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M Llinás

Publications and source records attributed to M Llinás.

At least 73 records · Page 4Linked to original sources
PubMed1985

Proton Overhauser experiments on kringle 4 from human plasminogen. Implications for the structure of the kringles' hydrophobic core.

1H-NMR Overhauser experiments at 300 and 600 MHz have been implemented on the isolated kringle 4 fragment of human plasminogen. This study shows that Leu46 and Leu77 CH3 delta,delta' groups, as well as two threonine CH3 gamma and a methionine S-CH epsilon (probably Met48) groups, are in efficient dipolar contact with histidine and aromatic side-chains. In particular, the experiments reveal that of the two Leu46 CH3 delta,delta' groups, one is in efficient contact with tryptophan (Trp25 and Trp62) indole rings while the other interacts with a tyrosine (probably Tyr41) phenol. Leu46 appears also to be close to an Ala CH3 beta group. Such a hydrophobic cluster appears to be contiguous to Trp72, hence to Arg71, residues that are through to be part of the lysine-binding site. Acid-base titration experiments show that the buried methionine S-CH3 epsilon group senses a neighboring ionizable group of pK*1 = 3.76, suggesting presence of a carboxyl anionic group (probably an aspartic acid side-chain) in the vicinity of the hydrophobic core. A preliminary model is proposed for the overall folding of the kringle polypeptide chain.

Amino Acid Sequence↗
PubMed1985

Analysis and identification of aromatic signals in the proton magnetic resonance spectrum of the kringle 4 fragment from human plasminogen.

The aromatic 1H NMR spectrum of the kringle 4 domain from human plasminogen has been reexamined in order to identify signals stemming from individual residues. Acid-base titration, nuclear Overhauser effect experiments, and two-dimensional correlated spectroscopies have been implemented in order to analyze the spectrum both in the presence and in the absence of ligands. All six histidyl imidazole singlets have been recognized and paired according to their common side-chain origin. A similar identification has been achieved for the three sets of tryptophanyl resonances, and for Trp-I, the correspondence between indole singlet and multiplets is unambiguously established. The single phenylalanyl side chain and all tyrosyl phenol spin systems have been identified. Titration experiments indicate that one or two of the tryptophans are in the vicinity of carboxyl groups. It is shown that the spectrum for one tyrosyl ring, Tyr-V, undetectable at approximately 300 MHz, becomes visible at 600 MHz, reflecting slow motion on the NMR time scale and a constrained location within the kringle. A simulation of the complete kringle 4 aromatic spectrum is included.

Humans↗
PubMed1985

Macro- and micro-stabilities of the kringle 4 domain from plasminogen. The effect of ligand binding.

1H-NMR spectra of kringle 4 from human plasminogen have been recorded over wide pH* and temperature ranges, both in the presence and in the absence of p-benzylaminesulfonic acid (BASA). Several resonances exhibit chemical shift differences between kringle folded and unfolded forms which are sufficiently well resolved to allow for a determination of equilibrium Van't Hoff enthalpies and entropies for unfolding. The interaction with BASA shifts the kringle unfolding temperature from approximately 335 degrees K to approximately 343 degrees K. The pH* range of stability is also wider for the complex than for the free kringle: in the acidic range the pH* of half-unfolding, pHu*, is decreased from 2.8 for the unligated polypeptide to approximately 2.0 in the presence of BASA, while in the basic range pHu*, shifts from approximately 10.8 to 11.5. However, in contrast with what is observed at acidic pH*, unfolding at basic pH* leads to irreversible denaturation and exhibits a sharp, order-disorder transition both in the presence and in the absence of ligand. The structural stabilization conferred by the ligand is accompanied by a drastic reduction of the average rate of 1H-2H exchange in 2H2O under conditions that preclude a major cooperative unfolding. Thus, macro- and micro-stabilities of kringle domains appear to be highly correlated.

Benzylamines↗
PubMed1984

Characterization of the aromatic proton magnetic resonance spectrum of crambin.

The hydrophobic protein crambin (Mr 4715) has an aromatic content of one phenylalanyl residue (site 13) and two tyrosyl residues (sites 29 and 44). The aromatic residues have been studied by 1H NMR spectroscopy at 300 and 600 MHz for crambin dissolved in deuterated glacial acetic acid and in aqueous organic media. In particular, a 3:1 acetone/water mixture affords a solvent system of low viscosity, which yields very narrow line protein spectra. The aromatic proton spectrum is unusual in that signals are doubled. Spectral simulation of modified and unmodified crambin aromatic spin systems can be accomplished only by assuming that the protein is a mixture of two species. Dynamic 1H-[1H] Overhauser experiments centered on the aromatic doublets of Tyr29 indicate that the two species do not interconvert within 2 s. NMR spectra of crambin samples obtained by repeated crystallization in 17:3 acetone/water indicate that the process enriches the mixture with one component that does not convert to the other, even upon heating of the sample to 351 K. The combined evidence strongly favors the view that the doubled spectrum results from a compositional heterogeneity, most likely a mixture of two homologues, rather than from an equilibrium between interconvertible forms. This is important in view of the fact that its crystallographic structure, solved to 1.5-A resolution [Hendrickson, W.A., & Teeter, M. M. (1981) Nature (London) 290, 107-113], is based on samples containing the two species.(ABSTRACT TRUNCATED AT 250 WORDS)

Cystine↗
PubMed1982

Proton magnetic resonance studies of barley and wheat thionins: structural homology with crambin.

The thionins comprise a group of very basic proteins of Mr approximately 5000 found in the seeds of Gramineae. They each contain 45 amino acid residues arranged along a single polypeptide chain that is constrained by four disulfide bridges. Five thionins of known sequence, from barley and wheat, have been investigated and compared by 1H NMR spectroscopy at 600 MHz. From their spectral characteristics it is concluded that the five proteins have very similar, nonrandom conformations in 2H2O solution. Moreover, on the basis of selective nuclear Overhauser experiments at 300 MHz, features of their secondary and tertiary structures are shown to be similar to those of crambin, a related, hydrophobic protein extracted from seeds of the crucifer Crambe abyssinica. The strong compositional homology of the thionins facilitates the assignment of methyl and aromatic resonances, as only a few residues are replaced and these are at known sites. The substitution of leucine for an isoleucine does not affect significantly the local magnetic environment, suggesting that those isomeric side chains easily accommodate the same spatial constraints. A fast hydrogen-deuterium exchange is observed at pH 6.25, 25 degrees C. This indicates that, although of folded conformation, the thionins are structurally flexible polypeptides that efficiently expose all amides to the solvent.

Amino Acid Sequence↗
PubMed1982

Siderochromes from Pseudomonas fluorescens. I. Isolation and characterization.

Several iron-binding pigments (siderochromes) produced by Pseudomonas fluorescens have been isolated and partially characterized. They include ferribactin and various forms of pyoverdine, as well as some previously unreported compounds. In particular, the existence of ferribactin has been independently confirmed for the first time. Column and thin layer chromatographic procedures have been developed to fractionate, purify, and identify the siderochromes. We find ferribactin to contain nine amino acids, one residue each of glutamine, tyrosine, and glycine, and two each of serine, lysine, and N-hydroxyornithine, rather than 10 as earlier reported. Pyoverdine is a peptide with the same composition as ferribactin except for the absence of glutamine and the substitution of a fluorescent chromophore for tyrosine. Paper electrophoresis reveals an extra ionizable group in ferric pyoverdine relative to pyoverdine or ferribactin which provides that complex a definite cathodic mobility at pH 3. Optical spectra of the pyoverdine fluorescent component indicate that, in conjunction with the two hydroxamate groups, it is involved in the metal ion coordination, conferring on pyoverdine a dramatically increased affinity for Fe(III) relative to ferribactin.

Chromatography, Thin Layer↗
PubMed1982

Siderochromes from Pseudomonas fluorescens. II. Structural homology as revealed by NMR spectroscopy.

Ferribactin and the pyoverdines, siderochromes that are obtained from liquid cultures of Pseudomonas fluorescens cells, have been studied and compared by 1H and 13C NMR spectroscopy. The proton spectra of the iron-free compounds show that the pyoverdines share with ferribactin a common feature, formyl hydroxamic acid groups, that previously had only been observed in hadacidin, and antitumor antibiotic produced by Penicillium frequentans. The 1H and 13C NMR data confirm that ferribactin is a nonapeptide that contains two residues each of lysine and N6-formyl-N6-hydroxyornithine. This corrects an earlier report (Maurer, B., Müller, A., Keller-Schierlein, W., and Zähner, H. (1968) Arch. Mikrobiol. 60, 326-339) ascribing two acetyl hydroxamic acid groups and three lysyl residues to ferribactin. Similarly, the spectroscopic data show that pyoverdine lacks the Glx and Tyr residues present in ferribactin. On the basis of the compositional analogy exhibited by pyoverdine and ferribactin, it is suggested that the two siderochromes may be metabolically related. The 13C NMR spectra of pyoverdine indicate that its fluorescent component is a nine-carbon aromatic heterocycle, probably identical with an o-dihydroxyquinoline chromophore found in pseudobactin, a fluorescent siderophore produced by Pseudomonas B10.

Iron Chelating Agents↗
PubMed1979

Complete assignment of carbon signals in a stereospecific peptide via selective and single off-resonance proton decoupling experiments. Analysis of the carbon-13 nuclear magnetic resonance spectrum of alumichrome at 67.88 MHz.

Polypeptides and proteins in native conformation exhibit 13C NMR spectra which are highly nondegenerate. Assignment of resonances to carbons in particular residues is hence a prerequisite for a structural analysis of the spectroscopic data. For nonprotonated carbonyl carbons, the assignment can be achieved by selective (1H alpha)13C' 2J decoupling. Using this method, we have assigned the Orn1 and Gly2 carbonyl resonances in alumichrome at 67.9 MHz. We show that a single off-resonance experiment with the decoupling frequency centered in the aliphatic proton spectrum is sufficient to assign unequivocally all the protonated carbon resonances via analysis of the reduced 1J heteronuclear splittings. Alumichrome thus becomes the first complex polypeptide spin system whose 1H, 15N, and now 13C nuclear resonances have been fully identified to date. 13C chemical shifts and 1H--13C spin--spin couplings are discussed in terms of structural strain leading to specific orbital hybridizations and on the basis of polarization effects due to electron density shifts toward hydrogen-bonding and metal-binding sites. A number of 3J(13C--C--C--1H) coupling constants measured on selected multiplets after resolution enhancement were used to derive the x-related Karplus relationship 3J(theta) = (10.2 cos2 theta -- 1.3 cos theta + 0.2) Hz.

Aluminum↗
PubMed1978

Amide proton spin-lattice relaxation in polypeptides. A field-dependence study of the proton and nitrogen dipolar interactions in alumichrome.

The proton nuclear magnetic resonance (NMR) spin-lattice relaxation of all six amides of deferriferrichrome and of various alumichromes dissolved in hexadeutero-dimethylsulfoxide have been investigated at 100, 220, and 360 MHz. We find that, depending on the type of residue (glycyl or ornithyl), the amide proton relaxation rates are rather uniform in the metal-free cyclohexapeptide. In contrast, the (1)H spinlattice relaxation times (T(1)'s) are distinct in the Al(3+)-coordination derivative. Similar patterns are observed in a number of isomorphic alumichrome homologues that differ in single-site residue substitutions, indicating that the spin-lattice relaxation rate is mainly determined by dipole-dipole interactions within a rigid molecular framework rather than by the specific primary structures. Analysis of the data in terms of (1)H-(1)H distances (r) calculated from X-ray coordinates yields a satisfactory linear fit between T(1) (-1) and Sigmar(-6) at the three magnetic fields. Considering the very sensitive r-dependence of T(1), the agreement gives confidence, at a quantitative level, both on the fitness of the crystallographic model to represent the alumichromes' solution conformation and on the validity of assuming isotropic rotational motion for the globular metallopeptides. An extra contribution to the amide proton T(1) (-1) is proposed to mainly originate from the (1)H-(14)N dipolar interaction: this was supported by comparison with measurements on an (15)N-enriched peptide. The nitrogen dipolar contribution to the peptide proton relaxation is discussed in the context of {(1)H}-(1)H nuclear Overhauser enhancement (NOE) studies because, especially at high fields, it can be dominant in determining the amide proton relaxation rates and hence result in a decreased effectiveness for the (1)H-(1)H dipolar mechanism to cause NOE's. From the slope and intersect values of T(1) (-1) vs. Sigmar(-6) linear plots, a number of independent estimates of tau(r), the rotational correlation time, were derived. These and the field-dependence of the T(1)'s yield a best estimate approximately 0.37 ns, in good agreement with 0.38 ns [unk] [unk] 0.41 ns, previously determined from (13)C and (15)N spin-lattice relaxation data.

Aluminum↗
PubMed1976

The structure of two alanine containing ferrichromes: sequence determination by proton magnetic resonance.

Metal coordination confers an extraordinary structural stability to the ferrichromes which, independent of their variable amino acid composition, results in a basically unperturbed conformation for all the homologous peptides in the series. The proton magnetic resonance (pmr) characteristics for Al3+ analogues (alumichromes) reflect this conformational isomorphism in usual solvents so that single site substitutions are clearly recognized in the pmr spectra. Thus, the substitution of glycine by L-alanine or L-serine introduce new resonances characteristic of the sidechains and alter the pattern of the amide NH pmr region in that doublets substitute for glycyl triplets at the same site. Since for glycine- and L-serine containing alumichromes the resonances have already been identified, it is possible to unequivocally establish the primary structure of the two L-alanyl homologues ferrichrome C (see article) and sake colorant A (see article) on the basis of the comparative pmr spectra of their Al3+ analogues, namely, alumichrome C and alumisake. The resonance assignment, and hence the site occupancy, is substantiated by the temperature coefficients of the NH chemical shifts, rates of 1H-2H exchange and homonuclear proton spin decoupling experiments centered on the NH spectral region. Occupancy of site 1 by a glycine residue is observed for all known ferrichromes, which serves to conserve a "hairpin" turn. This method of obtaining sequence information should prove of general use for other systems of homologous polypeptides, provided their conformations are not affected by the residue substitutions.

Alanine↗