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summary of the identified exchange peaks observed to- gether with their chemical shifts is reported in Table 1. Their assignment was achieved assuming that each resonance of the native state is in exchange with the resonance of the correspond- ing proton in the molten globule state (e. g. the H2,6 resonance will interconvert directly with the corresponding H2’,6’ reso- nance, where the prime notation refers to protons in the molten globule state). A comparison between the chemical shifts of the molten globule species and the corresponding native resonances shows that, as previously observed by Shimizu et al. (1993), the former move towards the random coil values (Table l), with H4 of Trpl04 as the only exception. Exchange is observed for Trp26, Phe31, Tyr103, Trpl04 and Trpll8. The observation of these exchange peaks has also been reported by Baum et al. (1989) in a two-dimensional exchange spectrum of guinea pig a-lactalbumin and by Shimizu et al. (1993) for bovine a-lactal- bumin. The presence of exchange involving other residues, such as Tyrl8 and Phe53, could not be excluded from the ROESY, but could not be detected because of peak overlap. An estimate of the exchange rate was derived for the well separated resonances of Tyr103 H2,6/H2’,6’, Phe3 1 H2,6/H2’,6’ and Trpl04 H4/H4’ from the ratio between cross and diagonal peaks in the NOESY spectrum at 40 ms mixing time (Emst et al., 1987). Simplified equations were used based on the following assumptions: (a) the two sites are assumed equally populated; (b) both cross and spin lattice relaxation terms are equal for the two species; (c) an initial rate approximation is assumed. While the first two assumptions are rather arbitrary in a general case, the last one is expected to be reliable at a mixing time of 40 ms. Comparable interconversion rates are shown by Phe31 and Trpl04 (1.5 s-’ and 1.7

We have suggested elsewhere the use of surface mapping by spin label probes (Esposito et al., 1992). According to this approach, soluble nitroxides are added to a protein solution. Resonances of protons that are accessible to the nitroxide are broadened and bleached out of the spectrum, while resonances in the protein interior remain unaffected. This approach is, in principle, complementary to another technique, photochemically induced dynamic nuclear polarization, which maps the position of aromatic protons on the protein surface. A detailed comparison between the two techniques is necessary for a confident use of the more recent suggested nitroxide perturbation approach. In the present study, we show that the results obtained by the two techniques for the native state of bovine alpha-lactalbumin are fully consistent and may therefore be combined for the study of protein surfaces.

Nuclear Magnetic Resonance (NMR) is an analytical technique extensively used in almost every chemical laboratory for structural identification. This technique provides statistically equivalent signals in spite of using spectrometer with different hardware features and is successfully used for the traceability and quantification of analytes in food samples. Nevertheless, to date only a few internationally agreed guidelines have been reported on the use of NMR for quantitative analysis. The main goal of the present study is to provide a methodological pipeline to assess the reproducibility of NMR data produced for a given matrix by spectrometers from different manufacturers, with different magnetic field strengths, age and hardware configurations. The results have been analyzed through a sequence of chemometric tests to generate a community-built calibration system which was used to verify the performance of the spectrometers and the reproducibility of the predicted sample concentrations.

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.

An interlaboratory comparison (ILC) was organized with the aim to set up quality control indicators suitable for multicomponent quantitative analysis by nuclear magnetic resonance (NMR) spectroscopy. A total of 36 NMR data sets (corresponding to 1260 NMR spectra) were produced by 30 participants using 34 NMR spectrometers. The calibration line method was chosen for the quantification of a five-component model mixture. Results show that quantitative NMR is a robust quantification tool and that 26 out of 36 data sets resulted in statistically equivalent calibration lines for all considered NMR signals. The performance of each laboratory was assessed by means of a new performance index (named Qp-score) which is related to the difference between the experimental and the consensus values of the slope of the calibration lines. Laboratories endowed with a Qp-score falling within the suitable acceptability range are qualified to produce NMR spectra that can be considered statistically equivalent in terms of relative intensities of the signals. In addition, the specific response of nuclei to the experimental excitation/relaxation conditions was addressed by means of the parameter named NR. NR is related to the difference between the theoretical and the consensus slopes of the calibration lines and is specific for each signal produced by a well-defined set of acquisition parameters. (Graph Presented).

This study investigated whether the pre-surgical plasma levels of TAT and F1 + 2 of patients undergoing major surgery for localized tumours could identify patients at higher risk of thrombosis, and how heparin prophylaxis affected in vivo coagulation after cancer surgery. We measured the pre- and post-operative levels of TAT, F1 + 2, total factor VII (FVIIt) and zymogen FVII (FVIIz) in 117 cancer patients, with and without heparin prophylaxis. The end points of this study were DVT, initially detected by I-125-fibrinogen uptake test and confirmed by ascending venography. Pre-operative [TAT] and [F1 + 2] of the cancer patients were significantly higher than those of age-matched control subjects (n = 50) (P3.5 ng/ml identified patients at higher risk for post-operative DVT. Heparin reduced plasma TAT levels and FVII consumption following surgery, suggesting that heparin modulates coagulation associated with cancer surgery. The results of this study also suggest that the pre-operative [TAT] may identify patients with higher risk for post-operative DVT

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.

The synthesis and characterization of a new series of substituted polythiophenes containing an electron acceptor anthraquinone moiety in the side chain are reported. The acceptor molar content was varied by the co-polymerization of both alkylthiophene and thiophene bearing anthraquinone monomers in different ratios. NMR analysis shows a good correlation between the monomer feed composition at the beginning of the polymerization and the actual unit composition of the backbone. The conjugation length and the chemical composition of the copolymers as a function of the molecular weight have been studied by size exclusion chromatography. Small angle X-ray scattering and UV-Vis absorption spectra have been used to monitor the degree of order and chain organization in the solid state. The materials exhibit a lamellar organization, in which the anthraquinone units of neighboring side chains are also organized to some degree in layered structures parallel to the polythiophene main chains. The photoluminescence measurements in solution suggest that, upon photoexcitation of the polythiophene backbone, the anthraquinone moieties act as electron acceptors and the conjugated backbone as electron donor. The tunability of the donor-acceptor ratio and the morphology in the solid state make these photoactive copolymers interesting candidates for organic photoelectric conversion devices.

Sso7d is a small basic protein consisting of 62 amino acids isolated from the thermoacidophilic archeobacterium Sulfolobus solfataricus. The protein is endowed with DNA binding properties, RNase activity, and the capability of rescuing aggregated proteins in the presence of ATP. In this study, the electrostatic properties of Sso7d are investigated by using the Poisson-Boltzmann calculation of the surface potential distribution and following by NMR spectroscopy the proton chemical shift pH titration of acidic residues. Although the details of the catalytic mechanism still have to be defined, the results from NMR experiments confirm the possible involvement of Glu35 as the proton acceptor in the catalytic reaction, as seen by its abnormally high pK(a) value. Poisson-Boltzmann calculations and NMR titration shifts suggest the presence of a possible hydrogen bond between Glu35 and Tyr33, with a consequent rather rigid arrangement at these positions. Comparison with RNase T1 suggests that Tyr7 may be a good candidate for acting as a proton donor in the active site of Sso7d as shown by its low phenolic pK(a) of approximately 9.3. Titration experiments performed with the UpA, a RNA dinucleotide model, showed that the protein residues affected by the interaction are mainly located in a different region with respect to the surface affected by DNA recognition, in good agreement with the surface potential distribution found with electrostatic calculations.

Sso7d is a small, basic, abundant protein from the thermoacidophilic archaeon Sulfolobus solfataricus. Previous research has shown that Sso7d can bind double-stranded DNA without sequence specificity by placing its triple-stranded P-sheet across the minor groove. We previously found RNase activity both in preparations of Sso7d purified from its natural source and in recombinant, purified protein expressed in Escherichia coli, This paper provides conclusive evidence that supports the assignment of RNase activity to Sso7d, shown by the total absence of activity in the single-point mutants E35L and K12L, despite the preservation of their overall structure under the assay conditions. In keeping with our observation that the residues putatively involved in RNase activity and those playing a role in DNA binding are located on different surfaces of the molecule, the activity was not impaired in the presence of DNA, If a small synthetic RNA was used as a substrate, Sso7d attacked both predicted double- and single-stranded RNA stretches, with no evident preference for specific sequences or individual bases. Apparently, the more readily attacked bonds were those intrinsically more unstable. (C) 2001 Federation of European Biochemical Societies.

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.