Human centrin 2 (HsCen2) is an EF-hand protein that plays a critical role in the centrosome duplication and separation during cell division. We studied the structural and Ca(2+)-binding properties of two C-terminal fragments of this protein: SC-HsCen2 (T94-Y172), covering two EF-hands, and LC-HsCen2 (M84-Y172), having 10 additional residues. Both fragments are highly disordered in the apo state but become better structured (although not conformationally homogeneous) in the presence of Ca(2+) and depending on the nature of the cations (K(+) or Na(+)) in the buffer. Only the longer C-terminal domain, in the Ca(2+)-saturated state and in the presence of Na(+) ions, was amenable to structure determination by nuclear magnetic resonance. The solution structure of LC-HsCen2 reveals an open two EF-hand structure, similar to the conformation of related Ca(2+)-saturated regulatory domains. Unexpectedly, the N-terminal helix segment (F86-T94) lies over the exposed hydrophobic cavity. This unusual intramolecular interaction increases considerably the Ca(2+) affinity and constitutes a useful model for the target binding.

The Stokes-Einstein-Debye equation is currently used to obtain information on protein size or on local viscosity from the measurement of the rotational correlation time. However, the implicit assumptions of a continuous and homogeneous solvent do not hold either in vivo, because of the high density of macromolecules, or in vitro, where viscosity is adjusted by adding viscous cosolvents of various size. To quantify the consequence of nonhomogeneity, we have measured the rotational Brownian motion of three globular proteins with molecular mass from 66 to 4000 kD in presence of 1.5 to 2000 kD dextrans as viscous cosolvents. Our results indicate that the linear viscosity dependence of the Stokes-Einstein relation must be replaced by a power law to describe the rotational Brownian motion of proteins in a macromolecular environment. The exponent of the power law expresses the fact that the protein experiences only a fraction of the hydrodynamic interactions of macromolecular cosolvents. An explicit expression of the exponent in terms of protein size and cosolvent's mass is obtained, permitting definition of a microscopic viscosity. Experimental data suggest that a similar effective microviscosity should be introduced in Kramers' equation describing protein reaction rates.

Human centrin 2 (HsCen2), a member of the EF-hand superfamily of Ca 2؉ -binding proteins, is commonly associated with centrosome-related structures. The protein is organized in two domains, each containing two EFhand motifs, but only the C-terminal half exhibits Ca

Centrin and calmodulin (CaM) are closely related four-EF-hand Ca 2+ -binding proteins. While CaM is monomeric, centrin 2 is dimeric and binds only two Ca 2+ per dimer, likely to site IV in each monomer. Ca 2+ binding to centrin 2 displays pronounced negative cooperativity and a [Ca 2+ ] 0X5 of 30 W WM. As in CaM, Ca 2+ binding leads to the exposure of a hydrophobic probe-accessible patch on the surface of centrin 2. Provided Ca 2+ is present, centrin 2 forms a 1:1 peptide:monomer complex with melittin with an affinity of 100 nM. The complex binds four instead of two Ca 2+ . Our data point to surprising differences in the mode of activation of these homologous proteins.z 2000 Federation of European Biochemical Societies.

In order to obtain a transgenic mouse model of sickle cell disease, we have synthesized a novel human beta‐globin gene, beta SAD, designed to increase the polymerization of the transgenic human hemoglobin S (Hb S) in vivo. beta SAD (beta S‐Antilles‐D Punjab) includes the beta 6Val substitution of the beta S chain, as well as two other mutations, Antilles (beta 23Ile) and D Punjab (beta 121Gln) each of which promotes the polymerization of Hb S in human. The beta SAD gene and the human alpha 2‐globin gene, each linked to the beta‐globin locus control region (LCR) were co‐introduced into the mouse germ line. In one of the five transgenic lines obtained, SAD‐1, red blood cells contained 19% human Hb SAD (alpha 2 human 1 beta 2SAD) and mouse‐human hybrids in addition to mouse hemoglobin. Adult SAD‐1 transgenic mice were not anemic but had some abnormal features of erythrocytes and slightly enlarged spleens. Their erythrocytes displayed sickling upon deoxygenation in vitro. SAD‐1 neonates were anemic and many did not survive. In order to generate adult mice with a more severe sickle cell syndrome, crosses between the SAD progeny and homozygous for beta‐thalassemic mice were performed. Hemoglobin SAD was increased to 26% in beta‐thal/SAD‐1 mice which exhibited: (i) abnormal erythrocytes with regard to shape and density; (ii) an enlarged spleen and a high reticulocyte count indicating an increased erythropoiesis; (iii) mortality upon hypoxia; (iv) polymerization of hemolysate similar to that obtained in human homozygous sickle cell disease; and (v) anemia and mortality during development.

Human centrin 2 (HsCen2) is a member of the EF-hand superfamily of calcium-binding proteins, often associated with the centrosomes and basal bodies. These organelles exhibit different morphological aspects, including a variety of centrin-containing fibers that connect the two centrioles or other structural elements of the pericentriolar space. The molecular basis of the Ca 2؉ -sensitive fibers and their precise role in centrosome duplication are not known. To explore the possible structural role of HsCen2, we initiated a physicochemical study of the self-assembly properties of the purified protein in vitro. Using light scattering experiments, we investigated the temporal evolution of the assembly process and characterized the dependence on various chemical and physical factors, including temperature, di-cation concentration, ionic strength, protein concentration, and pH. The reversible self-assembly revealed many features of a large-size protein polymerization, with nucleation and elongation steps. Kinetic and equilibrium experiments show that a hydrophobic fluorescent probe (ANS) inhibits the polymerization by interfering with the nucleation step, probably through interactions with the apolar exposed sites on the protein surface. A truncated form of HsCen2, lacking the first 25 residues (⌬25HsCen2), shows no detectable self-assembly, pointing to the critical role played by the N-terminal fragment in the supermolecular organization of HsCen2. As revealed by isothermal titration experiments, the isolated N-terminal domains bind with a significant affinity (2 ؋ 10 5 M ؊1 ) to preformed oligomers of ⌬25HsCen2 through an entropy-driven mechanism.

Evidence for ligand migration toward the xenon-binding cavities in myoglobin comes from a number of laser photolysis studies of MbO2 including mutants and from cryo- and time-resolved crystallography of MbCO. To explore ligand migration in greater detail, we investigated the rebinding kinetics of both MbO2 and MbCO under a xenon partial pressure ranging from 1 to 16 atm over the temperature range (293-77 K). Below 180 K xenon affects to a significant, but minor, extent the thermodynamic parameters for rebinding from the primary docking site in each Mb taxonomic substate. Above 200 K the ligand migrates to the proximal Xe1 site but when the latter is occupied by xenon a new kinetic process appears. It is attributed to rebinding from transient docking sites located on the path between the primary and the secondary docking site of both ligands. Ligand escape exhibits a more complicated pattern than expected. At room temperature O2 and CO escape appears to take place exclusively from the primary site. In contrast, at T approximately 250 K, roughly 50% of the CO molecules that have escaped from the protein originate from the Xe1 secondary site.

There are four isoforms of centrin in mammals, with variable sequence, tissue expression, and functional properties. We have recently characterized a number of structural, ion, and target binding properties of human centrin isoform HsCen2. This paper reports a similar characterization of HsCen3, overexpressed in Escherichia coli and purified by phase-reversed chromatography. Equilibrium and dynamic binding studies revealed that HsCen3 has one mixed Ca(2+)/Mg(2+) binding site of high affinity (K(d) = 3 and 10 microM for Ca(2+) and Mg(2+), respectively) and two Ca(2+)-specific sites of low affinity (K(d) = 140 microM). The metal-free protein is fragmented by an unidentified protease into a polypeptide segment of 11 kDa, which was purified by HPLC, and identified by mass spectrometry as the segment of residues 21-112. Similarly, controlled trypsinolysis on Ca(2+)-bound HsCen3 yielded a mixture of segments of residues 1-124 and 1-125. The Ca(2+)/Mg(2+) site could be assigned to this segment and thus resides in the N-terminal half of HsCen3. Temperature denaturation experiments, circular dichroism, and utilization of fluorescence hydrophobic probes allowed us to propose that the metal-free protein has molten globule characteristics and that the dication-bound forms are compact with a polar surface for the Mg(2+) form and a hydrophobic exposed surface for the Ca(2+) form. Thus, HsCen3 could be classified as a Ca(2+) sensor protein. In addition, it is able to bind strongly to a model target peptide (melittin), as well as to peptides derived from the protein XPC and Kar1p, with a moderate Ca(2+) dependence.