in contrast to the uniformly distributed effects of temperature and denaturants that depend upon hydration of exposed surface area upon unfolding. Given its local nature, the extent to which pressure globally affects protein structure can inform on the degree of cooperativity and long-range coupling intrinsic to the folded state. We also show that the energetics of the protein's conformations can significantly modulate their volumetric properties, providing further insight into protein stability.

The ionization of internal groups in proteins can trigger conformational change. Despite this being the structural basis of most biological energy transduction, these processes are poorly understood. Small angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy experiments at ambient and high hydrostatic pressure were used to examine how the presence and ionization of Lys-66, buried in the hydrophobic core of a stabilized variant of staphylococcal nuclease, affect conformation and dynamics. NMR spectroscopy at atmospheric pressure showed previously that the neutral Lys-66 affects slow conformational fluctuations globally, whereas the effects of the charged form are localized to the region immediately surrounding position 66. Ab initio models from SAXS data suggest that when Lys-66 is charged the protein expands, which is consistent with results from NMR spectroscopy. The application of moderate pressure (

This paper presents a salient analog phase-locked loop that adaptively controls the loop bandwidth according to the locking status. An extended loop bandwidth enhancement is achieved by the adaptive control on the charge pump current. First of all, when the phase error is large, such as in the locking mode, the PLL increases the loop bandwidth and achieves fast locking. On the other hand, when the phase error is small, this PLL decreases the loop bandwidth and minimizes output jitters. The relationships of performance aspects to design variables are presented and the basic tradeoffs of the new concept are discussed. A circuit implementation of the adaptive PLL is described in detail and simulation result of a 50 MHz PLL in a 0.15 mum CMOS technology is presented.

A new adaptation scheme for low noise and fast settling 50 MHz analog phase-locked loop (PLL) is presented. According to the locking status, an extended loop bandwidth enhancement is achieved by the adaptive control on the charge pump current . First of all, when the phase error is large, such as in the locking mode, the PLL increases the loop bandwidth and achieves fast locking. On the other hand, when the phase error is small, this PLL decreases the loop bandwidth and minimizes output jitters. The relationships of performance aspects to design variables are presented and the basic tradeoffs of the new concept are discussed. A circuit implementation of the adaptive PLL is described in detail and simulation result of a 50 MHz PLL in a 0:15 mum CMOS technology is presented.

La cartographie des densites spectrales a differentes valeurs d'induction magnetique est certainement la meilleure methode pour decrire le comportement dynamique d'une proteine en solution, a travers l'analyse des parametres de relaxation heteronucleaire 15 N. Cependant, de telles analyses ne sont que rarement reportees dans la litterature, probablement parce que cette methode est excessivement consommatrice de temps machine. En effet, si l'on utilise n valeurs differentes de champs magnetiques, l'echantillonnage discret de la fonction de densite spectrale par 2n + 1 points demande la mesure de 3n parametres de relaxation (n R 1 , n R 2 et n 15 N{ 1 H}NOEs), dans l'hypothese de validite de l'approximation des hautes frequences. Nous avons propose une nouvelle strategie qui permet de decrire la fonction densite spectrale par n + 2 points en ne mesurant qu'un total de n +2 parametres de relaxation heteronucleaire, moyennant quelques approximations dont nous avons verifie la validite par simulation. Appliquee a l'analyse de la proteine C12A-pB MTCP1 a cinq inductions differentes de champ magnetique, cette approche permet de diviser au minimum par deux le temps de mesure experimentale, sans alterer l'interpretation dynamique des fonctions de densite spectrale par le modele de Lipari-Szabo.

A design of a 3.3 Volts BiCMOS buffer with current consumption and linearity control is presented. This buffer with a low output impedance is fully differential and designed to be used with RF (radio frequency) mixer in low IF (intermediate frequency) architecture. The proposed buffer is directly connected to the output of the RF mixer and designed to drive off- chip low impedance load as SAW (surface acoustic wave) or discrete differential filters. A good flexibility is obtained about the control of the linearity performance with the possibility to adjust the power consumption. The linearity and current consumption are independently adjustable and an optimum of the 3rd order linearity performance is achieved for each current consumption value. A schematic of the differential Bi-CMOS 3.3 volts buffer is proposed and some linearity simulation results are presented and discussed in terms of output IM3 level and current consumption. The results show a saving of half power in comparison with a buffer with no linearity control for the same output IM3 level.

The feasibility of low noise sensitivity 2.4-GHz Phase Locked Loop for use in wireless communications as well as in clock generation circuit is demonstrated. The system uses low- gain-multi-band Voltage Controlled Oscillator which achieves a phase noise of -98 dBc/Hz @ 1MHz offset while a lock time of 150 - mus has been obtained from the PLL loop. The design has been implemented on standard CMOS technology.

This paper presents an analog phase-locked loop (PLL) that adaptively controls the loop bandwidth according to the locking status. When the phase error is large, such as in the locking mode, the PLL increases the loop bandwidth and achieves fast locking. On the other hand, when the phase error is small, this PLL decreases the loop bandwidth and minimizes output jitters. The relationships of performance aspects (settling time, phase noise, and spurious signals) to design variables (loop bandwidth, phase margin, and loop filter attenuation at the reference frequency) are presented and the basic tradeoffs of the new concept are discussed. A circuit implementation of the adaptive PLL is described in detail and a prototype 50 MHz PLL in a 0.18 - mum CMOS technology is tested.

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Since Bridgman's seminal experiments on high-pressure denaturation of albumen in 19141, the origin of pressure unfolding of proteins has remained unresolved. We report here a systematic study of the contribution of cavities to the volume difference between unfolded and folded states (ΔVu), using 10 single point variants of staphylococcus nuclease (SNase). Each mutation is localised in a strategic position and was designed to change a large buried hydrophobic side chain into alanine, thus opening tunable cavities in the SNase structure. For every variant, a crystal structure confirmed the presence of the designed cavity with no detectable presence of water molecules. High-pressure fluorescence experiments show significantly larger ΔVu values for the cavity mutants in comparison to the reference protein. This demonstrates that solvent-excluded cavities make a major contribution to ΔVu. Thus, pressure effects have their origin in a property of the folded states of proteins, unlike temperature and chemical denaturants, whose effects are governed by exposed surface area in the unfolded state. High-pressure NMR experiments on 4 cavity mutants, recording HSQCs peak intensities up to 2500 bar, allowed precise estimations of the apparent ΔVu monitored by more than two-thirds of the residues. An innovative combination of the site-specific NMR data and Go-model simulations revealed significant departures from the apparent two-state folding process for the SNase reference protein and the cavity mutants. This study opens up highly promising perspectives on the use of high pressure for characterization of folding landscapes inaccessible by other methods.1. Bridgman, P. W. J. Biol. Chem. 1914.19, 511–512.

Pressure unfolds many proteins. In contrast to acid, heat and chemical denaturation, which have been studied with many proteins and which are relatively well understood, the molecular determinants of pressure unfolding of proteins are not known. Volume is the conjugate variable of pressure; therefore, the pressure unfolding of proteins is governed by differences in volume between the native and the unfolded ensembles. What is less obvious is where the differences in volume originate. Among the possibilities that have been considered are volume changes related to conformational fluctuations, volume changes related to hydration, and volume changes related to the loss of cavities present in the folded state. We have examined the role of cavities as determinants of pressure unfolding systematically, using staphylococcal nuclease as a model system. Ten different variants of a highly stable form of nuclease were engineered with single site substitutions in the hydrophobic core, designed to create cavities in the interior of the protein. High resolution crystal structures of all proteins were obtained to examine the state of the cavities and as starting structures for calculations of internal void volume with MD and MC methods. Pressure unfolding monitored with Trp fluorescence was used to measure volume changes upon unfolding. Our ultimate goal was to determine if volume changes measured in the equilibrium thermodynamic experiments correlated with the size of the cavities observed in the proteins. The mapping of experimentally determined cavity persistence ratios, combined with simulation data, provides new insight into the subtle interplay between local dynamics, water penetration, and structural properties of cavities in determining volume changes upon unfolding. Overall, the data suggest that internal cavities are one of the main determinants of the pressure-induced unfolding of proteins.

The first step of HIV infection involves the fusion of the viral and target cell membranes, a process mediated by the viral envelope glycoproteins, gp120 and gp41. The binding of gp120 to the cell surface receptors CD4 triggers a cascade of conformational changes that disrupt the gp41-gp120 interactions and allows the insertion of the N-terminal fusion peptide of gp41 into the host cell membrane. The gp41 trimer then rapidly folds into a 6-helix bundle that pulls together the fusion peptide, inserted in the host cell membrane and the transmembrane domain, located in the viral membrane. Very little structural information is known about the pre-fusion state of gp41 notwithstanding its critical importance for the design of fusion inhibitors. To investigate the dynamics and structural properties of such metastable states, we designed a set of protein constructs mimicking the extracellular ectodomain of gp41. We found that the secondary structure of gp41 ectodomain is pronouncedly impacted by the presence of phospholipid vesicles or detergents containing phosphatidyl choline head groups. Our NMR and multi-angle light scattering data attribute these changes to the transition between a trimer in the absence of detergent to a monomer in the presence of a membrane-mimicking environment. The structure of gp41 in a monomeric state was determined by state-of-the-art NMR techniques, including measurements of NOE distance restraints and residual dipolar couplings in weakly aligned solutions. 15N relaxation data provided a detailed view at the backbone dynamics of both the trimeric and monomeric conformations. This stable monomeric state may represent a crucial structural intermediate facilitating both the gp41 conformational change during fusion and the local apposition of the viral and cellular membranes.