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Background: How proteins approach surrounding molecules is fundamental to our understanding of the specific interactions that occur at the surface of proteins. The enhanced surface accessibility of small molecules such as organic solvents and paramagnetic probes to protein binding sites has been observed; however, the molecular basis of this finding has not been fully established. Recently, it has been suggested that hydration dynamics play a predominant role in controlling the distribution of hot spots on surface of proteins. Results: In the present study, the hydration of the archaeal multifunctional protein Sso7d from Solfolobus solfataricus was investigated using a combination of computational and experimental data derived from molecular dynamics simulations and ePHOGSY NMR spectroscopy. Conclusions: We obtained a convergent protein hydration landscape that indicated how the shape and stability of the Sso7d hydration shell could modulate the function of the protein. The DNA binding domain overlaps with the protein region involved in chaperon activity and this domain is hydrated only in a very small central region. This localized hydration seems to favor intermolecular approaches from a large variety of ligands. Conversely, high water density was found in surface regions of the protein where the ATP binding site is located, suggesting that surface water molecules play a role in protecting the protein from unspecific interactions. © 2011 Bernini et al; licensee BioMed Central Ltd.

Understanding how proteins are approached by surrounding molecules is fundamental to increase our knowledge of life at atomic resolution. Here, the surface accessibility of a multifunctional small protein, the archaeal protein Sso7d from Sulfolobus solfataricus, has been investigated by using TEMPOL and Gd(III)(DTPA-BMA) as paramagnetic probes. The DNA binding domain of Sso7d appears very accessible both to TEMPOL and Gd(III)(DTPA-BMA). Differences in paramagnetic attenuation profiles of (1)H-(15)N HSQC protein backbone amide correlations, observed in the presence of the latter paramagnetic probes, are consistent with the hydrogen bond acceptor capability of the N-oxyl moiety of TEMPOL to surface exposed Sso7d amide groups. By using the gadolinium complex as a paramagnetic probe a better agreement between Sso7d structural features and attenuation profile is achieved. It is interesting to note that the protein P-loop region, in spite of the high surface exposure predicted by the available protein structures, is not approached by TEMPOL and only partially by Gd(III)(DTPA-BMA).

Sso7d is a small protein especially attractive as a model for characterizing by NMR, the structural determinants responsible for thermal stability. With this aim, structural parameters of the Sso7d protein have been compared with those of single point mutants, in particular with K12L, more stable (T-m around 100 degrees C) and F31A, less stable (T-m 61 degrees C) than the wild-type. In addition, a mutant lacking eight residues of the C-terminal helix (T-m 50 degrees C) has been studied to investigate the role of the helix in structure stabilization. Melting temperatures, Delta Delta G's of unfolding, cavities, electrostatic interactions, solvent accessible areas, hydrophobic cores, and aromatic cluster geometries have been analyzed in order to gain insights into the key structural factors determining the thermostability differences. Our data suggest that absence of cavities and a larger salt bridge network represent the major determinants contributing to the enhanced stability of K12L relative to the Sso7d protein and its mutant F31A.

Ras proteins are small G proteins playing a major role in eukaryotic signal transduction. Guanine nucleotide exchange factors (GEF) stimulate GDP/GTP exchange, resulting in the formation of the active Ras-GTP complex. In mammalian cells, two major Ras-specific GEF exist: Sos-like and Cdc25-like. To date, structural data are available only for Cdc25(Mm). We designed and synthesized Cdc25(Mm)-derived peptides spanning residues corresponding to the hSos1 HI helical hairpin that has been implicated in the GEF catalytic mechanism. NMR experiments on a chemically synthesized Cdc25(1178-1222)(Mm) peptide proved that helix I readily reaches a conformation very similar to the corresponding helix in hSos 1, while residues corresponding to helix H in hSos1 show higher conformational flexibility. Molecular dynamics studies with the appropriate solvent model showed that different conformational spaces are available for the peptide. Since helix H is making several contacts with Ras and a Cdc25(1178-1222)(Mm) peptide is able to bind nucleotide-free Ras in a BIAcore assay, the peptide must be able to obtain the proper Ras-interacting conformation, at least transiently. These results indicate that rational design and improvement of the Ras-interacting peptides should take into account conformational and flexibility features to obtain molecules with the appropriate biochemical properties.

Malignancy is a risk factor for thromboembolism and anti-cancer chemotherapy can increase this risk. Prophylaxis of thrombosis with very-low-dose warfarin given concurrently with chemotherapy has a significantly reduced rate of thromboembolism in a randomized trial in women with stage TV breast cancer. In a group of 32 patients randomized in one center (16 subjects on warfarin and 16 on placebo), we have prospectively studied the plasma levels of: 1. Markers of 'in vivo' clotting activation (thrombin-antithrombin complex [TAT], prothrombin fragment 1+2 [F1+2] and D-dimer), 2. Factor VII (FVII), and 3. Natural anticoagulants (protein C [PC] and antithrombin [ATI). The aims of this study were: 1. to examine whether laboratory tests predict ed those patients who developed thrombosis, and 2. to evaluate the effect of very-low-dose warfarin on hemostatic variables. The patients' hemostatic parameters were evaluated before entry into the study and after starting chemotherapy +/- prophylaxis, before each course for nine courses. Before-treatment results were compared to those of a sex and age-matched non-cancer control group. There was a significant elevation of plasma levels of TAT (p < 0.001), F1+2 (p < 0.001), D-dimer (p < 0.0001) and FVIIa (p < 0.05), as well as an increase of FVII proteolysis (p < 0.05), whereas plasma PC and AT concentrations were not different from controls. After starting chemotherapy, markers of clotting activation were progressively lower in the group receiving warfarin prophylaxis compared to the group on placebo. Differences between the groups became statistically significant (p < 0.01) after the 4th course of chemotherapy. Deep vein thrombosis occurred in two patients in the placebo arm. The results of this study indicate that before therapy, an hypercoagulable state is present in stage IV breast cancer, and after starting chemotherapy, abnormalities of hypercoagulation markers persist, however they are reduced by very-low-do

This study reports the characterization of the recombinant 7-kDa protein P2 from Sulfolobus solfataricus and the mutants F31A and F31Y with respect to temperature and pressure stability, As observed in the NMR, FTIR, and CD spectra, wild-type protein and mutants showed substantially similar structures under ambient conditions. However, midpoint transition temperatures of the denaturation process were 361, 334, and 347 K for wild type, F31A, and F31Y mutants, respectively: thus, alanine substitution of phenylalanine destabilized the protein by as much as 27 K. Midpoint transition pressures for wild type and F31Y mutant could not be accurately determined because they lay either beyond (wild type) or close to (F31Y) 14 kbar, a pressure at which water undergoes a phase transition. However, a midpoint transition pressure of 4 kbar could be determined for the F31A mutant, implying a shift in transition of at least 10 kbar, The pressure-induced denaturation was fully reversible; in contrast, thermal denaturation of wild type and mutants was only partially reversible. To our knowledge, both the pressure resistance of protein P2 and the dramatic pressure and temperature destabilization of the F31A mutant are unprecedented, These properties may be largely accounted for by the role of an aromatic cluster where Phe31 is found at the core, because interactions among aromatics are believed to be almost pressure insensitive; furthermore, the alanine substitution of phenylalanine should create a cavity with increased compressibility and flexibility, which also involves an impaired pressure and temperature resistance

All-trans-retinoic acid (ATRA) induces complete remission (CR) in up to 90% of acute promyelocytic leukemia (APL) patients with rapid amelioration of the bleeding syndrome. Previous studies indicate that ATRA treatment in vitro of the APL NB4 cell line can affect their procoagulant activity (PCA). To assess whether ATRA has this effect also in vivo, we prospectively studied the PCA of bone marrow blasts from APL patients on therapy with ATRA alone or associated with chemotherapy. Samples were obtained before, during, and after ATRA. To characterize the coagulopathy, we measured a series of plasma hemostatic variables before and during the first two weeks of therapy, as follows: (1) markers of hypercoagulability; (2) natural anticoagulants; (3) fibrinolysis proteins; and (4) elastase. The results by enzymatic and immunologic methods show that both total (tissue factor-like) and factor VII- independent (cancer procoagulant-like) blast cell PCAs, present before therapy, were reduced during (69% and 65% decrement, respectively) and virtually undetectable after ATRA. The plasma hemostatic assessment of patients before treatment was elevated hypercoagulability markers, low mean protein C, normal fibrinolysis proteins, and increased elastase. After starting ATRA, hypercoagulability markers were reduced within 4 to 8 days, protein C augmented, the overall fibrinolytic balance was unmodified, and elastase remained elevated. These results were not different either with or without chemotherapy and are consistent with the clinical findings of rapid improvement of the coagulopathy.

Investigations were performed on recombinant ribonuclease P2 from Sulfolobus solfataricus, previously cloned and expressed in Escherichia coli [Fusi, P., Grisa, M., Mombelli, E., Consonni, R., Tortora, P. and Vanoni, M. (1995) Gene 154, 99-103], MMR and photo-CIDNP spectroscopies showed that the enzyme possesses an aromatic cluster constisting of Phe(5), Tyr(7), Phe(31) and Tyr(33) while Trp(23) is fully exposed to solvent, Phe(31), Tyr(33) and Trp(23) located within a triple stranded antiparallel beta-sheet, each one being part of an amino acid stretch matching consensus sequences for RNA binding, Phe(31) and Trp(23) are exposed to and specifically interact with a flavin dye used as a model ligand, with a topology reminiscent of that found in several eubacterial and eukariotic RNA-binding proteins

This work reports the molecular cloning and expression of a synthetic gene encoding P2, a 7-kDa ribonuclease (RNase) previously isolated in our laboratory from the archaebacterium Sulfolobus solfataricus [Fusi et al., Eur. J. Biochem. 211 (1993) 305-310]. The P2-encoding synthetic gene was expressed in E. coli and in Saccharomyces cerevisiae. The recombinant (re-) protein was produced to approx. 1.5% of the total protein content in S. cerevisiae using the galactose-inducible GALI promoter and to 3% (tac/lac tandem promoters) or 6.5% (T7 promoter) in E. coli as judged by immunological and biochemical criteria. E. coli-produced P2 was purified to electrophoretic homogeneity through a one-step procedure, i.e., DEAE-Sephacel chromatography at pH 9.3. S. cerevisiae-produced P2 additionally required filtration through a Centricon-10 microconcentrator to obtain the same purity. The re-P2 was found to be indistinguishable from the Su. solfataricus enzyme on the basis of heat stability, pH optimum and RNA digestion pattern. Furthermore, monodimensional nuclear magnetic resonance showed that the E. coli- and Su. solfataricus-produced enzymes were structurally identical, the only exceptions being that Lys4 and Lys6 were not methylated in the re-enzyme, thus showing that lysine methylation does not play a role in P2 thermostabilization