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M Rico

Publications and source records attributed to M Rico.

At least 91 records · Page 5Linked to original sources
PubMed1995

1H-nmr studies on the structure of a new thionin from barley endosperm.

A new thionin from barley, omega-hordothionin, has been shown to exist in aqueous solution as a mixture of two different isoforms in a 3:2 ratio, as revealed by a complete analysis of its two-dimensional 1H-nmr spectra. The conformational heterogeneity arises from cis-trans isomerism about the Phe 12-Pro 13 peptide bond, where the major form corresponds to the cis conformation. The complete assignment of chemical shifts and nuclear Overhauser effects (NOEs) of the two isoforms allows a detailed comparative analysis of their conformational properties, even though a complete calculation of their solution structures is not possible because of a somewhat limited number of NOE constraints. Structures for the two isomers could be modeled, however, on the basis of the high structural homology between omega-hordothionin and related gamma-thionins, and under the conditions of satisfying all observed experimental data. The two isoforms adopt practically identical global folds and the structural changes imposed by cis-trans isomerization are confined to the region proximal to Pro 13. The cis-trans isomerism occurs in a conserved loop connecting the first beta-strand of the triple-stranded antiparallel beta-sheet and the alpha-helix. A comparative analysis of the sequences of this loop in the different thionins suggests that the cis-trans equilibrium about the X-Pro peptide bond depends on the size of the side chain of X (X = Gly in gamma-thionins and Phe in omega-thionin). The structural homology of this new thionin with gamma-thionins as well as with some scorpion toxins and insect defensins suggests that these proteins may share a common mode of functional activity.

Amino Acid Sequence↗
PubMed1995

Effects of acute administration of growth hormone-releasing hormone (GHRH) and oxytocin on somatotroph cells in sheep: morphometric study and growth hormone (GH) secretion.

In this work, the combined use of the morphometric study of somatotroph cells and plasma GH levels provided new data for the interpretation of the role played by OT and GHRH on GH cells. GHRH 1-29 (15 micrograms/kg), oxytocin (2.5 IU animal) or 1 ml saline solution were administered to male lambs. The GH plasma concentration was measured by RIA and for the morphometric study the cellular area, nuclear area and volume density of the somatotroph cells were measured in 1 micron semi-thin sections immunolabeled with avidin-biotin technique (ABC). The area under the GH response curve for 3 hours after injection was similar in both saline and OT-treated animals (24.8 +/- 9.1 and 31.4 +/- 14.7 micrograms/ml, respectively) and much lower than that observed in GHRH-treated animals (445.5 +/- 126.7 micrograms/ml). The cell area of somatotrophs was smaller in the GHRH-treated lambs and larger in the OT-treated lambs than in the control lambs (71.47 +/- 1.56, 91.42 +/- 1.72 and 83.1 +/- 1.74 microns 2, respectively). A similar change was observed in the nuclear area; it decreased in the GHRH-treated lambs (21.61 +/- 0.52 microns 2) and increased in the OT-treated lambs (25.45 +/- 0.68 microns 2) with respect to the control group (23.75 +/- 0.44 microns 2). No significant differences were found in volume density.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

[Orthotopic heart transplantation in a patient with previously undiagnosed persistence of the left superior vena cava].

The amount of transplants has gone through a remarkable increase during the last years. As a result, congenital anomalies of little prevalence appear more and more often while performing the transplants. The persistence of upper cava vein is one of them and, if not linked to other anomalies, it usually remains unnoticed. This article presents a case of an orthotopic cardiac transplant in a patient with an upper cava vein duplication not diagnosed before undergoing surgery, and it compares the way it was handled to diverse techniques previously described.

Adult↗
PubMed1994

NMR solution structure of the C-terminal fragment 255-316 of thermolysin: a dimer formed by subunits having the native structure.

The solution structure of the C-terminal fragment 255-316 of thermolysin has been determined by two-dimensional proton NMR methods. For this disulfide-free fragment there was a previous proposal according to which it would fold into a stable helical structure forming a dimer at concentrations above 0.06 mM. A complete assignment of the proton NMR resonances of the backbone and amino acid side chains of the fragment was first performed using standard sequential assignment methods. On the basis of 729 distance constraints derived from unambiguously assigned nuclear Overhauser effect (NOE) proton connectivities, the three-dimensional structure of a monomeric unit was then determined by using distance geometry and restrained molecular dynamic methods. The globular structure of fragment 255-316 of thermolysin in solution, composed of three helices, is largely coincident with that of the corresponding region in the crystallographic structure of intact thermolysin [Holmes, M. A., & Matthews, B. W. (1982) J. Mol. Biol. 160, 623-639]. This fact allowed identification as intersubunit of up to 52 NOE cross correlations, which were used to dock the two subunits into a symmetric dimer structure. The obtained dimeric structure served as the starting structure in a final restrained molecular dynamic calculation subjected to a total of 1562 distance constraints. In the resulting dimeric structure, the interface between the two subunits, of a marked hydrophobic character, coincides topologically with the one between the 255-316 fragment and the rest of the protein in the intact enzyme. The present work decisively shows that the thermolysin fragment 255-316 can attain a stable and nativelike structure independently of the rest of the polypeptide chain. Considering that the thermolysin molecule is constituted of two structural domains of equal size (residues 1-157 and 158-316), the results of this study show that autonomously folding units can be substantially smaller than entire domains.

Amino Acid Sequence↗
PubMed1994

Helix stop and start signals in peptides and proteins. The capping box does not necessarily prevent helix elongation.

Recently, several papers have addressed the existence of helix stop signals at the beginning of alpha-helices. It has been indicated that the existence of a reciprocal backbone-side-chain hydrogen-bond interaction, designated the capping box, could be one of these signals. The fingerprint sequence of this capping box is Ser/Thr-X-X-Glu/Gln. In the fifth alpha-helix of the chemotactic alpha/beta parallel protein CheY there is such a sequence in the middle of the helix. In a peptide corresponding to this alpha-helix the capping box is bypassed, as deduced from NMR analysis. However, making the peptide shorter so that the capping box fingerprint is closer to the beginning of the peptide results in the formation of the capping box. These results indicate that, although the capping box could play a role in stabilizing and nucleating helical peptides in solution, it is not necessarily a stop signal and can be bypassed when favourable interactions exist between the surrounding residues.

Amino Acid Sequence↗
PubMed1994

1H-NMR assignment and solution structure of human acidic fibroblast growth factor activated by inositol hexasulfate.

A major fragment of human acidic fibroblast growth factor of 132 amino acid residues is shown to be as active and stable as the 139 residue molecule initially described, and commonly used in physiological studies. It is shown that inositol hexasulfate is a good substitute for heparin in both activating and protecting acidic fibroblast growth factor. The complex between the shortened form of the protein and inositol hexasulfate was used to determine the structure of activated acidic fibroblast growth factor in solution. The 1H-NMR spectrum of the complex was totally assigned, and a low-resolution, three-dimensional structure of the protein computed. The global fold of the activated acidic fibroblast growth factor is similar to that proposed for a crystallized variant of the protein obtained by genetic engineering whose activity is not dependent on heparin. The inositol hexasulfate binds to the protein through the positively charged groups of Lys126, Lys127, Arg133 and Lys142 side-chains. The computed three-dimensional structure suggests that inositol hexasulfate may stabilize and activate the protein by conferring rigidity to the hairpin involving beta-strands 10 and 11.

Amino Acid Sequence↗
PubMed1994

NMR solution structure of the isolated N-terminal fragment of protein-G B1 domain. Evidence of trifluoroethanol induced native-like beta-hairpin formation.

The solution structure of the isolated N-terminal fragment of streptococcal protein-G B1 domain has been investigated in H2O and TFE/H2O solution by CD and NMR to gain insight into the possible role that native beta-hairpin secondary structure elements may have in early protein folding steps. The fragment also has been studied under denaturing conditions (6 M urea), and the resulting NMR chemical shifts were used as a reference for the disordered state. On the basis of CD and NMR data, it is concluded that in aqueous solution the fragment is basically flexible, with two local low populated chain bends involving residues 8-9 and 14-15, respectively, in close agreement with secondary structure predictions, a structure that is different from the final folded state of that segment of the protein. The changes in the CD spectrum, the presence of several medium-range NOEs plus two long-range NOEs, and the sign of the H alpha conformational shifts reveal that the addition of TFE facilitates the formation of a set of transient beta-hairpins involving essentially the same residues that form the native beta-hairpin found in the final three-dimensional structure of the B1 domain. The stabilization of native-like structures by TFE is known to occur for helices, but, to our knowledge, this is the first time the stabilization of a native-like beta-hairpin structure by TFE is reported. Since long-range tertiary interactions are absent in the isolated fragment, our results support the idea that, in addition to helices, beta-hairpins may play an active role in directing the protein folding process.

Amino Acid Sequence↗
PubMed1994

The residues Leu(Ile)475-Ile(Leu, Val, Ala)476, contained in the extended carboxyl cytoplasmic tail, are critical for targeting of the resident lysosomal membrane protein LIMP II to lysosomes.

LIMP II, a type II lysosomal integral membrane protein, and the CD36/LIMP II construct are targeted to lysosomes by means of a signal expressed in the tyrosine-lacking carboxyl cytoplasmic tail of LIMP II (Vega, M. A., Rodriguez, F., Seguí, B., Calés, C., Alcalde, J., and Sandoval, I. V. (1991) J. Biol. Chem. 266, 16269-16272; Vega, M. A., Seguí-Real, B., Garcia, J. A., Calés, C., Rodriguez, F., Vandekerckhove, J., and Sandoval, I. V. (1991) J. Biol. Chem. 266, 16818-16824). Substitution of Leu475 with Ile resulted in a decreased efficiency of targeting. Mutant forms produced by substituting Leu475 by hydrophobic residues with either large (Val) or small (Ala, Gly) side chains, or by a charged residue (Asp), showed inhibited targeting. In contrast, the contiguous Ile476 residue could be replaced by either Leu, without loss in the efficiency of targeting, or by Val or Ala, with some impediment. Substitution of Ile476 by either Gly or Asp inhibited completely the targeting. The addition of the sequence Ser-Trp-Asp to the carboxyl end of the construct did not interfere with targeting. Data from 1H NMR analysis of the icosapeptide corresponding to the carboxyl cytoplasmic tail of LIMP II indicated the predominance of structures with extended random coil conformations, suggesting that the targeting signal is contained in a domain with an extended configuration.

Alanine↗
PubMed1994

Helix formation by the phospholipase A2 38-59 fragment: influence of chain shortening and dimerization monitored by nmr chemical shifts.

The solution structure of a peptide fragment corresponding to the 38-59 region of porcine phospholipase A2 has been investigated using CD, nmr chemical shifts, and nuclear overhauser effects (NOEs). This isolated fragment of phospholipase forms an alpha-helix spanning residues 38-55, very similar to the one found in the native protein, except for residues 56-58, which were helical in the crystal but found random in solution. Addition of triflouroethanol (TFE) merely increased helix population but it did not redefine helix limits. To investigate how the folding information, in particular that concerning eventual helix start and stop signals, was coded in this particular amino acid sequence, the helices formed by synthetic peptides reproducing sections of this phospholipase 38-59 fragment, namely 40-59, 42-59, 38-50, and 45-57, were characterized using NOEs and helix populations quantitatively evaluated on different peptide chain segments using nmr chemical shifts in two solvents (H2O and 30% TFE/H2O). A set of nmr spectra was also recorded and assigned under denaturing conditions (6M urea) to obtain reliable values for the chemical shifts of each peptide in the random state. Based on chemical shift data, it was concluded that the helix formed by the phospholipase 38-59 fragment was not abruptly, but progressively, destabilized all along its length by successive elimination of residues at the N end, while the removal of residues at the C end affected helix stability more locally and to a lesser extent. These results are consistent with the idea that there are not single residues responsible for helix initiation or helix stability, and they also evidence an asymmetry for contributions to helix stability by residues located at the two chain ends. The restriction of molecular mobility caused by linking with a disulphide bridge at Cys 51 two identical 38-59 peptide chains did not increase helix stability. The helix formed by the covalently formed homodimer was very similar in length and population to that formed by the monomer.

Amino Acid Sequence↗
PubMed1993

1H- and 15N-NMR assignment and solution structure of the chemotactic Escherichia coli Che Y protein.

Che Y is a 129-residue parallel alpha/beta protein involved in bacterial chemotaxis. We have used this protein as a model to study the folding reaction of parallel alpha/beta proteins. As a first step we carried out the complete assignment of the 1H and 15N spectra from Escherichia coli Che Y protein on the basis of two-dimensional 1H homonuclear and 1H-15N heteronuclear experiments by using sequence-specific methods. Our assignments differ from the preliminary assignments made by Kar et al. [Kar, L., Matsumura, P. & Johnson, M.E. (1992) Biochem. J. 287, 521-531] of aromatic residues obtained by comparison of NOEs with short proton-proton distances in the crystal structure of Che Y. The analysis of the extension of the secondary elements, as well as a preliminary calculation of the three-dimensional structure, indicate that the solution structure is closely coincident with the single crystal structure determined by X-ray diffraction.

Amino Acid Sequence↗
PubMed1993

Three-dimensional structure of omega-conotoxin GVIA determined by 1H NMR.

omega-Conotoxin GVIA, a peptide of 27 amino acid residues and three disulfide bridges, has been studied by NMR techniques. The complete assignment of the corresponding proton NMR spectra was performed by two-dimensional sequence specific methods at 288 K and pH 3.5. On the basis of 169 distance restraints derived from this analysis, the three-dimensional structure was obtained. A total of 30 initial structures were generated by distance geometry methods and further refined by restrained energy minimization techniques yielding a final set of 8 structures. The mean root-mean-square deviation between each of the 8 structures and the mean atomic coordinates for all residues is 0.82 +/- 0.06 A for the backbone atoms and 1.45 +/- 0.18 A for all non-H atoms. The structure shows a globular folding pattern that is stabilized by the three disulfide linkages and a number of intramolecular hydrogen bonds. A total of 14 hydroxyl groups are found at the periphery fully exposed to the solvent. These groups, together with the charged side chains of Lys and Arg residues emerging radially from the peptide core, provide specific recognition elements for the interaction of this toxin with neuronal calcium channels.

Amino Acid Sequence↗
PubMed1993

High-resolution three-dimensional structure of ribonuclease A in solution by nuclear magnetic resonance spectroscopy.

High-resolution three-dimensional structures of bovine pancreatic ribonuclease A in aqueous solution have been determined by nuclear magnetic resonance (NMR) spectroscopy combined with restrained molecular dynamics calculations. The structures are based on: (1) 464 interproton distance constraints with accurate upper and lower limits, determined from build-up rates of nuclear Overhauser effects (NOE) by using the complete relaxation matrix; (2) 999 more approximate upper limits for interproton distances; and (3) 42 dihedral angle constraints (37 for phi and 5 for chi 1). A total of 16 structures were calculated, which show a root-mean-square (r.m.s.) deviation of 0.66 A for the backbone atoms and 1.68 A for all heavy-atoms. The converged structures are highly similar to those found in the crystal state. r.m.s. deviation of backbone atom positions in the crystal as compared to those in the average solution structure is 0.92 A. Observed differences are concentrated in loop regions and in the neighborhood of His119 and His48 side-chains. Dynamic aspects, such as H/D amide proton exchange and side-chain mobility have been examined.

Amides↗
PubMed1993

CD and 1H-NMR studies on the conformational properties of peptide fragments from the C-terminal domain of thermolysin.

The propensity of the peptide fragments 233-248, 245-260, 258-276, 279-298 and 299-316 from the thermolysin C-terminal domain to form non-random structures has been examined by CD and two-dimensional NMR spectroscopy. The conformational properties of these fragments have been studied in aqueous solution and in the mixed solvent trifluoroethanol/H2O (3:7 by vol.). Small but detectable populations of helical structures (up to 10-20%) in aqueous solution have been found for the fragments 233-248, 279-298 and 299-316. These populations are remarkably enhanced (50-70%) in the more hydrophobic mixed solvent, where the fragment 258-276 also forms a comparable helical population. These four fragments are helical in the native crystal structure and the spanning of the corresponding helices in the isolated peptides in solution matches very closely the ones in the native structure. In contrast, the fragment 245-260, an omega-loop in the crystal, remains unstructured in both solvents. Medium-range NOE between protons in sidechains indicate the adoption of preferred sidechain conformations accompanying helix formation. Results are in agreement with the framework model of folding, in which native elements of secondary structure are formed first and folding follows from the collapse of these structural elements.

Amino Acid Sequence↗
PubMed1993

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.

Amino Acid Sequence↗
PubMed1992

Periodic properties of proton conformational shifts in isolated protein helices. An experimental study.

In this work, the helix-forming residues in fragments of several proteins (ribonuclease, thermolysin, tendamistat and angiogenin) were identified by NOE and the helix proton shifts were measured as delta changes associated with helix-population increments driven by trifluoroethanol addition. When estimated in this way, a regular pattern of helix conformational shifts was clearly seen in the delta delta versus sequence profiles of all the peptides studied. The helix periodicity of the H alpha and H beta resonances was especially clear, an observation that earlier statistical studies of protein delta values failed to predict. Amide protons showed the largest helix shifts, but with a less-sharply defined periodic character. Aromatic residues considerably distorted the periodicity of the helix amide shifts in some peptides, as evidenced by the delta shifts of a RNase A fragment 1-15 analog in which the two aromatic residues were replaced by Ala. The relationship between helix periodicity and peptide amphiphatic character is discussed.

Amino Acid Sequence↗
PubMed1992

The homologous angiogenin and ribonuclease N-terminal fragments fold into very similar helices when isolated.

The solution structure of the N-terminal hexadecapeptide of human angiogenin, a protein of unknown tertiary structure, has been precisely delineated by the combined use of CD, NOE and secondary shift data. A helix that starts just after Ser 3 and ends at Asp 14 was stabilized in 30% trifluoroethanol. This helix is strikingly similar in origin and length to the one formed by its homologous, the S-peptide of Ribonuclease (conformationally reexamined here), despite their quite different sequences (only four conserved residues). These results support the idea that individual start and stop signals indeed govern the location and size of natural isolated helices.

Amino Acid Sequence↗
PubMed1991

Tendamistat (12-26) fragment. NMR characterization of isolated beta-turn folding intermediates.

In order to determine whether regions of a protein that are turns in the native structure are able to maintain such a structure when isolated, we have studied the conformational properties of various peptide fragments corresponding to the 12-26-peptide region of the alpha-amylase inhibitor tendamistat, by NMR. Amide solvent accessibility, NOE spectroscopy (NOESY) and rotating-frame NOE spectroscopy (ROESY) data strongly support the conclusion that the 12-26 and 15-23 peptides adopt in aqueous solution, a set of turn-like structures located around the central region of their corresponding polypeptidic chains, the same region where a beta turn exists in the native protein. Such a set of structures are destabilized when one residue located within the native beta turn of the 15-23 peptide is modified Trp18----Ser. Our results indicate that the tendency to bend in a predetermined region of a protein chain seems to exist from the very beginning of the folding process and therefore it could drive the folding instead of being a consequence of the tertiary assembly of the protein.

Amides↗