Biomedical subjects

K H Mayo

Publications and source records attributed to K H Mayo.

At least 55 records · Page 3Linked to original sources

PubMed1996

Integrin receptor GPIIb/IIIa bound state conformation of the fibrinogen gamma-chain C-terminal peptide 400-411: NMR and transfer NOE studies.

The C-terminal dodecapeptide from human fibrinogen gamma-chain, residues 400-411, HHLG-GAKQAGDV (gamma12), is known to inhibit fibrinogen-mediated platelet cell aggregation via competitive interactions with platelet glycoprotein integrin receptor GPIIb/IIIa. NMR studies of gamma12 in the presence of purified GPIIb/IIIa (230 kDa) demonstrate that two gamma12 binding states (gamma12-I and gamma12-II) are present on the integrin receptor. The N-terminal sequence HHLG is crucial to formation of gamma12 state I since in a shorter gamma-chain octapeptide, GAKQAGDV, gamma12-I is not observed. Addition of the hexapeptide GRGDSP to the gamma12-receptor preparation effectively removes the gamma12-I population, suggesting either that gamma12 and GRGDSP share one binding site or that their binding sites are allosterically linked. Distance geometry calculations using transfer NOEs from gamma12-I (gamma12-II shows practically no NOEs) indicate the presence of helix conformation when bound to the receptor. Line broadening and chemical shift changes relative to free gamma12 suggest that gamma12 interacts with GPIIb/IIIa primarily through N-terminal residues H400 to Q407.

Amino Acid Sequence↗

PubMed1996

Interactions of integrin GPIIb/IIIa-derived peptides with fibrinogen investigated by NMR spectroscopy.

Three peptides derived from platelet receptor glycoprotein alphaIIbBeta3 (GPIIb/IIIa) have been identified recently as fibrinogen-binding sequences: GPIIb 300-314 and 656-667 and GPIIIa 211-223. NMR spectroscopy has been used here to investigate the interactions of these peptides with parent fibrinogen. Based on resonance broadening and chemical-shift changes of peptides in the presence and absence of fibrinogen, interactions in the fast ligand-exchange regime are apparent and interfacial residues can be proposed. Positively charged arginines and histidines, along with several hydrophobic residues, are implicated as being crucial to the binding process. Transferred nuclear Overhauser effects and distance geometry calculations allow discussion of probable conformations in peptide-'bound' states. These identifications are consistent with other biological/chemical data and provide the basis for further studies aimed at understanding fibrinogen-mediated platelet aggregation on the molecular level.

Amino Acid Sequence↗

PubMed1996

Direct binding of ethanol to bovine serum albumin: a fluorescent and 13C NMR multiplet relaxation study.

Molecular mechanisms of ethanol interaction with proteins are not well-understood. In the present study, direct interaction of ethanol with hydrophobic binding sites on fatty acid free bovine serum albumin (BSA) was determined using the fluorescent probe 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS), cis-parinaric acid, and 13C NMR. The affinity of ethanol for BSA (Kd) was (5.21 +/- 0.31) x 10(-2) mol. Ethanol (25-200 mmol) competitively inhibited 1,8-ANS binding to BSA in a concentration-dependent manner with a Ki (concentration of ethanol that decreased 1,8-ANS binding by 50%) of 658 mmol. Preincubation of BSA with ethanol significantly decreased cis-parinaric acid binding to BSA, indicating interaction of ethanol with hydrophobic fatty acid-binding site(s) on BSA. Furthermore, ethanol was found to act on three of the five fatty acid-binding sites on BSA. These data indicated selectivity in the interaction of ethanol with hydrophobic sites on BSA. 13C NMR multiplet relaxation was used to characterize the interaction of ethanol with binding sites on BSA. Detailed analysis of [13C]ethanol relaxation data obtained in the presence of increasing BSA concentrations (25-200 mg/mL) led to the conclusion that the ethanol methyl group, as opposed to its hydroxyl group, binds in a hydrophobic pocket(s) on the protein. Ethanol-induced changes in activity of certain proteins may result from direct binding of ethanol to specific hydrophobic binding sites and/or displacement of endogenous ligands from those sites.

Anilino Naphthalenesulfonates↗

PubMed1996

RGD induces conformational transition in purified platelet integrin GPIIb/IIIa-SDS system yielding multiple binding states for fibrinogen gamma-chain C-terminal peptide.

Fibrinogen gamma-chain C-terminal peptide HHLG-GAKQAGDV (gamma 12) and alpha-chain peptide GRGDSP are known to inhibit fibrinogen-mediated platelet cell aggregation via competitive interactions with platelet integrin receptor GPIIb/IIIa. NMR studies of gamma 12 in the presence of purified GPIIb/IIIa in SDS/water solution have demonstrated the presence of two gamma 12 binding states, one of which is eliminated by GRGDSP (RGD) up to a RGD: gamma 12 ratio of 2:1. RGD: gamma 12 ratios greater than 2:1 produce multiple sets of gamma 12 NMR signals in TOCSY spectra. At a ratio of 4:1, two to four such resonance sets can be resolved for A405, Q407, A408, G409, D410 and V411 spin systems. The number of multiple resonances remains unchanged at ratios of 6:1 and 8:1. Addition of gamma 12 to reverse the ratio to 8:8 (1:1) has no apparent effect on the RGD-induced distribution. Results suggest that RGD irreversibly induces a conformational transition(s) in GPIIb/IIIa to produce multiple gamma 12 binding sites on the receptor.

Amino Acid Sequence↗

PubMed1996

NMR and x-ray studies of collagen model peptides.

The main structural component in collagen is the triple helix which is generally composed of the amino acid sequence repeat (X-Y-Gly)n with proline and hydroxyproline often present at positions X and Y. Non-globular, fibrillar proteins like most collagens are difficult to work with from a structural perspective. An alternative approach to collagen structural elucidation is to study considerably shorter fragments of the triple helix. To date, various triple helical model peptides such as (Pro-Pro-Gly)n and (Pro-Hyp-Gly)n have been investigated by various physical and spectroscopic techniques. The advent of easy solid phase peptide synthetic methodology and the development of multi-dimensional heteronuclear and high field NMR technologies have promoted significant advances in the structure elucidation of a number of triple helix peptides. Here, the main focus is to review and to address the current state of knowledge in the field of NMR and x-ray analysis of triple helical model peptides.

Amino Acid Sequence↗

PubMed1996

A recipe for designing water-soluble, beta-sheet-forming peptides.

Based on observations of solubility and folding properties of peptide 33-mers derived from the beta-sheet domains of platelet factor-4 (PF4), interleukin-8 (IL-8), and growth related protein (Gro-alpha), as well as other beta-sheet-forming peptides, general guidelines have been developed to aid in the design of water soluble, self-association-induced beta-sheet-forming peptides. CD, 1H-NMR, and pulsed field gradient NMR self-diffusion measurements have been used to assess the degree of folding and state of aggregation. PF4 peptide forms native-like beta-sheet tetramers and is sparingly soluble above pH 6. IL-8 peptide is insoluble between pH 4.5 and pH 7.5, yet forms stable, native-like beta-sheet dimers at higher pH. Gro-alpha peptide is soluble at all pH values, yet displays no discernable beta-sheet structure even when diffusion data indicate dimer-tetramer aggregation. A recipe used in the de novo design of water-soluble beta-sheet-forming peptides calls for the peptide to contain 40-50% hydrophobic residues, usually aliphatic ones (I, L, V, A, M) (appropriately paired and mostly but not always alternating with polar residues in the sheet sequence), a positively charged (K, R) to negatively charged (E, D) residue ratio between 4/2 and 6/2, and a noncharged polar residue (N, Q, T, S) composition of about 20% or less. Results on four de novo designed, 33-residue peptides are presented supporting this approach. Under near physiologic conditions, all four peptides are soluble, form beta-sheet structures to varying degrees, and self-associate. One peptide folds as a stable, compact beta-sheet tetramer, whereas the others are transient beta-sheet-containing aggregates.

Amino Acid Sequence↗

PubMed1995

Heparin binding to platelet factor-4. An NMR and site-directed mutagenesis study: arginine residues are crucial for binding.

Native platelet factor-4 (PF4) is an asymmetrically associated, homo-tetrameric protein (70 residues/subunit) known for binding polysulphated glycosaminoglycans like heparin. PF4 N-terminal chimeric mutant M2 (PF4-M2), on the other hand, forms symmetric tetramers [Mayo, Roongta, Ilyina, Milius, Barker, Quinlan, La Rosa and Daly (1995) Biochemistry 34, 11399-11409] making NMR studies with this 32 kDa protein tractable. PF4-M2, moreover, binds heparin with a similar affinity to that of native PF4. NMR data presented here indicate that heparin (9000 Da cut-off) binding to PF4-M2, while not perturbing the overall structure of the protein, does perturb specific side-chain proton resonances which map to spatially related residues within a ring of positively charged side chains on the surface of tetrameric PF4-M2. Contrary to PF4-heparin binding models which centre around C-terminal alpha-helix lysines, this study indicates that a loop containing Arg-20, Arg-22, His-23 and Thr-25, as well as Lys-46 and Arg-49, are even more affected by heparin binding. Site-directed mutagenesis and heparin binding data support these NMR findings by indicating that arginines more than C-terminal lysines, are crucial to the heparin binding process.

Amino Acid Sequence↗

PubMed1995

Effect of pH on the conformation and backbone dynamics of a 27-residue peptide in trifluoroethanol. An NMR and CD Study.

The C-terminal fragment, residues 385-411, from human fibrinogen gamma-chain, i.e. KIIPFNRLTIGEGQQHHLG-GAKQAGDV, shows multiple turn conformations in aqueous solution (Mayo, K. H., Burke, C., Lindon, J. N., and Kloczewiak, M. (1990) Biochemistry 29, 3277-3286). The present study investigates the effect of pH and trifluoroethanol on the conformation and backbone dynamics of this 27-residue peptide. Both circular dichroism (CD) and 1H-NMR data indicate the normally observed increased helical conformations as a function of increasing trifluoroethanol. 1H-NMR structural studies done in the presence of 40% trifluoroethanol, pH 5.3, yield a network of nuclear Overhauser effects consistent with significant populations of helix-like conformation. Distance geometry calculations based on nuclear Overhauser effect-derived distance constraints yield a family of structures with relatively well defined N- and C-terminal conformations and an ill defined mid-peptide region from Gly397 to Gly403. Similar conformational populations are observed at pH 2.5. CD studies, however, indicate an increase in average alpha-helix content on decreasing the pH from 6 to 2. This apparent conflict between CD and NMR results may be explained by a transition from multiple beta-turn character at pH 5.3 to increased alpha-helix structure at pH 2.5. 13C alpha NMR relaxation data analyzed with the Lipari-Szabo model-free approach provide order parameters that demonstrate little if any influence of pH on backbone motional restrictions within the more flexible mid-peptide domain. At low pH, however, motions become less restricted within N-terminal residues Lys385-Phe389 and more restricted within C-terminal residues Ala405-Val411.

Amino Acid Sequence↗

PubMed1995

NMR solution structure of the 32-kDa platelet factor 4 ELR-motif N-terminal chimera: a symmetric tetramer.

Native human platelet factor 4 (PF4) is a homotetrameric protein (70 residues/subunit) known for its anticoagulant heparin binding activity. 2D 15N--1H HSQC NMR experiments of native PF4 in solution show the presence of conformational heterogeneity consistent with the formation of asymmetric homo-tetramers as observed in the X-ray crystal structure of both human and bovine PF4. A chimeric mutant of PF4 (called PF4-M2) which substitutes the first 11 N-terminal residues for the first eight residues from homologous interleukin-8 forms symmetric homo-tetramers with essentially the same heparin binding activity as native PF4. The solution structure of PF4-M2 has been investigated by using two- and three-dimensional 1H- and 15N-NMR spectroscopy and NOE-restrained simulated annealing molecular dynamics. As with other members of the CXC chemokine family whose structures are known, the PF4-M2 subunit monomer consists of a mostly hydrophobic, triple-stranded antiparallel beta-sheet onto which is folded an amphipathic C-terminal helix and a less periodic N-terminal domain. Although N-terminal substitution with the less acidic interleukin-8 sequence most affects the quarternary structure relative to native PF4 at the AC and AD dimer interfaces, AB dimer stability is weakened as reflected in reduced equilibrium association binding constants.

Amino Acid Sequence↗

PubMed1995

Design and evaluation of nonpeptide fibrinogen gamma-chain based GPIIb/IIIa antagonists.

Two series of nonpeptide turn mimetics were designed by analysis of the solution NMR structure of the 385-411 sequence of the gamma-chain of fibrinogen. These compounds, based on the KQAGD (Lys-Gln-Ala-Gly-Asp, 406-410) sequence, were synthesized and studied in vitro. The most interesting compound from our study, RWJ 50042 (25), exhibits potent inhibition of fibrinogen binding to GPIIb/IIIa (IC50 = 0.009 microM), as well as thrombin- or collagen-induced platelet aggregation (IC50 = 0.76, 0.14 microM). Since the 400-411 sequence is required for gamma-chain bioactivity and is a unique recognition sequence among ligands for integrins, vis-a-vis other RGD (Arg-Gly-Asp)-presenting proteins, these turn mimetics may represent a new, selective approach to antagonism of the fibrinogen receptor.

Amino Acid Sequence↗

PubMed1995

B/PI-derived synthetic peptides: synergistic effects in tethered bactericidal and endotoxin neutralizing peptides.

Human neutrophil bactericidal protein (B/PI) is known for its ability to kill bacteria and to neutralize the action of endotoxin. Short linear peptides derived from residues 80-109 have been synthesized and their bactericidal and endotoxin neutralizing activities have been assayed. A series of 'walk-through' decapeptides, overlapping 3 to 4 residues, indicates that endotoxin neutralizing and partial bactericidal activities can be localized within the N- and C-terminal portions, respectively, of the 80-109 sequence. Bactericidal activity toward Pseudomonas aeruginosa was localized in central peptides of the walk-through series and greatest in peptide 90-99. By using longer peptides, residues 86-104 and 82-108, both bactericidal and endotoxin neutralizing activities are significantly enhanced. Bactericidal activity of peptide 82-108 is now only 6-fold less than that of parent B/PI and 9-fold more potent than peptide 86-104. The 82-108 peptide was 7-fold more active at endotoxin neutralization than 86-104 but showed less enhanced activity, being approx. 470-times less active than B/PI. Cyclized 82-108 peptide retained bactericidal activity but did not improve in capacity to neutralize endotoxin.

Amino Acid Sequence↗

PubMed1995

Multiple native-like conformations trapped via self-association-induced hydrophobic collapse of the 33-residue beta-sheet domain from platelet factor 4.

Native platelet factor 4 (PF4) (70 residues) has a hydrophobic three-stranded anti-parallel beta-sheet domain on to which is folded an amphipathic C-terminal alpha-helix and an aperiodic N-terminal domain. The 33-amino acid beta-sheet domain from PF4 (residues 23-55) has been synthesized and studied by c.d. and n.m.r. At 10 degrees C and low concentration, peptide 23-55 appears to exist in aqueous solution in a random-coil distribution of highly flexible conformational states. Some preferred conformation, however, is observed, particularly within a relatively stable chain reversal from Leu-45 to Arg-49. As the peptide concentration and/or temperature is increased, a new conformational state(s) appears and intensifies as slowly exchanging (600 MHz 1H-n.m.r. chemical-shift time scale) random-coil resonances disappear. Hill plots of the concentration-dependence indicated mostly tetramer formation as found in native PF4. Although apparent resonance linewidths in aggregate state(s) are of the order of 100 Hz, sequence-specific assignments for most resonances could be made. N.m.r./nuclear Overhauser effect structural analysis indicates the formation of multiple native-like anti-parallel beta-sheet conformations, kinetically trapped via subunit-association-induced hydrophobic collapse and stabilized by low-dielectric electrostatic interactions among/between Gly-28 and Lys-50 in opposing subunits. Results are discussed in terms of protein folding.

Amino Acid Sequence↗

PubMed1995

Quarternary structure amplification of protein folding differences observed in 'native' platelet factor-4.

Platelet factor 4 (PF4), a protein of 70 residues, exists in solution as a distribution of monomer, dimer and tetramer (M-D-T) states in slow exchange on a 600 MHz 1H-NMR chemical shift time scale. Well-resolved Y60 ring proton resonances in each aggregate state allow derivation of M-D-T populations. Under the same set of solution conditions, M-D-T aggregate state distributions vary from prep to prep or with repeated freeze-drying or raising/lowering the solution pH. These variations are not the result of chemical modifications and reflect differences in the strength of subunit associations and therefore folding. Variations are greatest at pH values at or below the pKas of carboxylate groups, supporting the idea that electrostatic interactions modulate PF4 subunit interactions. Treating these distributions as true equilibria results in free energy differences of about 0.5 kcal/mol subunit. Quarternary structure amplifies free energy differences among various folded states of monomer PF4.

Humans↗

PubMed1995

Lysine side-chain dynamics derived from 13C-multiplet NMR relaxation studies on di- and tripeptides.

13C NMR relaxation data have been used to determine dipolar auto- and cross-correlation times for the di- and tripeptides GK, KG, and GKG, primarily to analyze lysine side-chain motional dynamics. In general, correlation times are largest for backbone positions and decrease on going through the lysine side chain, consistent with the idea of increased mobility at C delta and C episilon methylenes. Correlation times, however, vary with the peptide ionization state. In the zwitterionic state of GK, for example, both auto- and cross-correlation times are at their maximum values, indicating reduced internal motions probably resulting from intramolecular electrostatic interactions. Modifying the charge state increases motional fluctuations. Activation energies determined from the temperature dependence of CH rotational autocorrelation times at neutral pH are approximately equal for glycine and lysine C alpha and lysine C beta and C gamma positions (4.1 +/- 0.2 to 4.5 +/- 0.2 kcal/mol) and tend to decrease slightly for lysine C delta and C epsilon (3.8 +/- 0.2 to 4.3 +/- 0.2 kcal/mol). The sign of lysine side-chain cross-correlations could not be explained by using any available rotational model, including one parameterized for multiple internally restricted rotations and anisotropic overall tumbling. Molecular and stochastic dynamics calculations were performed to obtain insight into correlated internal rotations and coupled overall tumbling and internal motions. Relatively strong correlations were found for i,i+1 backbone and lysine side-chain internal bond rotations. Stochastic dynamics calculations were more successful at explaining experimentally observed correlation times. In the fully charged state, a preferred conformation was detected with an all-trans lysine side chain.

Amino Acid Sequence↗

PubMed1995

13C multiplet nuclear magnetic resonance relaxation-derived ring puckering and backbone dynamics in proline-containing glycine-based peptides.

13CH2-multiplet nuclear magnetic resonance relaxation studies on proline (P)-containing glycine (G)-based peptides, GP, PG, GPG, PGG, and GPGG, provided numerous dipolar auto- and cross-correlation times for various motional model analyses of backbone and proline-ring bond rotations. Molecular dynamics simulations and bond rotation energy profiles were calculated to assess which motions could contribute most to observed relaxation phenomena. Results indicate that proline restricts backbone psi 1, psi 2, and phi 2 motions by 50% relative to those found for a polyglycine control peptide. psi 1 rotations are more restricted in the trans-proline isomer state than in the cis form. A two-state jump model best approximates proline ring puckering which in water could occur either by the C gamma endo-exo or by the C2 interconversion mechanism. The temperature dependence (5 degrees to 75 degrees C) of C beta, and C gamma, and C delta angular changes is rather flat, suggesting a near zero enthalpic contribution to the ring puckering process. In lower dielectric solvents, dimethylsulfoxide and methanol, which may mimic the hydrophobic environment within a protein, the endo-exo mechanism is preferred.

Amino Acid Sequence↗

PubMed1995

Peptide derivatives of three distinct lipopolysaccharide binding proteins inhibit lipopolysaccharide-induced tumor necrosis factor-alpha secretion in vitro.

BACKGROUND: Bactericidal permeability increasing protein (BPI), Limulus anti-lipopolysaccharide factor (LALF), and lipopolysaccharide binding protein (LBP) are three distinct proteins that bind to lipopolysaccharide (LPS). Intriguingly, binding of BPI and LALF to LPS results in neutralization of LPS activity, whereas the binding of LBP to LPS creates a complex that results in augmentation of LPS activity. Despite their different effector functions, we hypothesized that peptides based on the sequences of the proposed LPS-binding motif from each protein would neutralize LPS in vitro. METHODS: Three peptide sequences, each 27 amino acids in length, of the proposed LPS-binding motif of BPI (BG38), LALF (BG42), and LBP (BG43) were synthesized. These peptides were then tested for their: (1) ability to inhibit macrophage secretion of TNF-alpha after stimulation by LPS derived from Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Serratia marcescens; and (2) bactericidal activity against these same four gram-negative bacteria in vitro. RESULTS: Synthetic peptides BG38 (BPI-derived), BG42 (LALF-derived), and BG43 (LBP-derived) but not control peptide significantly inhibited LPS-induced tumor necrosis factor-alpha secretion by macrophages and mediated the lysis of gram-negative bacteria in vitro. In addition, preincubation of LPS with peptide BG38 mediated complete protection subsequent to lethal endotoxin challenge. CONCLUSIONS: These data demonstrate that small peptides derived from BPI, LALF, and LBP retained significant endotoxin-neutralizing and bactericidal activity against many different gram-negative bacteria in vitro. Identification of this conserved LPS-binding region within each protein may aid in the development of new immunomodulatory reagents for use as adjuvant therapy in the treatment of gram-negative bacterial sepsis.

Acute-Phase Proteins↗

PubMed1994

Secondary structure of neutrophil-activating peptide-2 determined by 1H-nuclear magnetic resonance spectroscopy.

Neutrophil-activating protein-2 (NAP-2) is a 72 residue protein demonstrating a range of proinflammatory activities. The solution structure of monomeric NAP-2 has been investigated by two-dimensional 1H-n.m.r. spectroscopy. Sequence-specific proton resonance assignments have been made and secondary structural elements have been identified on the basis of nuclear Overhauser data, coupling constants and amide hydrogen/deuteron exchange. The NAP-2 monomer consists of a triple-stranded anti-parallel beta-sheet arranged in a 'Greek key' and a C-terminal helix (residues 59-70) and is very similar to that found in the n.m.r. solution conformation of dimeric interleukin-8 and the crystal structure of tetrameric bovine platelet factor-4. Results are discussed in terms of heparin binding and neutrophil-activation properties of NAP-2.

Amino Acid Sequence↗

PubMed1994

Synthetic peptides probe folding initiation sites in platelet factor-4: stable chain reversal found within the hydrophobic sequence LIATLKNGRKISL.

Platelet factor-4 (PF4) is a 70-residue protein which contains a 3-stranded antiparallel beta-sheet domain on to which is folded a C-terminal alpha-helix and an aperiodic N-terminal region. In this study, three peptides derived from the beta-sheet (residues 24-46 and 38-57) and helix (residues 57-70) domains have been synthesized and studied in aqueous solution by CD and NMR. While peptides 24-46 and 56-70 demonstrate some weak conformational preferences, peptide 38-57 maintains a relatively well-defined, NOE-rich chain reversal sequence, L45-K46-N47-G48-R49-K50, which apparently is stabilized by hydrophobic side-chain interactions from the flanking sequences L41-L45 and I51-L53. Some helix-like conformational populations are noted in the native PF4 I42-A43-T44-L45 beta-strand segment. NOE-based distance geometry calculations yield native-like conformations within the L45-K50 sequence. Among 40 structures, backbone RMS deviations range from 0.5 to 1.2 A, and compared to the same sequence in native PF4, the average RMS deviation is 1.1 A. These results suggest that beta-sheet/turn residues L41-L53 present a folding initiation site on the PF4 folding pathway.

Amino Acid Sequence↗