Biomedical subjects

M Llinás

Publications and source records attributed to M Llinás.

At least 55 records · Page 3Linked to original sources

PubMed1990

The solution conformations of ferrichrome and deferriferrichrome determined by 1H-NMR spectroscopy and computational modeling.

We have applied computational procedures that utilize nmr data to model the solution conformation of ferrichrome, a rigid microbial iron transport cyclohexapeptide of known x-ray crystallographic structure [D. van der Helm et al. (1980) J. Am. Chem. Soc. 102, 4224-4231]. The Al3+ and Ga3+ diamagnetic analogues, alumichrome and gallichrome, dissolved in d6-dimethylsulfoxide (d6-DMSO), were investigated via one- and two-dimensional 1H-nmr spectroscopy at 300, 600, and 620 MHz. Interproton distance constraints derived from proton Overhauser experiments were input to a distance geometry algorithm [T. F. Havel and K. Wüthrich (1984) Bull. Math. Biol. 46, 673-691] in order to generate a family of ferrichrome structures consistent with the experimental data. These models were subsequently optimized through restrained molecular dynamics/energy minimization [B. R. Brooks et al. (1983) J. Comp. Chem. 4, 187-217]. The resulting structures were characterized in terms of relative energies and conformational properties. Computations based on integration of the generalized Bloch equations for the complete molecule, which include the 14N-1H dipolar interaction, demonstrate that the x-ray coordinates reproduce the experimental nuclear Overhauser effect time courses very well, and indicate that there are no significant differences between the crystalline and solution conformations of ferrichrome. A similar study of the metal free peptide, deferriferrichrome, suggests that at least two conformers are present in d6-DMSO at 23 degrees C. Both are different from the ferrichrome structure and explain, through conformational averaging, the observed amide NH and CH alpha multiplet splittings. The occurrence of interconverting peptide backbone conformations yields an increased number of sequential NH-CH alpha and NH-NH Overhauser connectivities, which reflects the mean value of r-6 dependence of the dipolar interaction. Our results support the idea that, in the case of structurally rigid peptides, moderately accurate distance constraints define a conformational subspace encompassing the "true" structure, and that energy considerations reduce the size of this subspace. For flexible peptides, however, the straight-forward approach can be misleading since the nmr parameters are averaged over substantially different conformational states.

Amino Acid Sequence↗

PubMed1989

1H NMR structural characterization of a recombinant kringle 2 domain from human tissue-type plasminogen activator.

The kringle 2 domain of human tissue-type plasminogen activator (t-PA) has been characterized via 1H NMR spectroscopy at 300 and 620 MHz. The experiments were performed on the isolated domain obtained by expression of the 174-263 portion of t-PA in Escherichia coli [Cleary et al. (1989) Biochemistry 28, 1884-1891]. The spectrum of t-PA kringle 2 is characteristic of a globular structure and shows overall similarity to that of the plasminogen (PGN) kringle 4. Spectral comparison with human and bovine PGN kringle 4 identifies side-chain resonances from Leu46, which afford a fingerprint of kringle folding, and from most of the aromatic ring spin systems. Assignment of signals arising from the His13, His48a, and His64 side chains, which are unique to t-PA kringle 2, was assisted by the availability of a His64----Tyr mutant. Ligand-binding studies confirm that t-PA kringle 2 binds L-lysine with an association constant Ka approximately 11.9 mM-1. The data indicate that homologous or conserved residues relative to those that compose the lysine-binding sites of PGN kringles 1 and 4 are involved in the binding of L-lysine to t-PA kringle 2. These include Tyr36 and, within the kringle inner loop, Trp62, His64, Trp72, and Tyr74. Acid/base titration of aromatic singlets in the presence of L-lysine yields pKa* approximately 6.25 and approximately 4.41 for His13 and His64, respectively, and shows that the His48a imidazole group does not protonate down to pH* approximately 4.3. Thus, the His48a and His64 side chains are in solvent-shielded locations. As observed for the PGN kringles, the Trp62 indole group titrates with pKa* approximately 4.60, which indicates proximity of the side chain to a titratable carboxyl group, most likely that of Asp57 at the binding site. Several labile NH protons of t-PA kringle 2 exhibit retarded H-exchange kinetics, requiring more than a week in 2H2O for full deuteration in the presence of L-lysine at 37 degrees C. This reveals that kringle 2 is endowed with a compact, dynamically stable conformation. Proton Overhauser experiments in 1H2O, centered on well-resolved NH resonances between 9.8 and 12 ppm, identify signals arising from the His48a imidazole NH3 proton and the three Trp indole NH1 protons. A strong dipolar interaction was observed among the Trp25 indole NH1, the Tyr50 amide NH, and the His48a imidazole CH2 protons, which affords evidence for an aromatic cluster in t-PA kringle 2 similar to that found at the hydrophobic kernel of PGN kringles.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1989

1H NMR studies of aliphatic ligand binding to human plasminogen kringle 4.

A detailed 1H NMR analysis of ligand binding to the human plasminogen kringle 4 domain has been carried out at 300 MHz. The ligands that were investigated are N alpha-acetyl-L-lysine, L-lysine methyl ester, N alpha-acetyl-L-lysine methyl ester, L-lysine hydroxamic acid, trans-(aminomethyl)cyclohexanecarboxylic acid (AMCHA), and 4-(aminomethyl)bicyclo[2.2.2]octane-1-carboxylic acid (AMBOC). Specific ligand-binding effects were detected via two-dimensional COSY experiments. The side chains that are the most perturbed by ligand presence are those from Trp62, Phe64, and Trp72. Ligand-kringle saturation transfer (Overhauser) experiments show that the aromatic rings from these three residues, especially Trp72, are in direct contact with the ligand. These results add support to a previously reported model of the kringle 4 lysine-binding site [Ramesh, V., Petros, A. M., Llinás, M., Tulinsky, A., & Park, C. H. (1987) J. Mol. Biol. 198, 481-498] by which these aromatic groups are assigned a key role in establishing hydrophobic interactions with the ligand molecule. Equilibrium association constants (Ka) and kinetic rate constants (kon, koff) were determined for the binding of the various linear and cyclic ligands to kringle 4. We find that those ligands whose carboxylate function is blocked bind significantly weaker (Ka approximately less than 2 mM-1) than the corresponding analogues where the anionic center is present (Ka approximately greater than 20 mM-1), which underscores the relevance of the polar group in stabilizing the interaction with the kringle 4 binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Histidine↗

PubMed1989

Human plasminogen kringle 4. Crystallization and preliminary diffraction data of two different crystal forms.

Human plasminogen kringle 4 has been crystallized in two different crystal forms: monoclinic, a = 32.78(3), b = 49.17(2), c = 46.27(3) A, beta = 100.67 degrees, space group P2(1), four molecules/unit cell, two molecules/asymmetric unit; orthorhombic, a = 32.09(7), b = 49.14(6), c = 49.47(9) A, space group P2(1)2(1)2, four molecules/unit cell. Both crystal forms have a large protein fraction (66% for monoclinic and 62% for orthorhombic) and diffract x-rays to 2.0 A resolution. A self-rotation function has been calculated with monoclinic data indicating a non-crystallographic 2-fold rotation approximately parallel to a* (peak height of 14.3 x sigma). Cross-rotation function calculations are in progress utilizing the coordinates of the conserved structure of kringle 1 of prothrombin and plasminogen kringle 4.

Crystallization↗

PubMed1989

Ligand-binding effects on the kringle 4 domain from human plasminogen: a study by laser photo-CIDNP 1H-NMR spectroscopy.

Photo-chemically induced dynamic nuclear polarization (photo-CIDNP) one-dimensional and two-dimensional (2D) 1H-NMR techniques have been applied to the study of the kringle 4 domain of human plasminogen both ligand-free and complexed to the antifibrinolytic drugs epsilon-aminocaproic acid and p-benzylaminesulfonic acid (BASA). A number of aromatic side-chains (His3, Trp72, Tyr41, Tyr50 and Tyr74) appear to be exposed and accessible to 3-N-carboxymethyl-lumiflavin, the photopolarizing flavin dye, both in the presence and in the absence of ligands. A lesser exposure is observed for the Trp25 and Trp62 indole groups in the presence of BASA. The spin-spin (J-coupling) and dipolar (Overhauser) connectivities in the 2D experiments afford absolute assignment of aromatic resonances for the above residues, as well as of those stemming from the Trp72 ring in the presence of BASA. Moreover, a number of H beta resonances can be identified and sorted according to specific types of amino acid residues.

Aminocaproic Acid↗

PubMed1988

Analysis of the aromatic 1H-NMR spectrum of the kringle 5 domain from human plasminogen. Evidence for a conserved kringle fold.

A kringle 5 domain fragment from human plasminogen has been investigated by 1H-NMR spectroscopy at 300 MHz and 620 MHz. The study focuses on the kringle 5 aromatic spectrum as aromatic side chains appear to mediate the binding of benzamidine. Spin-echo experiments and acid/base-titration studies in conjunction with two-dimensional double-quantum and chemical-shift-correlated spectroscopies were used to identify individual spin systems. Sequence-specific assignments of aromatic resonances are derived from direct comparison of the kringle 5 spectrum with spectra of the homologous kringle 1 and kringle 4 domains of plasminogen. As previously observed for kringles 1 and 4, the pattern we detect for Tyr9 in kringle 5 reflects a slow conformational exchange between two states in equilibrium, one in which the Tyr9 ring is freely mobile and one in which its flip dynamics are constrained. Proton Overhauser experiments in 1H2O and in 2H2O have been used to probe aromatic ring interactions and to identify residues which are part of the hydrophobic core centered at the Leu46 side chain. Overall, the data indicate a strong structural homology among the three plasminogen kringles.

Histidine↗

PubMed1988

Analysis of the aliphatic 1H-NMR spectrum of plasminogen kringle 4. A comparative study of human, porcine, bovine and chicken homologs.

The aliphatic 1H-NMR spectrum of the kringle 4 domain of human plasminogen has been studied via two-dimensional chemical shift correlated (COSY) and nuclear Overhauser correlated (NOESY) experiments at 300 MHz and 620 MHz. A number of aliphatic proton spin systems have been identified and several definite assignments have been made. This was mainly achieved by comparison of the human kringle 4 spectrum with spectra of the porcine, bovine and chicken homologs and also with that of the kringle 1 from human plasminogen on which we have reported previously. The three valyl and two leucyl residues of human kringle 4 have been assigned. The eleven threonyl spin systems have been identified via a RELAYED-COSY experiment and Thr17 has been assigned. The three alanyl spin systems have been identified and assigned. Six seryl spin systems have been identified and the signals from the seven glycyl residues of human kringle 4 have been located with Gly45 assigned. Furthermore, 24 AMX spin systems have been mapped in the COSY spectrum of human kringle 4 and H alpha-H beta,beta' spin systems of Tyr2, Tyr41, Tyr50, Tyr74, Trp25 and Trp62 have been assigned. From the spectrum of a deglycosylated chicken homolog, the epsilon-methyl singlets of Met28 and Met48 have been assigned. Finally, ligand effects on selected aliphatic resonances were observed which could be analyzed in terms of residues likely to neighbor the kringle lysine-binding site.

Amino Acid Sequence↗

PubMed1988

Lysine/fibrin binding sites of kringles modeled after the structure of kringle 1 of prothrombin.

The Lys binding site of kringle 1 and 4 (K1 and K4) of plasminogen (PG) has been modeled on the basis of the three-dimensional structure of kringle 1 of prothrombin and 300- and 600-MHZ proton nuclear magnetic resonance observations. These structures were then compared to the corresponding regions of modeled kringle 1 and 2 of tissue plasminogen activator (PA). The coordinates of the modeled structures have been refined by energy minimization in the presence and absence of epsilon-aminocaproic acid ligand in order basically to remove unacceptable van der Waals contacts. The binding site is characterized by an apparent dipolar surface, the polar parts of which are separated by a hydrophobic region of highly conserved aromatic residues. Zwitterionic ligands such as Lys and epsilon-aminocaproic acid form ion pair interactions with Asp55 and Asp57 located on the dipolar surface; the latter are also conserved in all the Lys binding kringles. The cationic center of the dipolar surface is Arg71, in the case of PGK4, and is composed of Arg34 and Arg71 in PGK1. The doubly charged anionic/cationic interaction centers of the latter might account for the larger binding constants of PGK1 for like-ligands but the modeling suggests that PGK4 might be kinetically faster in binding bulkier ligands. The binding site region of PAK2, which also binds Lys, resembles those of PGK1 and PGK4. Since PAK2 lacks both cationic center Arg residues, ligand carboxylate binding appears to be accomplished though an imidazolium ion of His64, which is located just below the outer surface of the kringle.

Amino Acid Sequence↗

PubMed1988

Analysis of the aromatic 1H NMR spectrum of chicken plasminogen kringle 4.

The intact kringle 4 domain of chicken plasminogen has been characterized by 1H NMR spectroscopy at 300 and 620 MHz in both the presence and absence of epsilon-aminocaproic acid, an antifibrinolytic drug. The study focuses on the aromatic resonances. Comparisons with spectra from human, porcine and bovine kringle 4 homologs indicates a strict conservancy of conformation, reflecting the underlying primary sequence homology, and leads to an unambiguous assignment of all the aromatic resonances, including those of Phe15 and His40 which are unique to the chicken domain. Conclusive evidence is found that the Tyr9 ring fluctuates between two states, one in which it flips fast and other in which it is severely hindered. Similarly, the Tyr64 side chain finds itself in a structurally constrained locus. The Trp62, Tyr64, and Trp72 aromatic resonances are most sensitive to ligand presence, supporting a previously reported model of the kringle 4 lysine-binding site. His40, Phe41, and Tyr74 are also perturbed by ligand indicating proximity to the site. In contrast, the Phe15 aromatic spectrum indicates a rather mobile phenyl ring which is insensitive to ligand presence, thus confirming the lesser importance of the corresponding segment within the first kringle loop in determining kringle structure and/or function.

Amino Acid Sequence↗

PubMed1987

Proton magnetic resonance study of lysine-binding to the kringle 4 domain of human plasminogen. The structure of the binding site.

The binding of L-Lys, D-Lys and epsilon-aminocaproic acid (epsilon ACA) to the kringle 4 domain of human plasminogen has been investigated via one and two-dimensional 1H-nuclear magnetic resonance spectroscopy at 300 and 600 MHz. Ligand-kringle association constants (Ka) were determined assuming single site binding. At 295 K, pH 7.2, D-Lys binds to kringle 4 much more weakly (Ka = 1.2 mM-1) than does L-Lys (Ka = 24.4 mM-1). L-Lys binding to kringle 4 causes the appearance of ring current-shifted high-field resonances within the -1 approximately less than delta approximately less than 0 parts per million range. The ligand origin of these signals has been confirmed by examining the spectra of kringle 4 titrated with deuterated L-Lys. A systematic analysis of ligand-induced shifts on the aromatic resonances of kringle 4 has been carried out on the basis of 300 MHz two-dimensional chemical shift correlated (COSY) and double quantum correlated spectroscopies. Significant differences in the effect of L-Lys and D-Lys binding to kringle 4 have been observed in the aromatic COSY spectrum. In particular, the His31 H4 and Trp72 H2 singlets and the Phe64 multiplets appear to be the most sensitive to the particular enantiomers, indicating that these residues are in proximity to the ligand C alpha center. In contrast, the rest of the indole spectrum of Trp72 and the aromatic resonances of Trp62 and Tyr74, which are affected by ligand presence, are insensitive to the optical nature of the ligand isomer. These results, together with two-dimensional proton Overhauser studies and ligand-kringle saturation transfer experiments reported previously, enabled us to generate a model of the kringle 4 ligand-binding site from the crystallographic co-ordinates of the prothrombin kringle 1. The latter, although lacking recognizable lysine-binding capability, is otherwise structurally homologous to the plasminogen kringles.

Binding Sites↗

PubMed1987

Complete assignment of the aromatic proton magnetic resonance spectrum of the kringle 1 domain from human plasminogen: structure of the ligand-binding site.

The kringle 1 domain of human plasminogen has been investigated by 1H NMR spectroscopy at 300 and 600 MHz on the basis of a fragment obtained via controlled proteolysis of the zymogen with Staphylococcus aureus V8 protease. The aromatic spectrum has been fully analyzed and all resonances assigned. The Tyr ring signals were identified by reference to the recently reported spectra of the plasminogen kringle 4 homologues from human, bovine, and porcine origin [Ramesh, V., Gyenes, M., Patthy, L., & Llinás, M. (1986) Eur. J. Biochem. 159, 581-595]. In particular, Tyr74 was assigned on the basis of a proton Overhauser experiment showing cross-relaxation with the Trp-II (Trp62) indole ring, a connectivity previously observed in all the kringle 4 variants and that clearly represents a conserved feature of the kringle structure. Ligand binding was investigated by monitoring the effects of the antifibrinolytic drugs epsilon-aminohexanoic acid and p-benzylaminesulfonic acid (BASA) on the 1H NMR spectrum of kringle 1. It is observed that although most aromatic resonances are perturbed by ligand presence, the chemical shift response is significantly more marked for Phe36, Trp62, and Tyr72. Proton Overhauser experiments centered on aromatic transitions from these residues reveal efficient cross-relaxation with BASA, which indicates direct contacts between the hydrophobic side chain rings and the ligand hydrocarbon moiety at the binding site. A close interaction is also found between Tyr64 and Try72 which indicates that the residue 64 ring is positioned close to the binding site. Excellent overall agreement is found between the NMR data and the molecular folding of the prothrombin kringle 1 determined crystallographically [Park, C. H., & Tulinsky, A. (1986) Biochemistry 25, 3977-3982]. A structure is proposed here for the kringle 1 lysine-binding site which is based upon the NMR results, the X-ray structure, and computer graphics modeling. It is concluded that although features of the lysine-binding site are common to plasminogen kringles 1 and 4, in kringle 1 the binding site extends beyond the kringle inner loop as it encompasses residues Arg34 and Phe36 as well. Furthermore, it appears that in kringle 1 Arg34 and Asp55 are likely to play a direct role in the ligand-kringle 1 interaction by reinforcing the polarity of the cationic and anionic centers of the side chains of Arg71 and Asp57, which have been implicated to provide the electrostatic charges in kringle 4 that balance those of the ligand dipole at the binding site.

Amino Acid Sequence↗

PubMed1987

Isolation, purification and 1H-NMR characterization of a kringle 5 domain fragment from human plasminogen.

A scheme is proposed for generating the intact Val-448-Phe-545 polypeptide of human plasminogen which contains the fifth kringle domain of the plasmin heavy chain. The procedure is based on a pepsin fragmentation of miniplasminogen and involves the purification of the kringle 5-containing fragment by gel filtration and ion-exchange chromatography. The final product is characterized by amino acid analysis, N- and C-terminal analyses, and high-resolution 1H-NMR spectroscopy at both 300 MHz and 611 MHz. We detect a (40:60%) Asp/Asn heterogeneity at site 452 of the Glu-plasminogen molecule. In the conventional kringle numbering system, the kringle 5 domain extends from Cys-1 to Cys-80, which corresponds to Cys-461 to Cys-540 in plasminogen. A preliminary 1H-NMR characterization of kringle 5 focuses on the global conformational features of the polypeptide. Assignments are given for a number of resonances, including the Tyr-72, the His imidazoles' and the Trp indoles' spin systems. Comparison with human plasminogen kringles 1 and 4 shows that the kringle 5 conformation is highly structured and very similar to that of the homologous domains. This conservancy is particularly striking in the environment surrounding Leu-46 and in the overall features of the aromatic spectrum. There are some differences, particularly in the buried His-33 imidazole group, whose H2 resonance is shifted to 9.67 ppm. A preliminary study of benzamidine-binding shows that the ligand interacts weakly (Ka approximately equal to 1.7 mM -1) mainly through the amidino functional group. Trp-62 and Tyr-72 are significantly perturbed by benzamidine, suggesting that these residues are part of the ligand-binding site.

Benzamidines↗

PubMed1987

Proton magnetic resonance characterization of phoratoxins and homologous proteins related to crambin.

The mistletoe protein toxins ligatoxin, phoratoxins A and B, and viscotoxins A3 and B have been investigated by 1H NMR spectroscopy at 300 and 600 MHz. The five polypeptides define a set of closely related homologues, containing 46 amino acid residues each, in a structure constrained by three cystine bridges. Their methyl and aromatic spectra were analyzed and a number of signals identified and assigned via comparative criteria, two-dimensional chemical-shift correlated spectroscopy, acid-base titration, and proton Overhauser experiments in 1H2O. The spectra indicate a compact globular conformation and a common folding pattern for the toxins. In particular, use was made of well-resolved aliphatic and aromatic resonances in order to compare the mistletoe proteins with the thionins, a set of homologous toxins from gramineae, and with crambin, a closely related polypeptide from a crucifer, which we have previously studied by NMR. We observe that while all the investigated proteins have very similar secondary and tertiary structures, they differ widely in their dynamic characteristics as probed by the amide NH 1H-2H exchange kinetics in deuteriated solvents; thus, while crambin and the thionins exhibit very fast isotope exchange, the kinetics for the mistletoe toxins are slow, with some NH groups showing exchange half-lives that extend up to several days at pH* 5.8 or that are too long to be measurable at ambient temperature. The temperature dependence of the 1H NMR spectrum also indicates that the toxins are endowed with a thermally very stable native (ground-state) structure, with little evidence of large amplitude structural breathings up to approximately 370 K, although irreversible chemical degradation (denaturation) becomes evident at temperatures greater than or equal to 350 K. It is concluded that the mistletoe toxins afford valuable rigid structures for NMR conformational studies.

Amino Acid Sequence↗

PubMed1987

Analysis of ligand-binding to the kringle 4 fragment from human plasminogen.

The interaction of the isolated human plasminogen kringle 4 with the four omega-amino acid ligands epsilon-aminocaproic acid (epsilon ACA), N alpha-acetyl-L-lysine (AcLys), trans-aminomethyl(cyclohexane)carboxylic acid (AMCHA) and p-benzylaminesulfonic acid (BASA) has been further characterized by 1H-NMR spectroscopy at 300 and 600 MHz. Pronounced high-field shifts, reaching approximately 3 ppm, are observed for AMCHA resonances upon binding to kringle 4, which underscores the relevance of ligand lipophilic interactions with aromatic side chains at the binding site. Ligand titration curves for the nine His and Trp singlets found in the kringle 4 aromatic spectrum reveal a striking uniformity in the kringle response to the various ligands. The average binding curves exhibit a clear Langmuir absorption isotherm saturation profile and the data were analyzed under the assumption of one (high affinity) binding site per kringle. Equilibrium association constants (Ka) and first order dissociation rate constants (k off) were derived from linearized expressions of the Langmuir isotherm and of the spectral line-shapes, respectively. The results for the four ligands, at approximately 295 K, pH 7.2, indicate that: AMCHA exhibits the strongest binding (Ka = 159 mM-1) and epsilon ACA the weakest (Ka = 21 mM-1) with AcLys and BASA falling in between; epsilon ACA dissociates readily (k off = 5.3 X 10(3) s-1) and AMCHA associates the fastest (k on = 2.0 X 10(8) M-1 s-1) while the kinetics for BASA exchange is relatively slow (k off = 0.8 X 10(3) s-1, k on = 0.6 X 10(8) M-1 s-1); the ligand-binding kinetics is close to diffusion-controlled.

Aminocaproates↗

PubMed1986

Kringle 4 from human plasminogen: a proton magnetic resonance study via two-dimensional photochemically induced dynamic nuclear polarization spectroscopy.

Two-dimensional (2D) proton magnetic resonance techniques used in conjunction with laser photochemically induced dynamic nuclear polarization (photo-CIDNP) spectroscopy have been applied to studying the kringle 4 domain from human plasminogen at 360 MHz. Out of 11 potential CIDNP-sensitive aromatic side chains, only 5 (His3, Tyr41, Tyr50, Trp72, and Tyr74) appear to be accessible to 3-(carboxymethyl)lumiflavin, the dye used to photogenerate spin polarization. Of these, Trp72 and Tyr74 are known to be at, or near, the lysine-binding site. The spin-spin scalar (J) and phase-sensitive dipolar (Overhauser) connectivities in the 2D experiments yield absolute assignments for the aromatic signals stemming from the exposed tyrosyl and tryptophanyl rings. Moreover, a number of side-chain H beta resonances can be identified and assigned to specific types of aromatic amino acid residues.

Amino Acid Sequence↗

PubMed1986

Characterization of the low-field proton magnetic resonance spectrum of plasminogen kringle 4 via selective Overhauser experiments in 1H2O.

The low-field 1H NMR spectrum of the kringle 4 domain of human plasminogen has been investigated at 300 and 600 MHz for the protein dissolved in 1H2O. The spectrum exhibits six well-resolved resonances, spanning the 9.8 approximately less than delta approximately less than 13 ppm chemical shift range, which arise from exchange-labile H atoms. The acid-base response of the six resonances was monitored in order to characterize the signals in terms of their pH titration profiles. The sensitivity of the low-field resonances to kringle binding the antifibrinolytic ligands N alpha-acetyl-L-lysine and p-benzylaminesulfonic acid was also investigated. The lowest field resonance, at 12.6 ppm, is a doublet of J approximately 7.9 Hz, a splitting that is unprecedented for His or Trp ring NH signals. Selective Overhauser experiments centered on the exchangeable proton transitions identify four of the other resonances as stemming from the His31, His33, Trp I, and Trp II side-chain NH groups, where the latter two are, as yet, not definitely assigned to the specific residues, Trp25 and Trp62. The relative narrowness of the His imidazole NH signals indicates that the two rings are sterically shielded from direct water accessibility. In particular, the His33 NH site appears to be the most protected. The Overhauser evidence conclusively shows that the two identified exchangeable His ring proton signals arise from imidazole NH3 sites rather than from the NH1 tautomers. Similarly, these experiments lead to an unambigous characterization of the corresponding Trp aromatic CH spin systems.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1986

Proton magnetic resonance study of kringle 1 from human plasminogen. Insights into the domain structure.

The aromatic H NMR spectrum of the kringle 1 domain from human plasminogen has been investigated by proton Overhauser experiments, acid-base titration, and two-dimensional chemical shift correlated spectroscopy. Spin-echo and pH response experiments lead to the identification of the N-terminal Tyr-3 phenol ring signals. The connectivities among the tryptophanyl aromatic protons have been established and sets of singlet-doublet-triplet resonances stemming from each of the two indole groups sorted according to their common side chain origin. Similarly, the four histidyl singlets have been identified and paired per imidazole group. From their pH responses, it is indicated that a histidyl (His31) and a tryptophanyl (Trp-II) residue are placed in the neighborhood of carboxyl groups. The high-field chemical shifts observed for proton resonances of the ligand epsilon-aminocaproic acid upon binding to kringle 1 indicate that the ligand-binding site is rich in aromatic components. Overhauser experiments reveal that Leu46 is surrounded by a cluster of interacting aromatic side chains, which includes Trp25, Phe36, His41, Trp62, and Tyr64, and define a hydrophobic region contiguous to the kringle lysine-binding site. Relative internuclear distances have been estimated for aromatic H-atoms in the vicinity of Leu46 by reference to one of the latter's CH3 sigma, sigma' groups. Some of the connectives have previously been found for Leu46 in kringle 4 which further supports the idea of a common structure for the homologous domains.

Amino Acid Sequence↗

PubMed1986

The aromatic 1H-NMR spectrum of plasminogen kringle 4. A comparative study of human, porcine and bovine homologs.

The isolated kringle 4 domain of human plasminogen has been compared with homologous structures from bovine and porcine sources, both free and in the presence of the ligand 6-aminohexanoic acid, by two-dimensional 1H-NMR spectroscopies at 300 MHz and 600 MHz. The chemical-shift-correlated, spin-echo-correlated, and double-quantum-correlated aromatic spectra of the three proteins reveal that the globular conformation of the fourth kringle is closely maintained throughout the set of homologs. Direct comparison shows that the three conserved Trp residues (at sites 25, 62 and 72) which exhibit highly non-degenerate subspectra, find themselves in similar intramolecular environments. In particular, proton Overhauser experiments reveal that the close steric interaction between the Trp-II (Trp62 or Trp25) indole group and the aromatic ring at site 74 (Tyr74 or Phe74) is strictly preserved. This feature forces the kringle inner loop, closed by the Cys51-Cys75 link, to fold back onto itself so as to place the site 74 residue proximal to the Cys22-Cys63 bridge. Single-residue substitutions enable unambiguous assignments of His-I to His3, Tyr-III to Tyr41 and Tyr-IV to Tyr74. From this direct evidence, comparison with the kringle 1 spectrum, and the previously reported chemical modification of Tyr-II (Tyr50) [Trexler M., Bányai L., Patthy L., Pluck N. D. & Williams R. J. P. (1985) Eur. J. Biochem. 152, 439-446], Tyr-I and Tyr-V (the latter, an immobile ring on the 600-MHz time scale) could be assigned to Tyr2 and Tyr9, respectively. Since Trp-III has previously been assigned to Trp72 at the lysine-binding site, the present study completes the assignment of 10 out of 12 aromatic spin systems in the kringle 4 1H-NMR spectrum; the only ambiguity which remains concerns the Trp-I and Trp-II indole spin systems, which are totally identified but as yet only tentatively assigned to Trp25 and Trp62, respectively.

Animals↗