Biomedical subjects

H J Vogel

Publications and source records attributed to H J Vogel.

At least 37 records · Page 2Linked to original sources

PubMed2000

Peptide and metal ion-dependent association of isolated helix-loop-helix calcium binding domains: studies of thrombic fragments of calmodulin.

Calmodulin (CaM), the ubiquitous, eukaryotic, bilobal calcium-binding regulatory protein, has been cleaved by thrombin to create two fragments. TM1 (1-106) and TM2 (107-148). NMR and CD results indicate that TMI and TM2 can associate in the presence of Ca2+ to form a complex similar to native CaM, even though the cleavage site is not in the linker region between two helix-loop-helix domains, but rather within an alpha-helix. Cadmium-113 NMR results show that this complex has enhanced metal-ion binding properties when compared to either TM1 or TM2 alone. This complex can bind several CaM-binding target peptides, as shown by gel bandshift assays, circular dichroism spectra, and 13C NMR spectra of biosynthetically methyl-13C-Met-labeled TM1 and TM2; moreover, gel bandshift assays show that the addition of a target peptide strengthens the interactions between TM1 and TM2 and increases the stability of the complex. Cadmium-113 NMR spectra indicate that the TM1:TM2 complex can also bind the antipsychotic drug trifluoperazine. However, in contrast to CaM:peptide complexes, the TM1:TM2:peptide complexes are disrupted by 4 M urea; moreover, TM1 and TM2 in combination are unable to activate CaM-dependent enzymes. This suggests that TM1:TM2 mixtures cannot bind target molecules as tightly as intact CaM, or perhaps that binding occurs but additional interactions with the target enzymes that are necessary for proper activation are perturbed by the proteolytic cleavage. The results presented here reflect the importance of the existence of helix-loop-helix Ca2+-binding domains in pairs in proteins such as CaM, and extend the understanding of the association of such domains in this class of proteins in general.

Animals↗

PubMed1999

Structure of the antimicrobial peptide tritrpticin bound to micelles: a distinct membrane-bound peptide fold.

Tritrpticin is a member of the cathelicidin family, a group of diverse antimicrobial peptides found in neutrophil granules. The three Trp and four Arg residues in the sequence VRRFPWWWPFLRR make this a Trp-rich cationic peptide. The structure of tritrpticin bound to membrane-mimetic sodium dodecyl sulfate micelles has been determined using conventional two-dimensional NMR methods. It forms two adjacent turns around the two Pro residues, a distinct fold for peptide-membrane interaction. The first turn involves residues 4-7, followed immediately by a second well-defined 3(10)-helical turn involving residues 8-11. The hydrophobic residues are clustered together and are clearly separated from the basic Arg residues, resulting in an amphipathic structure. Favorable interactions between the unusual amphipathic fold and the micelle surface are probably key to determining the peptide structure. NMR studies of the peptide in the micelle in the presence of the spin-label 5-doxylstearic acid determined that tritrpticin lies near the surface of the micelle, where its many aromatic side chains appear to be equally partitioned into the hydrophilic-hydrophobic interface. Additional fluorescence studies confirmed that the tryptophan residues are inserted into the micelle and are partially protected from the effects of the soluble fluorescence quencher acrylamide.

Anti-Infective Agents↗

PubMed1999

Diversity of antimicrobial peptides and their mechanisms of action.

Antimicrobial peptides encompass a wide variety of structural motifs. Many peptides have alpha-helical structures. The majority of these peptides are cationic and amphipathic but there are also hydrophobic alpha-helical peptides which possess antimicrobial activity. In addition, some beta-sheet peptides have antimicrobial activity and even antimicrobial alpha-helical peptides which have been modified to possess a beta-structure retain part of their antimicrobial activity. There are also antimicrobial peptides which are rich in a certain specific amino acid such as Trp or His. In addition, antimicrobial peptides exist with thio-ether rings, which are lipopeptides or which have macrocyclic Cys knots. In spite of the structural diversity, a common feature of the cationic antimicrobial peptides is that they all have an amphipathic structure which allows them to bind to the membrane interface. Indeed, most antimicrobial peptides interact with membranes and may be cytotoxic as a result of disturbance of the bacterial inner or outer membranes. Alternatively, a necessary but not sufficient property of these peptides may be to be able to pass through the membrane to reach a target inside the cell. The interaction of these peptides with biological membranes is not just a function of the peptide but is also modulated by the lipid components of the membrane. It is not likely that this diverse group of peptides has a single mechanism of action, but interaction of the peptides with membranes is an important requirement for most, if not all, antimicrobial peptides.

Amino Acids↗

PubMed1999

Surface exposure of the methionine side chains of calmodulin in solution. A nitroxide spin label and two-dimensional NMR study.

Binding of calcium to calmodulin (CaM) causes a conformational change in this ubiquitous calcium regulatory protein that allows the activation of many target proteins. Met residues make up a large portion of its hydrophobic target binding surfaces. In this work, we have studied the surface exposure of the Met residues in the apo- and calcium-bound states of CaM in solution. Complexes of calcium-CaM with synthetic peptides derived from the CaM-binding domains of myosin light chain kinase, constitutive nitric-oxide synthase, and CaM-dependent protein kinase I were also studied. The surface exposure was measured by NMR by studying the effects of the soluble nitroxide spin label, 4-hydroxyl-2,2,6, 6-tetramethylpiperidinyl-1-oxy, on the line widths and relaxation rates of the Met methyl resonances in samples of biosynthetically 13C-methyl-Met-labeled CaM. The Met residues move from an almost completely buried state in apo-CaM to an essentially fully exposed state in Ca2+4-CaM. Binding of two Ca2+ to the C-terminal lobe of CaM causes full exposure of the C-terminal Met residues and a partial exposure of the N-terminal Met side chains. Binding of the three target peptides blocks the access of the nitroxide surface probe to nearly all Met residues, although the mode of binding is distinct for the three peptides studied. These data show that calcium binding to CaM controls the surface exposure of the Met residues, thereby providing the switch for target protein binding.

Animals↗

PubMed1999

The structure of the antimicrobial active center of lactoferricin B bound to sodium dodecyl sulfate micelles.

Lactoferricin B (LfcinB) is a 25-residue antimicrobial peptide released from bovine lactoferrin upon pepsin digestion. The antimicrobial center of LfcinB consists of six residues (RRWQWR-NH2), and it possesses similar bactericidal activity to LfcinB. The structure of the six-residue peptide bound to sodium dodecyl sulfate (SDS) micelles has been determined by NMR spectroscopy and molecular dynamics refinement. The peptide adopts a well defined amphipathic structure when bound to SDS micelles with the Trp sidechains separated from the Arg residues. Additional evidence demonstrates that the peptide is oriented in the micelle such that the Trp residues are more deeply buried in the micelle than the Arg and Gln residues.

Animals↗

PubMed1999

Calcium-dependent and -independent interactions of the calmodulin-binding domain of cyclic nucleotide phosphodiesterase with calmodulin.

The ubiquitous Ca2+-binding regulatory protein calmodulin (CaM) binds and activates a wide range of regulatory enzymes. The binding is usually dependent on the binding of Ca2+ to CaM; however, some target proteins interact with CaM in a calcium-independent manner. In this work, we have studied the interactions between CaM and a 20-residue synthetic peptide encompassing the major calmodulin-binding domain of cyclic nucleotide phosphodiesterase (PDE1A2). The binding was studied in the absence and presence of Ca2+ by far-UV and near-UV circular dichroism, fluorescence, and infrared spectroscopy. In addition, two-dimensional heteronuclear NMR studies with 13C-methyl-Met-CaM and uniformly 15N-labeled CaM were performed. Competition assays with smooth muscle myosin light chain kinase revealed a Kd of 224 nM for peptide binding to Ca2+-CaM, while binding of the peptide to apo-CaM is weaker. The peptide binds with an alpha-helical structure to both lobes of Ca2+-saturated CaM, and the single Trp residue is firmly anchored into the C-terminal lobe of CaM. In contrast, the Trp residue plays a minor role in the binding to the apo-protein. Moreover, when bound to apo-CaM, the PDE peptide is only partially helical, and it interacts solely with the C-terminal lobe of CaM. These results show that the Ca2+-induced activation of PDE involves a significant change in the structure and positioning of the CaM-bound PDE peptide domain.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1999

Lentivirus-derived antimicrobial peptides: increased potency by sequence engineering and dimerization.

We have previously described a family of cationic amphipathic peptides derived from lentivirus envelope proteins that have properties similar to those of naturally occurring antimicrobial peptides. Here, we explored the effects of amino acid truncations and substitutions on the antimicrobial potency and selectivity of the prototype peptide, LLP1. Removal of seven residues from the C-terminus of LLP1 had little effect on potency, but abrogated haemolytic activity. Replacement of the two glutamic acid residues of LLP1 with arginine resulted in a peptide with greater bactericidal activity. We discovered that the cysteine-containing peptides spontaneously formed disulphide-linked dimers, which were 16-fold more bactericidal to Staphylococcus aureus. Monomeric and dimeric LLP1 possessed similar alpha helical contents, indicating that disulphide formation did not alter the peptide's secondary structure. The dimerization strategy was applied to magainin 2, enhancing its bactericidal activity eight-fold. By optimizing all three properties of LLP1, a highly potent and selective peptide, named TL-1, was produced. This peptide is significantly more potent than LLP1 against gram-positive bacteria while maintaining high activity against gram-negative organisms and low activity against eukaryotic cells. In addition to new antimicrobial peptides, these studies contribute useful information on which further peptide engineering efforts can be based.

Amino Acid Sequence↗

PubMed1999

Substitution of the methionine residues of calmodulin with the unnatural amino acid analogs ethionine and norleucine: biochemical and spectroscopic studies.

Calmodulin (CaM) is a 148-residue regulatory calcium-binding protein that activates a wide range of target proteins and enzymes. Calcium-saturated CaM has a bilobal structure, and each domain has an exposed hydrophobic surface region where target proteins are bound. These two "active sites" of calmodulin are remarkably rich in Met residues. Here we have biosynthetically substituted (up to 90% incorporation) the unnatural amino acids ethionine (Eth) and norleucine (Nle) for the nine Met residues of CaM. The substituted proteins bind in a calcium-dependent manner to hydrophobic matrices and a synthetic peptide, encompassing the CaM-binding domain of myosin light-chain kinase (MLCK). Infrared and circular dichroism spectroscopy show that there are essentially no changes in the secondary structure of these proteins compared to wild-type CaM (WT-CaM). One- and two-dimensional NMR studies of the Eth-CaM and Nle-CaM proteins reveal that, while the core of the proteins is relatively unaffected by the substitutions, the two hydrophobic interaction surfaces adjust to accommodate the Eth and Nle residues. Enzyme activation studies with MLCK show that Eth-CaM and Nle-CaM activate the enzyme to 90% of its maximal activity, with little changes in dissociation constant. For calcineurin only 50% activation was obtained, and the K(D) for Nle-CaM also increased 3.5-fold compared with WT-CaM. These data show that the "active site" Met residues of CaM play a distinct role in the activation of different target enzymes, in agreement with site-directed mutagenesis studies of the Met residues of CaM.

Amino Acid Substitution↗

PubMed1998

Calcium-calmodulin-induced dimerization of the carboxyl-terminal domain from petunia glutamate decarboxylase. A novel calmodulin-peptide interaction motif.

The acidic, bilobed protein calmodulin (CaM; molecular mass of 16.7 kDa) can activate some 40 distinct proteins in a calcium-dependent manner. The majority of the CaM-binding domain regions of the target proteins are basic and hydrophobic in nature, are devoid of multiple negatively charged residues, and have a propensity to form an alpha-helix. The CaM-binding domain in the C-terminal region of petunia glutamate decarboxylase (PGD) is atypical because it contains five negatively charged residues. Therefore, we chose to study the binding of calcium-CaM to a 26-residue synthetic peptide encompassing the C-terminal region of PGD. Gel band shift assays, fluorescence spectroscopy, and NMR titration studies showed that a single unique complex of calcium-CaM with two PGD peptides is formed. The formation of a 1:2 protein-peptide complex is unusual; normally, calcium-CaM forms 1:1 complexes with the majority of its target proteins. Circular dichroism spectroscopy showed that the bound PGD peptides have an alpha-helical structure. NMR studies of biosynthetically [methyl-13C]methionine-labeled CaM revealed that all the Met side chains in CaM are involved in the binding of the PGD peptides. Analysis of fluorescence spectra showed that the single Trp residue of the two peptides becomes bound to the N- and C-terminal lobes of CaM. These results predict that binding of calcium-CaM to PGD will give rise to dimerization of the protein, which may be necessary for activation. Possible models for the structure of the protein-peptide complex, such as a dimeric peptide structure, are discussed.

Amino Acid Sequence↗

PubMed1998

Characterization of the Ca2+ -dependent and -independent interactions between calmodulin and its binding domain of inducible nitric oxide synthase.

Most interactions of calmodulin (CaM) with its target proteins are Ca2+-dependent, but a few Ca2+-independent CaM-target protein interactions have been identified. One example is the inducible isoform of nitric oxide synthase (iNOS) expressed in macrophages. We describe here the characterization of the Ca2+-independent interaction between CaM and a synthetic peptide corresponding to the CaM-binding domain of murine macrophage iNOS using circular dichroism (CD) spectroscopy. The CD spectrum of free iNOS peptide indicated a beta-sheet conformation. The interaction of iNOS peptide with apo-CaM in the absence of Ca2+ resulted in the peptide acquiring a type II beta-turn structure. This is in contrast to the situation in the presence of Ca2+ in which case the peptide acquired an alpha-helical conformation upon interaction with CaM, i.e. similar to the Ca2+-dependent interactions of CaM with numerous targets such as myosin light chain kinase (MLCK). Consistent with this similar structural change, iNOS peptide inhibited the Ca2+-CaM-dependent activation of smooth muscle MLCK by competing with MLCK for binding to Ca2+-CaM. The Kd of Ca2+-CaM for iNOS peptide was calculated from competition assays to be 0.3 nM. These results indicate that the structure of the CaM-binding domain of iNOS is quite different when bound to apo-CaM than Ca2+-CaM.

Amino Acid Sequence↗

PubMed1998

Activation of calcineurin and smooth muscle myosin light chain kinase by Met-to-Leu mutants of calmodulin.

The effects of replacement of each of the individual Met in calmodulin (CaM) with Leu on the activation of two CaM target enzymes [smooth muscle myosin light chain kinase (smMLCK) and calcineurin (CN)] were investigated. The KD and Pmax (percentage maximal activation) values for activation of both enzymes by M76L-CaM were indistinguishable from wild-type (wt)-CaM, which is consistent with the location of Met-76 in the central linker that is not involved in target protein interaction. The other eight Met in CaM are exposed in the hydrophobic surfaces that are involved in target-enzymes binding, and in general equivalent effects are observed for substitutions of Leu for Met residues in homologous positions in the two CaM domains. However, the importance of the interaction of specific Met residues with the target enzyme depends on the particular enzyme. Leu substitution at Met-36 or Met-109 reduced the affinity of MLCK for the mutant and the maximal activation of CN. MLCK had a higher KD for M51L-CaM whereas M124L-CaM activated the kinase to only 68% of maximal activity induced by wt-CaM; these mutants were indistinguishable from wt-CaM in activation of CN. M71L- and M144L-CaMs behaved like wt-CaM in activation of MLCK, but activated the phosphatase to only about 80% of maximal activity induced by wt-CAM. M72L-CaM exhibited an increased affinity for MLCK compared to wt-CaM and slightly decreased maximal activation, whereas M145L-CaM exhibited maximal activation significantly greater than that due to wt-CaM; these mutants behaved like wt-CaM with respect to CN activation. Finally, a mutant CaM in which all four C-terminal Met were replaced by Leu (M4-CT-L4-CaM) had similar affinities for MLCK and CN as wt-CaM but maximal activation of these enzymes by this mutant was only 60-70% of that achieved with wt-CaM. These results imply that, in addition to removing the autoinhibitory domain from the active site of the target enzyme, CaM must induce a conformational change in the active site itself.

Animals↗

PubMed1998

Three-dimensional solution structure of lactoferricin B, an antimicrobial peptide derived from bovine lactoferrin.

The solution structure of bovine lactoferricin (LfcinB) has been determined using 2D 1H NMR spectroscopy. LfcinB is a 25-residue antimicrobial peptide released by pepsin cleavage of lactoferrin, an 80 kDa iron-binding glycoprotein with many immunologically important functions. The NMR structure of LfcinB reveals a somewhat distorted antiparallel beta-sheet. This contrasts with the X-ray structure of bovine lactoferrin, in which residues 1-13 (of LfcinB) form an alpha-helix. Hence, this region of lactoferricin B appears able to adopt a helical or sheetlike conformation, similar to what has been proposed for the amyloidogenic prion proteins and Alzheimer's beta-peptides. LfcinB has an extended hydrophobic surface comprised of residues Phe1, Cys3, Trp6, Trp8, Pro16, Ile18, and Cys20. The side chains of these residues are well-defined in the NMR structure. Many hydrophilic and positively charged residues surround the hydrophobic surface, giving LfcinB an amphipathic character. LfcinB bears numerous similarities to a vast number of cationic peptides which exert their antimicrobial activities through membrane disruption. The structures of many of these peptides have been well characterized, and models of their membrane-permeabilizing mechanisms have been proposed. The NMR solution structure of LfcinB may be more relevant to membrane interaction than that suggested by the X-ray structure of intact lactoferrin. Based on the solution structure, it is now possible to propose potential mechanisms for the antimicrobial action of LfcinB.

Amino Acid Sequence↗

PubMed1998

Melatonin and serotonin interactions with calmodulin: NMR, spectroscopic and biochemical studies.

It has been reported that the hormone melatonin binds tightly to the ubiquitous calcium-regulatory protein, calmodulin (CaM) with a Kd value around 0.1 nM [Benítez-King et al., Biochim. Biophys. Acta, 1290 (1993) 191-196]. Normally CaM only binds to target proteins and various 20-residue synthetic peptides encompassing the CaM-binding domain of these target proteins with K(d) values ranging between 1.0 microM and 0.1 nM. Here we have studied the interaction of melatonin and several structurally related compounds--serotonin, 5-hydroxytryptophan, and tryptophan--to CaM through gel band shift assays, enzymatic competition assays with calcineurin, fluorescence spectroscopy, far and near UV circular dichroism spectropolarimetry and NMR spectroscopy. Fluorescence spectra show that the binding is calcium dependent. NMR studies with biosynthetically labelled methyl-13C-Met CaM show that melatonin and the other compounds interact with the hydrophobic cleft regions of the protein. Our NMR data show that melatonin binds to both domains of the dumbbell-shaped CaM, while serotonin appears to bind only to the C-terminal domain. This binding mode is further substantiated by fluorescence and gel band shift competition experiments with synthetic peptides from myosin light chain kinase and constitutive nitric oxide synthase. Circular dichroism spectra indicate that the secondary structure of CaM is not altered by addition of melatonin. Our data are internally consistent and reveal Kd values in the mM range for melatonin. Thus the binding of these compounds to CaM is substantially weaker than was previously reported and is unlikely to be of physiological significance.

Binding, Competitive↗

PubMed1998

Tryptophan fluorescence quenching by methionine and selenomethionine residues of calmodulin: orientation of peptide and protein binding.

The two interaction surfaces of the dumbbell-shaped calcium-regulatory protein calmodulin (CaM) are rich in the amino acid Met. In this work we have used fluorescence spectroscopy to study the role of these Met residues in binding the single Trp residue that is found in many CaM-binding domain peptides. This approach is facilitated by the absence of Trp residues in CaM. In addition to the wild-type protein, we studied CaM containing the unnatural amino acid selenomethionine (SeMet), which was biosynthetically substituted for its nine Met residues. Furthermore, a CaM mutant protein in which all four Met residues in the C-terminal domain were mutated to Leu, and the N-terminal domain contained either Met or the unnatural SeMet, was studied. The Trp fluorescence quantum yield of many Trp-containing CaM-binding peptides increases upon binding to calcium-CaM. Moreover, the emission wavelength of the Trp fluorescence is blue-shifted from 353 to 325-333 nm. These parameters indicate movement of Trp from a solvent exposed to a hydrophobic environment. The fluorescence results obtained with these four CaM variants showed that Se is very effective at quenching Trp fluorescence in the calmodulin-bound peptides from myosin light chain kinase (MLCK) and CaM kinase I, while S is somewhat effective (Se > S > C). The quenching effect is markedly distance dependent, as it only influences the Trp residue of the bound peptide (<=7 A) but has little effect on the two Tyr residues in the C-terminal domain of CaM (>=10 A). Since the Trp fluorescence quenching is very dramatic, the protein containing Leu's in the C-terminal domain and SeMet's in the N-terminal domain allowed us to directly determine the orientation of the MLCK and CaM kinase I peptides bound to CaM; in both cases the Trp residue binds to the C-terminal domain of CaM. Our data indicate that SeMet quenching of Trp fluorescence could become a simple and useful tool for studies of protein folding, and protein-protein and protein-peptide interactions.

Amino Acid Sequence↗

PubMed1998

Metal ion binding to calmodulin: NMR and fluorescence studies.

Calmodulin is an important second messenger protein which is involved in a large variety of cellular pathways. Calmodulin is sensitive to fluctuations in the intracellular Ca2+ levels and is activated by the binding of four Ca2+ ions. In spite of the important role it plays in signal transduction pathways, it shows a surprisingly broad specificity for binding metal ions. Using 15N-Gly biosynthetically-labelled calmodulin, we have studied the binding of different metal ions to calmodulin, including K+, Na+, Ca2+, Mg2+, Zn2+, Cd2+, Pb2+, Hg2+, Sr2+, La3+ and Lu3+, by 1H,15N HMQC NMR experiments. The effects of these ions on the substrate-binding ability of calmodulin have also been studied by fluorescence spectroscopy of the single tryptophan residue in a 22-residue synthetic peptide encompassing the skeletal muscle myosin light chain kinase calmodulin-binding domain. Most of these metal ions can activate a calmodulin target enzyme to some extent, though they bind to calmodulin in a different manner. Mg2+, which is of direct physiological interest, has a distinct site-preference for calmodulin, as it shows the highest affinity for site I in the N-terminal domain, while the C-terminal sites III and IV are the high affinity binding sites for Ca2+ (as well as for Cd2+). At a high concentration of Mg2+ and a low concentration of Ca2+, calmodulin can bind Mg2+ in its N-terminal lobe while the C-terminal domain is occupied by Ca2+; this species could exist in resting cells in which the Mg2+ level significantly exceeds that of Ca2+. Moreover, our data suggest that the toxicity of Pb(2+)--which, like Sr2+, binds with an equal and high affinity to all four sites--may be related to its capacity to tightly bind and improperly activate calmodulin.

Amino Acid Sequence↗

PubMed1998

A flexible triangulation method to describe the solvent-accessible surface of biopolymers.

A relatively simple protein solvent-accessible surface triangulation method for continuum electrostatics applications employing the boundary element method is presented. First, the protein is placed onto a three-dimensional lattice with a specified lattice spacing. To each lattice point, a box is assigned. Boxes located in the solvent region and in the interior of the protein are removed from the set. Improper connections between boxes and the possible existence of cavities in the interior of the protein which would destroy the proper connectivity of the triangulated surface are taken care of. The remaining set of boxes define the outer contour of the protein. Each free face exposed to the solvent of the remaining set of boxes is triangulated after the surface defined by the free faces has been smoothed to follow the shape of the macromolecule more accurately. The final step consists of a mapping of triangle vertices onto a set of surface points which define the solvent-accessible surface. Normal vectors at triangle vertices are obtained also from the free faces which define the orientation of the surface. The algorithm was tested for six molecules including four proteins; a dipeptide, a helical peptide consisting of 25 residues, calbindin, lysozyme, calmodulin and cutinase. For each molecule, total areas have been calculated and compared with the result computed from a dotted solvent-accessible surface. Since the boundary element method requires a low number of vertices and triangles to reduce the number of unknowns for reasons of efficiency, the number of triangles should not be too high. Nevertheless, credible results are obtained for the total area with relative errors not exceeding 12% for a large lattice spacing (0.30 nm) while close to zero for a smaller lattice spacing (down to 0.16 nm). The output of the triangulation computer program (written in C++) is rather simple so that it can be easily converted to any format acceptable for any molecular graphics programs.

Biopolymers↗

PubMed1998

Quadrupolar metal ion NMR studies of metalloproteins.

We present a summary of the quadrupolar metal ion NMR studies of metalloproteins conducted in our laboratory in recent years. The approaches we employ can be subdivided into two categories: (i) the use of low-frequency metal nuclei to probe metal ion binding sites in small proteins, exemplified by 43Ca NMR studies of alpha-lactalbumins and calcium-binding lysozymes, and (ii) the novel detection of the central transition of half-integer quadrupolar nuclei of moderate frequency bound to large metalloproteins, typified by 27Al, 45Sc, 69,71Ga, and 51V NMR studies of the transferrins. We highlight the chemical information regarding the nature of metal ion binding sites that can be obtained from this technique and emphasize the salient parameters that an investigator must consider to successfully apply quadrupolar NMR to the study of biological macromolecules.

Animals↗

PubMed1998

Structure-function relationships of antimicrobial peptides.

Antimicrobial peptides are ubiquitously produced throughout nature. Many of these relatively short peptides (6-50 residues) are lethal towards bacteria and fungi, yet they display minimal toxicity towards mammalian cells. All of the peptides are highly cationic and hydrophobic. It is widely believed that they act through nonspecific binding to biological membranes, even though the exact nature of these interactions is presently unclear. High-resolution nuclear magnetic resonance (NMR) has contributed greatly to knowledge in this field, providing insight about peptide structure in aqueous solution, in organic cosolvents, and in micellar systems. Solid-state NMR can provide additional information about peptide-membrane binding. Here we review our current knowledge about the structure of antimicrobial peptides. We also discuss studies pertaining to the mechanism of action. Despite the different three-dimensional structural motifs of the various classes, they all have similar amphiphilic surfaces that are well-suited for membrane binding. Many antimicrobial peptides bind in a membrane-parallel orientation, interacting only with one face of the bilayer. This may be sufficient for antimicrobial action. At higher concentrations, peptides and phospholipids translocate to form multimeric transmembrane channels that seem to contribute to the peptide's hemolytic activity. An understanding of the key features of the secondary and tertiary structures of the antimicrobial peptides and their effects on bactericidal and hemolytic activity can aid the rational design of improved analogs for clinical use.

Amino Acid Sequence↗