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R Badii

Publications and source records attributed to R Badii.

At least 19 recordsLinked to original sources
PubMed2003

Dynamics of multimode diode lasers with strong, frequency-selective optical feedback.

The dynamical behavior of a class of multimode semiconductor diode lasers with emission wavelength around 980 nm is investigated both experimentally and numerically in the presence of strong, frequency-selective optical feedback provided by a fiber Bragg grating. The focus is set on the switching between broad- and narrow-band optical spectra, on chaotic transitions, and on the loss of frequency locking between laser and grating. Laser and feedback parameters are chosen in the typical ranges pertaining to wavelength stabilization in erbium-doped fiber amplifiers for telecommunication applications. An improved set of rate equations, which allows for arbitrary feedback levels and includes experimentally measured gain and linewidth enhancement factor, is studied analytically and numerically.

Journal Article↗
PubMed2001

Refined scaling hypothesis for breakdown coefficients in turbulence.

We study the statistics of Novikov's breakdown coefficients, which represent ratios between energy dissipation rates at different length scales in turbulence. The distribution of their logarithms is shown to be very closely reproduced by an analytic function that we obtain from a hierarchical stochastic process for the turbulent cascade. Correlations and deviation from Gaussianity in the model are accounted for by two parameters, one of which can be interpreted as a generalized dimension. Finally, we illustrate the lack of power-law scaling in the moments of the breakdown coefficients and propose an analytical approximation scheme for them.

Journal Article↗
PubMed2001

Structure-activity relationships in flexible protein domains: regulation of rho GTPases by RhoGDI and D4 GDI.

The guanine dissociation inhibitors RhoGDI and D4GDI inhibit guanosine 5'-diphosphate dissociation from Rho GTPases, keeping these small GTPases in an inactive state. The GDIs are made up of two domains: a flexible N-terminal domain of about 70 amino acid residues and a folded 134-residue C-terminal domain. Here, we characterize the conformation of the N-terminal regions of both RhoGDI and D4GDI using a series of NMR experiments which include (15)N relaxation and amide solvent accessibility measurements. In each protein, two regions with tendencies to form helices are identified: residues 36 to 58 and 9 to 20 in RhoGDI, and residues 36 to 57 and 20 to 25 in D4GDI. To examine the functional roles of the N-terminal domain of RhoGDI, in vitro and in vivo functional assays have been carried out with N-terminally truncated proteins. These studies show that the first 30 amino acid residues are not required for inhibition of GDP dissociation but appear to be important for GTP hydrolysis, whilst removal of the first 41 residues completely abolish the ability of RhoGDI to inhibit GDP dissociation. The combination of structural and functional studies allows us to explain why RhoGDI and D4GDI are able to interact in similar ways with the guanosine 5'-diphosphate-bound GTPase, but differ in their ability to regulate GTP-bound forms; these functional differences are attributed to the conformational differences of the N-terminal domains of the guanosine 5'-diphosphate dissociation inhibitors. Therefore, the two transient helices, appear to be associated with different biological effects of RhoGDI, providing a clear example of structure-activity relationships in a flexible protein domain.

Amides↗
PubMed2001

Structural consequences of site-directed mutagenesis in flexible protein domains: NMR characterization of the L(55,56)S mutant of RhoGDI.

The guanine dissociation inhibitor RhoGDI consists of a folded C-terminal domain and a highly flexible N-terminal region, both of which are essential for biological activity, that is, inhibition of GDP dissociation from Rho GTPases, and regulation of their partitioning between membrane and cytosol. It was shown previously that the double mutation L55S/L56S in the flexible region of RhoGDI drastically decreases its affinity for Rac1. In the present work we study the effect of this double mutation on the conformational and dynamic properties of RhoGDI, and describe the weak interaction of the mutant with Rac1 using chemical shift mapping. We show that the helical content of the region 45-56 of RhoGDI is greatly reduced upon mutation, thus increasing the entropic penalty for the immobilization of the helix, and contributing to the loss of binding. In contrast to wild-type RhoGDI, no interaction with Rac1 could be identified for amino-acid residues of the flexible domain of the mutant RhoGDI and only very weak binding was observed for the folded domain of the mutant. The origins of the effect of the L55S/L56S mutation on the binding constant (decreased by at least three orders of magnitude relative to wild-type) are discussed with particular reference to the flexibility of this part of the protein.

Cell Membrane↗
PubMed2000

Mapping the binding site for the GTP-binding protein Rac-1 on its inhibitor RhoGDI-1.

BACKGROUND: Members of the Rho family of small GTP-binding proteins, such as Rho, Rac and Cdc42, have a role in a wide range of cell responses. These proteins function as molecular switches by virtue of a conformational change between the GTP-bound (active) and GDP-bound (inactive) forms. In addition, most members of the Rho and Rac subfamilies cycle between the cytosol and membrane. The cytosolic guanine nucleotide dissociation inhibitors, RhoGDIs, regulate both the GDP/GTP exchange cycle and the membrane association/dissociation cycle. RESULTS: We have used NMR spectroscopy and site-directed mutagenesis to identify the regions of human RhoGDI-1 that are involved in binding Rac-1. The results emphasise the importance of the flexible regions of both proteins in the interaction. At least one specific region (residues 46-57) of the flexible N-terminal domain of RhoGDI, which has a tendency to form an amphipathic helix in the free protein, makes a major contribution to the binding energy of the complex. In addition, the primary site of Rac-1 binding on the folded domain of RhoGDI involves the beta4-beta5 and beta6-beta7 loops, with a slight movement of the 3(10) helix accompanying the interaction. This binding site is on the same face of the protein as the binding site for the isoprenyl group of post-translationally modified Rac-1, but is distinct from this site. CONCLUSIONS: Isoprenylated Rac-1 appears to interact with three distinct sites on RhoGDI. The isoprenyl group attached to the C terminus of Rac-1 binds in a pocket in the folded domain of RhoGDI. This is distinct from the major site on this domain occupied by Rac-1 itself, which involves two loops at the opposite end to the isoprenyl-binding site. It is probable that the flexible C-terminal region of Rac-1 extends from the site at which Rac-1 contacts the folded domain of RhoGDI to allow the isoprenyl group to bind in the pocket at the other end of the RhoGDI molecule. Finally, the flexible N terminus of RhoGDI-1, and particularly residues 48-58, makes a specific interaction with Rac-1 which contributes substantially to the binding affinity.

Amino Acid Sequence↗
PubMed1999

Direct measurement of the pKa of aspartic acid 26 in Lactobacillus casei dihydrofolate reductase: implications for the catalytic mechanism.

The ionization state of aspartate 26 in Lactobacillus casei dihydrofolate reductase has been investigated by selectively labeling the enzyme with [13Cgamma] aspartic acid and measuring the 13C chemical shifts in the apo, folate-enzyme, and dihydrofolate-enzyme complexes. Our results indicate that no aspartate residue has a pKa greater than approximately 4.8 in any of the three complexes studied. The resonance of aspartate 26 in the dihydrofolate-enzyme complex has been assigned by site-directed mutagenesis; aspartate 26 is found to have a pKa value of less than 4 in this complex. Such a low pKa value makes it most unlikely that the ionization of this residue is responsible for the observed pH profile of hydride ion transfer [apparent pKa = 6.0; Andrews, J., Fierke, C. A., Birdsall, B., Ostler, G., Feeney, J., Roberts, G. C. K., and Benkovic, S. J. (1989) Biochemistry 28, 5743-5750]. Furthermore, the downfield chemical shift of the Asp 26 (13)Cgamma resonance in the dihydrofolate-enzyme complex provides experimental evidence that the pteridine ring of dihydrofolate is polarized when bound to the enzyme. We propose that this polarization of dihydrofolate acts as the driving force for protonation of the electron-rich O4 atom which occurs in the presence of NADPH. After this protonation of the substrate, a network of hydrogen bonds between O4, N5 and a bound water molecule facilitates transfer of the proton to N5 and transfer of a hydride ion from NADPH to the C6 atom to complete the reduction process.

Apoenzymes↗
PubMed1999

Analysis of energy cascade models of turbulence.

The exponent tau(p) that describes the scaling of the order-p moment of the energy dissipation field in fully developed turbulence is studied in a range which extends to negative values of p. The curve tau(p) vs p and its derivative tau(')(p) are estimated in various ways, including a two-scale method. Predictions of recent cascade models agree with our findings, within the statistical errors, for moderately large, positive p. Evident discrepancies already appear, however, in the interval pin[0,1], and sometimes become dramatic for p<0. In the discussion of a class of cascade models, we present a scaling law which relates different moments to one another.

Journal Article↗
PubMed1999

Biasymptotic formula for the turbulent energy cascade.

We present a family of differential models for the scaling exponents tau(p) which characterize the moments of the energy dissipation rate in turbulence. This scheme interpolates between the asymptotic values of the derivative tau'p of tau(p) versus p in the limits p-->+/-infinity and reproduces the negative-p part of the exponents spectrum tau(p) as well, in contrast with other recent conjectures. Each member of the family is defined by a sigmoidal function, the form of which remains open to theoretical investigations.

Journal Article↗
PubMed1998

Bacterial expression and spectroscopic characterization of soybean leghaemoglobin a.

A gene encoding leghaemoglobin a from soybean has been constructed and the soluble recombinant protein expressed in E. coli. The integrity of the recombinant protein has been assessed by a range of spectroscopic techniques. Electrospray mass spectrometry of the protein indicates that the molecular mass of the protein corresponds to the predicted amino acid sequence. Circular dichroism spectra of the ferric derivative and UV-visible spectra of various ferric and ferrous derivatives (pH 6.99, mu = 0.10 M, 25.0 degrees C) are consistent with published data for the wild-type protein. For the ferric derivative, UV-visible (298 and 77 K) and EPR (10 K) spectra indicate the existence of a thermal equilibrium between high- and low-spin forms. Titration of the protein (0.10 M NaCl, mu = 0.10 M, 25.0 degrees C) between pHs 6.68 and 10.35 indicate formation (pKa = 8.3+/-0.03) of a 6-coordinate, hydroxide-bound form of the protein at high pH. All of the above data are consistent with the behaviour of the wild-type protein.

Amino Acid Sequence↗
PubMed1997

A modulator of rho family G proteins, rhoGDI, binds these G proteins via an immunoglobulin-like domain and a flexible N-terminal arm.

BACKGROUND: The rho family of small G proteins, including rho, rac and cdc42, are involved in many cellular processes, including cell transformation by ras and the organization of the actin cytoskeleton. Additionally, rac has a role in the regulation of phagocyte NADPH oxidase. Guanine nucleotide dissociation inhibitors (GDIs) of the rhoGDI family bind to these G proteins and regulate their activity by preventing nucleotide dissociation and by controlling their interaction with membranes. RESULTS: We report the structure of rhoGDI, determined by a combination of X-ray crystallography and NMR spectroscopy. NMR spectroscopy and selective proteolysis show that the N-terminal 50-60 residues of rhoGDI are flexible and unstructured in solution. The 2.5 A crystal structure of the folded core of rhoGDI, comprising residues 59-204, shows it to have an immunoglobulin-like fold, with an unprecedented insertion of two short beta strands and a 310 helix. There is an unusual pocket between the beta sheets of the immunoglobulin fold which may bind the C-terminal isoprenyl group of rac. NMR spectroscopy shows that the N-terminal arm is necessary for binding rac, although it remains largely flexible even in the complex. CONCLUSIONS: The rhoGDI structure is notable for the existence of both a structured and a highly flexible domain, both of which appear to be required for the interaction with rac. The immunoglobulin-like fold of the structured domain is unusual for a cytoplasmic protein. The presence of equivalent cleavage sites in rhoGDI and the closely related D4/Ly-GDI (rhoGDI-2) suggest that proteolytic cleavage between the flexible and structured regions of rhoGDI may have a role in the regulation of the activity of members of this family. There is no detectable similarity between the structure of rhoGDI and the recently reported structure of rabGDI, which performs the same function as rhoGDI for the rab family of small G proteins.

Amino Acid Sequence↗
PubMed1995

High-level expression and isotopic labeling of Lactobacillus casei dihydrofolate reductase for nuclear magnetic resonance spectroscopy.

Two efficient systems have been used for high-level expression of Lactobacillus casei dihydrofolate reductase in Escherichia coli, including the production of protein generally and specifically labeled with 13C and 15N. A system based on T7 RNA polymerase led to the production of dihydrofolate reductase at a level of 37% of the total soluble protein of the host strain: 50 mg of pure enzyme was obtained from a 1 liter of culture (or 14 mg/g wet weight of cells). In this system, a small amount of the enzyme (less than 5%) was identified as a catalytically active 21-kDa fusion protein. Introduction of a second in-frame (ochre) stop codon did not eliminate the production of this fusion protein. The same expression system was also used to prepare dihydrofolate reductase generally labeled with 15N and to prepare single and double mutants of the enzyme. In order to have an expression system which can be used with a range of auxotrophic strains of E. coli, a system based on the tac promoter was used. This led to the production of dihydrofolate reductase at a level of 29% of total soluble protein; a yield of 40 mg enzyme per liter of culture (or 11 mg/g wet weight of cells). This system was successfully used to produce mutants of the enzyme as well as the enzyme selectively labeled with [gamma-13C]aspartic acid.

Base Sequence↗