In this paper, a simulation of the folding process, based on a random perturbations of the ϕ, ψ, χ 1 dihedral angles, is proposed to approach the formation at the atom level of both principal elements of protein secondary structure, the α‐helix and the β‐hairpin structures. Expecting to understand what may happen in solution during the formation of such structures, the behaviour of large sets of random conformations that are generated for small oligopeptides was analysed. Different factors that may influence the folding (as conformational propensity, hydrophobic interactions and side‐chain mobility) were investigated. The difference between the corresponding theoretical folding and the real conformational diversity that is observed in solution is appraised by a comparison between the calculated and observed NMR secondary chemical shifts. From this study it appears that hydrophobic interactions and mobility represent the principal factors that initiate folding and determine the observed hydrogen‐bond pattern, which subsequently allows packing between the peptide side chains. Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd.

In this paper the NMR secondary chemical shifts, that are estimated from a set of 3D-structures, are compared with the observed ones to appraise the behaviour of a known x-ray diffraction structure (of the bovine pancreatic trypsin inhibitor protein) when various molecular dynamics are applied. The results of a 200 ps molecular dynamics under various conditions are analysed and different ways to modify the molecular dynamics are considered. With the purpose of avoiding the time-consuming explicit representation of the solvent (water) molecules, an attempt was made to understand the role of the solvent and to develop an implicit representation, which may be refined. A simulation of hydrophobic effects in an aqueous environment is also proposed which seems to provide a better approximation of the observed solution structure of the protein.

Rheumatoid arthritis is a common chronic inflammatory disease of the joints that leads to severe deformation and incapacitation of affected extremities.Adjuvant arthritis (AA) in the Lewis rat resembles in many aspects the human disease and, therefore, serves as a useful experimental model.It is an autoimmune disease that is driven by T-cells against specific epitopes on the Mycobacterium tuberculosis HSP65 (MT65) molecule.The role of the 65 Kd heat shock protein of Mt (MT65) in the pathogenesis of autoimmune arthritis, both in experimental animals as well as in humans has been investigated intensively in the past several years.In patients suffering from rheumatoid arthritis, an association between T-cell responses to MT65 and early stages of joint inflammation has been found.Pre-immunization of rats with the mycobacterial MT65 leads to resistance to induction of the disease by Mt, and this protective effect is believed to be mediated by T-cells specific for MT65.Understanding the processes at a molecular level requires knowledge of 3D structure of MT65 at high resolution.We have initiated the study of the MT65 protein structure using X-ray crystallographic techniques.We have succeeded in obtaining nearly pure protein.One particular difficulty in the project so far has been the proteins ability to form different oligomers.In the crystallization process this phenomenon is apparently critical.Usually we obtain a monomeric protein during protein purification.We have succeeded in obtaining small crystals of MT65.Currently we are working on modifications of protein purification procedure in order to improve a crystal quality.

The yeast Saccharomyces cerevisiae F1F0-ATPase epsilon-subunit (61 residues) was synthesized by the solid-phase peptide approach under both acidic and basic strategies. Only the latter strategy allowed us to obtain a pure epsilon-subunit. The strong propensity of the protein to produce few soluble dimeric species depending on pH has been proved by size-exclusion chromatography, electrophoresis and mass spectrometry. A circular dichroism study showed that an aqueous solution containing 30% trifluoroethanol or 200 mM sodium dodecyl sulphate is required for helical folding. In both solvents at acidic pH, the epsilon-subunit is soluble and monomeric.

Cubic F432 crystals of recombinant mouse L-chain apoferritin were obtained by the hanging-drop technique with ammonium sulfate and cadmium sulfate as precipitants. The structure was refined to 2.1 and 1.6 A resolution from data obtained at room temperature and under cryogenic conditions, respectively. The structure of an eight-amino-acid loop insertion in the mouse sequence is found to be highly disordered both at room temperature and at low temperature.

The HTLV‐I integrase N‐terminal domain [50‐residue peptide (IN50)], and a 35‐residue truncated peptide formed by residues 9–43 (IN35) have been synthesized by solid‐phase peptide synthesis. Formation of the 50‐residue zinc finger type structure through a HHCC motif has been proved by UV‐visible absorption spectroscopy. Its stability was demonstrated by an original method using RP‐HPLC. Similar experiments performed on the 35‐residue peptide showed that the truncation does not prevent zinc complex formation but rather that it significantly influences its stability. As evidenced by CD spectroscopy, the 50‐residue zinc finger is unordered in aqueous solution but adopts a partially helical conformation when trifluoroethanol is added. These results are in agreement with our secondary structure predictions and demonstrate that the HTLV‐I integrase N‐terminal domain is likely to be composed of an helical region (residues 28–42) and a β‐strand (residues 20–23), associated with a HHCC zinc‐binding motif. Size‐exclusion chromatography showed that the structured zinc finger dimerizes through the helical region. Copyright © 2000 European Peptide Society and John Wiley & Sons, Ltd.

Crystals of recombinant mouse L-chain apoferritin were obtained by the hanging-drop technique using ammonium sulfate as precipitant. Two crystal forms were observed in the same drop. The crystals belong to either the P2 monoclinic or to the P42(1)2 tetragonal space group. The monoclinic crystals diffracted to beyond 2.4 A resolution but were systematically twinned, while the tetragonal crystals diffracted to beyond 2.9 A. These crystallization conditions in the absence of metal salts should facilitate the study of the interaction between L-chain ferritins and heavy metals, particularly the iron core.

An 18-residue peptide, corresponding to the minimum sequence of the N-terminal zinc-finger domain in the nucleocapsid of human T-lymphotrophic virus type I, was synthesized by a solid-phase method and fully characterized. Its ability to complex metal ions (Co2+ and Zn2+) was clearly established by UV–visible spectroscopy and MS. The stability of these complexes was investigated by an original method with HPLC chromatography. Our results show that, even in the presence of air, the Zn2+ complex is highly stable. In contrast, the Co2+ complex undergoes a relatively fast degradation due to an intramolecular oxidation leading to the formation of a disulphide bridge between two cysteine residues. The 1H-NMR analysis indicates that Zn2+ binds to the Nδ atom of the histidine residue rather than to the Nε atom. Two-dimensional NMR techniques were used to determine the solution structure of the zinc-finger, illustrated by the existence of turns in the overall conformation.

The prokaryotic ferritin gene of Campylobacter jejuni was overexpressed in Escherichia coli under control of the bacteriophage T7 promoter and the protein (Cj-FTN) purified. Preliminary crystallization experiments have been performed using the hanging-drop vapour-diffusion method with ammonium sulfate as the precipitant. Diffraction studies show the crystals belong to the I432 space group (a = 151.52 A). Structure solution by molecular replacement is in progress while crystal quality improvement is carried out.

We refined the structure of the tetragonal form of recombinant horse L-chain apoferritin to 2.0 A and we compared it with that of the cubic form previously refined to the same resolution. The major differences between the two structures concern the cadmium ions bound to the residues E130 at the threefold axes of the molecule. Taking advantage of the significant anomalous signal (f" = 3.6 e-) of cadmium at 1.375 A, the wavelength used here, we performed anomalous Fourier difference maps with the refined model phases. These maps reveal the positions of anomalous scatterers at different locations in the structure. Among these, some are found near residues that were known previously to bind metal ions, C48, E57, C126, D127, E130, and H132. But new cadmium binding sites are evidenced near residues E53, E56, E57, E60, and H114, which were suggested to be involved in the iron loading process. The quality of the anomalous Fourier difference map increases significantly with noncrystallographic symmetry map averaging. Such maps reveal density peaks that fit the positions of Met and Cys sulfur atoms, which are weak anomalous scatterers (f" = 0.44 e-).

A recombinant human uroporphyrinogen decarboxylase (E.C. 4.1.1.37, UROD) has been expressed in Escherichia coli and purified to homogeneity. Crystals grew by the hanging-drop vapor-diffusion technique from a starting solution containing 1.5 mg ml-1 protein. The crystals belong to the trigonal space group P3121 or its enantiomer P3221 and diffract to 3 A resolution. The unit-cell parameters are a = b = 103.4, c = 75.7 A and gamma = 120 degrees. The asymmetric unit contains one molecule. Preliminary structural predictions suggest for the protein a TIM-barrel type tertiary structure.

Crystals of E. coli cytochrome b1, alias bacterioferritin, were grown fr om a low ionic strength solution. The resulting monoclniic P21 structure was solved by molecular replacement and refined using noncrystallographi c symmetries applied to the fundamental unit, consisting of two protein subunits and a single haem. From the Patterson self-rotation results it was shown that the asymmetric unit of the monoclinic crystal consists of 12 such dimers and corresponds to a complete, nearly spherical, molecule of bacterioferritin (M4 = 450 kDa) of 432 point-group symmetry. It is thus the most symmetrical cytochrome. As previously determined for the tetragonal form, the haem is located in a special position on a local twofold axis of the dimer. A bimetal centre is also observed within the four-helix bundle of each monomer; a metal-binding site is located on the fourfold axis.

Horse-spleen apoferritin is known to crystallize in three different space groups, cubic F432, tetragonal P42(1)2 and orthorhombic P2(1)2(1)2. A structure comparison of the cubic and tetragonal forms is presented here. Both crystal forms were obtained by the vapor-diffusion technique and data were collected at 2.26 A (cubic crystal) and 2.60 A (tetragonal crystal) resolution. Two main differences were observed between these crystal structures: (i) whereas intermolecular contacts only involve salt-bridge type interactions via cadmium ions in the cubic structure, two types of interactions are observed in the tetragonal crystal (cadmium-ion-mediated salt bridges and hydrogen-bonding interactions) and (ii) cadmium ions bound in the threefold axes of ferritin molecules exhibit lower site-occupation factors in the tetragonal structure than in the cubic one.