Manuel Rico

Solution structure of gamma 1-H and gamma 1-P thionins from barley and wheat endosperm determined by 1H-NMR: a structural motif common to toxic arthropod proteins.

The complete assignment of the proton NMR spectra of the homologous gamma 1-hordothionin and gamma 1-purothionin (47 amino acids, 4 disulfide bridges) from barley and wheat, respectively, has been performed by two-dimensional sequence-specific methods. A total of 299 proton-proton distance constraints for gamma 1-H and 285 for gamma 1-P derived from NOESY spectra have been used to calculate the three-dimensional solution structures. Initial structures have been generated by distance geometry methods and further refined by dynamical simulated annealing calculations. Both proteins show identical secondary and tertiary structure with a well-defined triple-stranded antiparallel beta-sheet (residues 1-6, 31-34, and 39-47), an alpha-helix (residues 16-28), and the corresponding connecting loops. Three disulfide bridges are located in the hydrophobic core holding together the alpha-helix and the beta-sheet and forming a cysteine-stabilized alpha-helical (CSH) motif. Moreover, a clustering of positive charges is observed on the face of the beta-sheet opposite to the helix. The three-dimensional structures of the gamma-thionins differ remarkably from plant alpha- and beta-thionins and crambin. However, they show a higher structural analogy with scorpion toxins and insect defensins which also present the CSH motif.

1H NMR detection of cerebral myo‐inositol

A previously unassigned group of prominent multiplets of the 360 MHz 1H NMR spectrum of acid stable metabolite extracts from rat brain is shown to arise from free myo‐inositol. This conclusion is derived from a systematic analysis of the high‐resolution 1H NMR spectra of brain acid extracts, in which appropriate conditions and optimal proton signals have been selected for the quantitative analysis of up to 15 metabolites. Developmental variations in the cerebral content of myo‐inositol could be readily detected using this approach, which provides a novel alternative to study myo‐inositol metabolism under physiological or pathological conditions.

Charge-charge interactions are key determinants of the pK values of ionizable groups in ribonuclease Sa (pI = 3.5) and a basic variant (pI = 10.2)

The pK values of the titratable groups in ribonuclease Sa (RNase Sa) (pI=3.5), and a charge-reversed variant with five carboxyl to lysine substitutions, 5K RNase Sa (pI=10.2), have been determined by NMR at 20 degrees C in 0.1M NaCl. In RNase Sa, 18 pK values and in 5K, 11 pK values were measured. The carboxyl group of Asp33, which is buried and forms three intramolecular hydrogen bonds in RNase Sa, has the lowest pK (2.4), whereas Asp79, which is also buried but does not form hydrogen bonds, has the most elevated pK (7.4). These results highlight the importance of desolvation and charge-dipole interactions in perturbing pK values of buried groups. Alkaline titration revealed that the terminal amine of RNase Sa and all eight tyrosine residues have significantly increased pK values relative to model compounds.A primary objective in this study was to investigate the influence of charge-charge interactions on the pK values by comparing results from RNase Sa with those from the 5K variant. The solution structures of the two proteins are very similar as revealed by NMR and other spectroscopic data, with only small changes at the N terminus and in the alpha-helix. Consequently, the ionizable groups will have similar environments in the two variants and desolvation and charge-dipole interactions will have comparable effects on the pK values of both. Their pK differences, therefore, are expected to be chiefly due to the different charge-charge interactions. As anticipated from its higher net charge, all measured pK values in 5K RNase are lowered relative to wild-type RNase Sa, with the largest decrease being 2.2 pH units for Glu14. The pK differences (pK(Sa)-pK(5K)) calculated using a simple model based on Coulombs Law and a dielectric constant of 45 agree well with the experimental values. This demonstrates that the pK differences between wild-type and 5K RNase Sa are mainly due to changes in the electrostatic interactions between the ionizable groups. pK values calculated using Coulombs Law also showed a good correlation (R=0.83) with experimental values. The more complex model based on a finite-difference solution to the Poisson-Boltzmann equation, which considers desolvation and charge-dipole interactions in addition to charge-charge interactions, was also used to calculate pK values. Surprisingly, these values are more poorly correlated (R=0.65) with the values from experiment. Taken together, the results are evidence that charge-charge interactions are the chief perturbant of the pK values of ionizable groups on the protein surface, which is where the majority of the ionizable groups are positioned in proteins.

Chemical structure and translation inhibition studies of the antibiotic microcin C7.

Escherichia coli microcin C7 (MccC7) is an antibiotic that inhibits protein synthesis in vivo. It is a heptapeptide containing unknown modifications at the N and C termini (García-Bustos, J. F., Pezzi, N., and Méndez, E. (1985) Antimicrob. Agents Chemoth. 27, 791-797). The chemical structure of MccC7 has been characterized by use of 1H homonuclear and heteronuclear (C, N, P) nuclear magnetic resonance spectroscopy as well as mass spectrometry (1177 ± 1 Da). The heptapeptide Met-Arg-Thr-Gly-Asn-Ala-Asp is substituted at the N terminus by a N-formyl group. The C-terminal substituent consists of the phosphodiester of 5′-adenylic acid and n-aminopropanol (AMPap), which is linked via the phosphorus atom to an amide group, thus forming a phosphoramide. The main chain carbonyl of the C-terminal aspartic acid residue is connected via this amide bond to the modified nucleotide unit. MccC7 and the peptide unit inhibit protein translation in vitro while a synthetic analog of the AMPap substituent is not active. Neither the peptide nor the AMPap molecule has an effect on the growth of MccC7-sensible cells. Our results strongly suggest that the peptide is responsible for MccC7 antibiotic activity while the C-terminal substituent is needed for MccC7 transport. Implications of the structure determined in this work for MccC7 synthesis and mode of action are discussed.

Conformational investigation of designed short linear peptides able to fold into β-hairpin structures in aqueous solution

BACKGROUND Formation of secondary structure plays an important role in the early stages of protein folding. The conformational analysis of designed peptides has proved to be very useful for identifying the interactions responsible for the formation and stability of alpha-helices. However, very little is known about the factors leading to the formation of beta-hairpins. In order to get a good beta-hairpin-forming model peptide, two peptides were designed on the basis of beta-sheet propensities and individual statistical probabilities in the turn sites, together with solubility criteria. The conformational properties of the two peptides were analyzed by two-dimensional NMR methods. RESULTS Long-range cross-correlations observed in NOE and ROE spectra, together with other NMR evidence, show that peptide IYSNPDGTWT forms a highly populated beta-hairpin in aqueous solution with a type I beta-turn plus a G1 beta-bulge conformation in the chain-bend region. The analogous peptide with a Pro5 substituted by Ser forms, in addition to the previous conformation, a second beta-hairpin with a standard type I beta-turn conformation, and the two forms are in fast dynamic equilibrium with one another. The effect of pH demonstrates the existence of a stabilizing interaction between the Asn and Asp sidechains. The populations of beta-hairpin conformations increase in the presence of trifluoroethanol (a structure-enhancing solvent). On the other hand, some residual structure persists at a high denaturant concentration (8 M urea). CONCLUSIONS This work highlights the importance of the beta-turn residue composition in determining the particular type of beta-hairpin adopted by a peptide, though a role of interstrand sidechain interactions in the stabilization of the formed beta-hairpin is not discarded. The fact that trifluoroethanol can stabilize alpha-helices or beta-hairpins depending on the intrinsic properties of the peptide sequence is again shown. An additional example of the presence of residual structure under denaturing conditions is also presented.

1H NMR and CD evidence of the folding of the isolated ribonuclease 50-61 fragment

In our search for potential folding intermediates we have prepared and characterized the fragment of RNase A corresponding to residues 50–61. Proton chemical shift variations with temperature, addition of stabilizing (TFE) or denaturing agents (urea) provide a strong experimental basis for concluding that in aqueous solution this RNase fragment forms an α‐helix structure similar to that in the intact RNase A crystal. This conclusion lends strong support to the idea that elements of secondary structure (mainly α‐helices) can be formed in the absence of tertiary interactions and act as nucleation centers in the protein folding process.

Low‐temperature 1H‐NMR evidence of the folding of isolated ribonuclease S‐peptide

The temperature (−7°C to 45°C, pH 5.4) and pH (0°C) dependence of 1H chemical shifts of ribonuclease S‐peptide (5 mM, 1 M NaCl) has been measured at 360 MHz. The observed variations evidence the formation of a partial helical structure, involving the fragment Thr‐3—Met‐13. Two salt‐bridges stabilize the helix: those formed by Glu‐9−…His‐12+ and Glu‐2−…Arg‐10+. The structural features deduced from the 1H‐NMR at low temperature for the isolated S‐peptide are compatible with the structure shown by the same molecule in the ribonuclease S crystal.

Factors involved in the stability of isolated β-sheets: Turn sequence, β-sheet twisting, and hydrophobic surface burial

We have recently reported on the design of a 20‐residue peptide able to form a significant population of a three‐stranded up‐and‐down antiparallel β‐sheet in aqueous solution. To improve our β‐sheet model in terms of the folded population, we have modified the sequences of the two 2‐residue turns by introducing the segment DPro‐Gly, a sequence shown to lead to more rigid type II′ β‐turns. The analysis of several NMR parameters, NOE data, as well as ΔδCαH, ΔδCβ, and ΔδCβ values, demonstrates that the new peptide forms a β‐sheet structure in aqueous solution more stable than the original one, whereas the substitution of the DPro residues by LPro leads to a random coil peptide. This agrees with previous results on β‐hairpin‐forming peptides showing the essential role of the turn sequence for β‐hairpin folding. The well‐defined β‐sheet motif calculated for the new designed peptide (pair‐wise RMSD for backbone atoms is 0.5 ± 0.1 Å) displays a high degree of twist. This twist likely contributes to stability, as a more hydrophobic surface is buried in the twisted β‐sheet than in a flatter one. The twist observed in the up‐and‐down antiparallel β‐sheet motifs of most proteins is less pronounced than in our designed peptide, except for the WW domains. The additional hydrophobic surface burial provided by β‐sheet twisting relative to a “flat” β‐sheet is probably more important for structure stability in peptides and small proteins like the WW domains than in larger proteins for which there exists a significant contribution to stability arising from their extensive hydrophobic cores.

NMR and SAXS characterization of the denatured state of the chemotactic protein Che Y: Implications for protein folding initiation

The denatured state of a double mutant of the chemotactic protein CheY (F14N/V83T) has been analyzed in the presence of 5 M urea, using small angle X‐ray scattering (SAXS) and heteronuclear magnetic resonance. SAXS studies show that the denatured protein follows a wormlike chain model. Its backbone can be described as a chain composed of rigid elements connected by flexible links. A comparison of the contour length obtained for the chain at 5 M urea with the one expected for a fully expanded chain suggests that ∼25% of the residues are involved in residual structures. Conformational shifts of the α‐protons, heteronuclear 15N‐{1H} NOEs and 15N relaxation properties have been used to identify some regions in the protein that deviate from a random coil behavior. According to these NMR data, the protein can be divided into two subdomains, which largely coincide with the two folding subunits identified in a previous kinetic study of the folding of the protein. The first of these subdomains, spanning residues 1–70, is shown here to exhibit a restricted mobility as compared to the rest of the protein. Two regions, one in each subdomain, were identified as deviating from the random coil chemical shifts. Peptides corresponding to these sequences were characterized by NMR and their backbone 1H chemical shifts were compared to those in the intact protein under identical denaturing conditions. For the region located in the first subdomain, this comparison shows that the observed deviation from random coil parameters is caused by interactions with the rest of the molecule. The restricted flexibility of the first subdomain and the transient collapse detected in that subunit are consistent with the conclusions obtained by applying the protein engineering method to the characterization of the folding reaction transition state.

Structural analysis of peptides encompassing all α-helices of three α/β parallel proteins: Che-Y, flavodoxin and P21-Ras: Implications for α-Helix stability and the folding of α/β parallel proteins

In an attempt to delineate the early folding events of structurally related proteins with no sequence homology, peptides including all five α-helices of three α/β parallel open-sheet proteins, Che-Y, flavodoxin and P21-ras, have been analyzed by circular dichroism (far-UV CD) and nuclear magnetic resonance (NMR) in water and 30% (v/v) trifluoroethanol (TFE). Comparison between the helical content estimations from far-UV CD and the results from the NMR analysis renders a reasonably good qualitative correlation, indicating that the same phenomenon is underlined by both methods. Helix limits, as indicated by the existence of ( i,i + 3) nuclear Overhauser effect (NOE) cross-correlations and significant up-field conformational shifts of the C α H protons, are practically coincident with those in the folded protein. On the other hand, the conformation of the side-chains differs markedly from those in the folded protein. Observation of NOE cross-correlations between pairs of residues at positions i,i + 3 has been used to statistically quantify free energies of i,i + 3 side-chain-side-chain interactions between the different pairs of residues in an α-helix. This analysis indicates that interactions between hydrophobic side-chains seem to be quite favorable for helix formation. The behaviour in aqueous solution of the structural equivalent peptides for the three proteins is quite unrelated except for the peptides corresponding to helices two and five. We postulate that, in the α/β parallel proteins, those helices that join two β-strands flanking another non-consecutive β-strand should not be stable for folding reasons.