Neurotoxins exert specific actions on molecular targets and thus provide a mechanism for characterizing the various ion channels and receptors in the nervous systems. Two types of scorpion toxins have been classified (alpha and beta), that are mammal-specific, which affect Na+ channels of excitable cells. The alpha-toxins modify mainly the inactivation mechanism of the channels whereas the activation mechanism of Na+ channels is modified by the beta-toxins. Several monoclonal antibodies have been raised against the new world Centruroides noxius 2 (Cn2) scorpion toxin as tools. One of these neutralizes the toxic action of the toxin, indicating that it binds to the toxin and obscures its channel-combining site. It was determined that the epitope recognized by this antibody is conformational, and includes residues at both the N-terminus and the C-terminus of the toxin; these segments of the toxin are bound together by one of the disulfide bonds. Several peptides have been synthesized that include this area of the toxin in an attempt to define the epitope precisely1). In this poster, we present the detailed conformational analysis of one of these peptides, namely Cn2(1-15)-NH2-S-S-Ac-Cn2(52-66)-NH2. The molecular structure of the synthetic peptide was determined using NMR and molecular dynamics calculations. The peptide has no significant secondary structure such as an alpha-helix or a beta-sheet, yet it has a fixed conformation for the first chain. The backbone structure of the well-defined loop involving residues 7 to 11 in this peptide shows an excellent overlap with the structures of natural neurotoxins from Centruroides sculpturatus Ewing. As this region in the Cetruroides toxins is one of highest sequence variability, then this region may account for the species specificity and/or fine mechanism of toxic action, hence the conformation of this region must play an important role in receptor recognition. 1) York, E. J., Stewart, J. M., Calderon-Aranda, E. S., and Possani, L. D. (1995) 14th American Peptide Society Meeting, Columbus, Ohio, June 1995, Poster P726.
Recent studies established that a diverse set of receptors for nicotine are present in mammalian central nervous system. The receptors activated by nicotine (nicotinic acetylcholine receptors) have subunits of two general types (apha and beta); a functional receptor is believed to have two alpha and three beta subunits. a-Conotoxin MII, isolated from Conus magus is a potent peptidic toxin which specifically targets the mammalian neuronal nicotinic acetylcholine receptor, a3b2 subtype. The three-dimensional structure of a-conotoxin MII in aqueous solution has been determined by two-dimensional 1H NMR spectroscopy. NOE-derived distances, refined by an iterative relaxation matrix approach, as well as dihedral and chirality restraints were used in high temperature biphasic simulated annealing calculations. Fourteen minimum energy structures out of fifty subjected to the SA simulations were chosen for evaluation; these fourteen structures have a final RMS deviation of 0.76 0.31 and 1.35 0.34 for the backbone and heavy atoms, respectively. The overall structure is unusually well-defined due to a large helical component around the two disulfide bridges. The principal backbone folding motif may be common to a subclass of a-conotoxins.
Peptide and protein hydration is a dominant factor in the stabilization of spatial molecular structures and plays an essential role in substrate binding and in the mechanisms of peptide and protein mediated reactions. There have been several developments in the past few years which promise to extend the NMR methodology as a powerful method for investigating the interaction of water with molecules of biological interest via 2D and 3D homonuclear NOE and ROE type dipolar cross-relaxation processes between water protons and polypeptide protons. However, to our knowledge, there are no reports in investigating the interaction of water with cis/trans isomers of peptides. In this communication we report the investigation of specific hydration phenomena of the neurohypophyseal peptide homone Lys-vasopressin which consists of a macrocyclic hexapeptide ring to which a tripeptide side chain is attached via a cystein6 - proline peptide bond. The combined use of 2D-WATERGATE-ROESY and NOESY gradient experiments resulted in an excellent alleviation of the water resonance and definition of the baseline in the NH region, This allows the estimation of differences in solvent accessibility of the NH protons on the basis of the ROE/NOE intensities and comparison with the classical methodoloy of delta(NH)/DELTA T temperature coefficients. However, the method suffers from low sensitivity, especially in the case of the minor cis-isomer, and complications due to proton exchange, even at low temperatures (273K). The sensitivity problem can be alleviated by the use of selective excitation techniques such as the off-resonance 1D-ROESY experiment which reduces the dimensionality of the spectra and, thus, the experimental time. In this case an off-resonance spin-lock field is applied where the transmitter frequency is shifted far to low (or high) frequency from the spectral region. The angle theta formed by the effective spin-lock axis and the Z axis in the rotating frame, represents an adjustable parameter with which one can vary the contribution of the longitudinal and transverse relaxation rates during the spin lock period.
Cross polarization and dipolar recoupling solid state NMR methods between specifically incorporated spins are proving powerful tools for obtaining new structural and mechanistic details for functional membrane proteins, in the absence of routine crystallographic, or indeed any other direct approaches. By focusing on the ligands which activate or inhibit membrane receptors, hitherto inaccessible information about protein-ligand contacts at the binding site and the specificity of ligand binding and functional consequences, can be uniquely gained. We have used two strategies with which to introduce NMR labels into large membrane proteins, chemical labelling (13C) at selected residues and uniform labelling (15N) in an expressed protein. In a P-type ATPase, 13C labels have been incorporated into accessible sites on the lumenal side of the membrane. Close (<7) contacts between labelled lysines in the Na+/K+-ATPase and labelled inhibitor, di-13C-ouabain, have been detected from observations of magnetization transfer in magic angle spinning NMR experiments. Solid state MAS NMR methods are used to identify the interactions between substrate (13C/19F-labelled sugars) and sites on the sugar transporter, galactose-H+ symport protein, GalP, uniformly 13C/15N-labelled and expressed at an amplified (50 60%) level of the total E. coli cytoplasmic membrane protein. For both proteins, details about these identified contacts provide information which can be used to test models for binding site composition and mechanisms of binding.
SH3 domains are small modules (~60aa) involved in numerous signaling pathways, which bind proline-rich sequences. This interaction localizes the SH3 module and covalently tethered catalytic domains (i.e. kinase, phosphatase) to specific cellular regions. The SH3 domain of Phosphatidylinositol-3-kinase (PI3K) has been shown to bind tightly to proline-rich peptides that share the motif RXLPPRPXX. Previous structural studies have identified a series of conserved aromatic residues on the SH3 surface as the peptide binding site. To further understand the molecular recognition of proline-rich ligands by SH3 domains we have determined the solution structure of the free 9 a.a. peptide RKLPPRPSK. The solution structure was determined in aqueous conditions using 2D NMR techniques on a Bruker DRX 600MHZ spectrometer a 5oC. Phase sensitive TOCSY, Magic-Angle-Gradient DQF COSY, NOESY and ROESY spectra were collected and analyzed. Initial structures were calculated with DYANA using upper distance restraints (47) derived from the NOESY and ROESY crosspeak intensities and phi angle restraints deduced from the coupling constants. The structures which best fit the DYANA target function were subjected to simulated annealing with OPAL. Superposition of the best 14 structures along the backbone atoms of residues 1-6 gave an RMSD value of .514. The data is consistent with 2 possible conformations with different psi angles at Pro-5 which gives rise to different orientations at Pro-7 near the C terminus. One of these conformations has significant type II-poly-proline helix characteristics and displays similarities to the observed conformation of the peptide bound to PI3K SH3. These results may aid in elucidating a mechanism for molecular recognition of proline-rich ligands by SH3 domains as well as be useful for drug design.
Two series of biologically active peptides, adrenocorticotropin hormones (ACTH) and Substance P (SP) peptides, in sodium dodecylsulfate (SDS) and dodecylphosphocholine (DPC) micelles have been studied by two-dimensional NMR, pulsed-field gradient diffusion techniques, and by molecular dynamics (MD) simulations involving explicit micelle-peptide complexes. The partition coefficients of these peptides in the micelles (as determined from diffusion measurements) and the secondary structures of the peptides upon bindng to the micelles have been determined and compared with the results when these peptides are partitioned in lipid bilayers. Major differences between the binding patterns of the peptides with micelles and with bilayers were found. PFG diffusion measurements were also made on the same peptides in POPC vesicles. The mode of binding of these peptides to the micelles was studied by transverse relaxation rates of the protons in individual segments, by intermolecular NOE between the peptides and the micelles, and by MD simulations. In addition, the thermodynamic functions (enthalpy and entropy) of partitioning of these peptides in DPC micelles was determined by temperature dependent measurement of the partition.
Conotoxins are small conformationally-constrained peptides, mostly with 12 to 27 amino acids with two or three disulfide bonds, produced by the venomous marine snails. Conotoxins are the major weapon that the sea snails use for capturing prey or defending themselves against predators. Alpha-conotoxins are one of the three major components of the sea snail cocktail venom targetting the neuromuscular nicotinic acetylcholine receptors. Most alpha-conotoxins whose structures have been determined preferentially bind to the alpha-gamma subunit interface of the receptor. The recently found alpha-conotoxin EI, RDOCCYHPTCNMSNPQIC-NH2, is the only one known to date that binds preferentially to the alpha-delta subunit interface and hence is a highly useful probe for "reverse mapping" the residues involved in the ligand-receptor interactions. A total of 291 restraints including 276 NOEs including 17 long-range NOEs, 15 torsion angles from 3JNa and 3Jab coupling constants were input for distance-geometry and restrained molecular dynamics calculations to produce a final structure of alpha-conotoxin EI with a backbone and heavy-atom rmsd values of 0.69 A and 1.02 A, respectively. Two R-factors, Ra =0.61 and Rb=0.16, were obtained from back-calculation of NOEs.
X-linked agammaglobulinemia (XLA), an inherited disease, is caused by mutations in the Bruton's tyrosine kinase (BTK). The absence of functional BTK leads to failure of B cell differentiation; this incapacitates antibody production in XLA patients, who suffer from recurrent, sometimes lethal, bacterial infections. BTK plays an important role in B cell development; it interacts with several proteins in the context of signal transduction. We have found that BTK SH3 domain binds to a class I proline-rich peptide and to the peptide of BTK TH domain1,2 . This suggests that BTK SH3 and TH domains may associate in inter- or intramolecular fashion, which raises the possibility that the kinase may be regulating its own activity by restricting the availability of both its ligand binding modules. To further understand the role of BTK and its binding peptides, we have synthesized a class I proline-rich peptide, TKRALPPLPQ, with 15N labelled at Alanine and Leucine and studied its backbone dynamics when bound to the SH3 domain of Bruton's tyrosine kinase by 15N relaxation techniques. (1) Chen, Y. J., Lin, S. J., Tzeng, S. Y., Patel, H., Lyu, P. C., & Cheng, J. W. Proteins 1996, 26, 465-471. (2) Patel, H. V., Tzeng, S. R., Liao, C. I., Chen, S. H., & Cheng, J. W. Proteins 1997, in press.
Hepatitis delta virus (HDV) is a satellite virus of the hepatitis B virus (HBV) which provides the surface antigen for the viral coat. Our results show that the N-terminal leucine-repeat region of hepatitis delta antigen (HDAg), encompassing residues 24~50, bind to the autolytic domain of HDV genomic RNA and inhibit its autolytic activity. Solution conformation of a synthetic peptide corresponding to residues 24~50 of HDAg as determined by two-dimensional 1H NMR and circular dichroism techniques is found to be an a-helix. Local helix content of this peptide were analyzed by NOEs and coupling constants. Mutagenesis study indicate that Lys38, Lys39, and Lys40 within this a-helical peptide are the RNA binding sites of HDAg. Structural knowledge of the N-terminal leucine-repeat region of HDAg, thus, provide a molecular basis for the understanding of its role in the interaction with RNA.
[Pen3,15-Nle7-(NMe)Ile20]ET-1 (Pen-2) is an antagonist of endothelin-1 (ET-1), the most potent mammalian vasocontrictor known. The solution structure of ET-1, which contains a semi-rigid bicylic core, shows an unstructured hydrophobic C-terminal region. The six residues of the C-terminus are known to be essential for binding and biological activity. NMR and CD studies of several ET-1 and Pen-2 analogs, shown below, have helped define structural preferences for the C-terminus of the antagonist. 1 5 10 15 20 C S C S S L M D K E C V Y F C H L D I I W ET-1 C S (Pen) S S L (Nle) D K E C V Y F (Pen) H L D I (NMe)I W Pen-2 D A E A V Y F A H L D I (NMe)I W NMe-14mer D A E A V Y F A H L D I I W 14mer G S H L D I (NMe)I W NMe-8mer G S H L D I I W 8mer Chemical shift, NOE profile and CD data indicate that both 14mers adopt a helical conformation from residues 9-15 in aqueous acetic acid media. Upon titration from 0 to 40% hexafluoroisopropanol (HFIP), the helical content of the analogs increase. The peptides containing N-methyl-Ile20 display unusual CD signatures and isoleucine chemical shifts, which are absent in the non-N-methylated-Ile20 analogs and, contrary to the recent findings of Cody et al. [J. Med. Chem. 1997, 40 (14), 2228-40], are not attributed to a cis-trans peptide bond isomerization. The trans configuration remains intact and is the major conformer in both Pen-2 and its fragment analogs.
A 21 residue synthetic peptide corresponding to amino aicds 84~104 of p16, the tumor suppressor has been synthesized and its strucuture was determined by 1H NMR spectroscopy. A p16-derived peptide (84~104 amino acids) has been known to form stable complexes with the cyclin D-dependent kinases, cdk4 and cdk6, inhibit their ability to phosphorate PRb in invitro, and block cell-cycle progression through G1/phase as shown for the full-length wild-type p16 protein. Its NMR spectral data including NOEs, 3JNH-Ha coupling constants, alpha H chemical shifts and temperature coefficients were compared to provide an indication of similarities and differences in local structural elements on the basis of secondary structure of full-length p16, which consists of helix-turn-helix structure. The 3D strucuture has been calculated from NOE-based distance restraints and torsion angle restraints, and will be used to interpret its activity data.
Aggregation of biomolecules such as peptides and proteins is an interesting and important problem. We are investigating the aggregation behavior of a fragment of the amyloid protein, beta(12-28) at low pH where hydrophobic aggregation predominates. In addition, the effect of primary structure on the aggregation of this peptide has been investigated through the synthesis of a series of analogs in which phenylalanines at residues 19 and 20 are selectively replaced with glycine or naphthylalanine. Because diffusion coefficients are indirectly proportional to the hydrodynamic volume, they reflect the aggregation state of the peptide. The measurement of the diffusion coefficients of this family of peptides by pulsed-field gradient NMR (PFG-NMR) will be presented. Additional information about intermolecular interactions can be obtained from the nuclear relaxation times of the peptides. Longitudinal (T1) and transverse (T2) relaxation times as well as correlation times calculated using these relaxation times will be presented. In addition, the rotational correlation times measured for the naphthylalanine-substituted peptides using time-resolved laser fluorescence spectroscopy will be compared to the results of the NMR experiments.
Abnormalities of growth factor receptors are critical to the origin of many breast cancers. Consequently, signal transduction initiated by tyrosine kinase activity represent an important target for the development of new therapeutic agents. The SH2 domains of signal transduction molecules that bind phospho-tyrosine have already been targeted by means of molecules that mimic the structure of phospho-tyrosine containing peptides. However, a novel new design of agents for inhibition of signal transduction indicates that SH2 domain antagonists need not contain phospho-tyrosine or its analogues. Both the phage library based lead cyclic disulfide-bridged (Cys1-Cys11)-amide peptide, G1, and a redox stable cyclic thioether (-Ac-Glu2-Cys11) peptide, G1TE, bind effectively to the Grb2-SH2 domain in the nonphosphorylated form. The G1 and G1TE peptides contain a Tyr-Xxx-Asn sequence similar to the consensus for peptides that bind the Grb2-SH2 domain suggesting that it may bind directly to the same site as the natural phospho-peptides. Preliminary binding studies on the several cyclic and linear peptides indicate that there is a clear conformational preference in the active peptides for Grb2-SH2 binding. Hence, it will be interesting and important to determine structural information on these initial cyclic peptides by NMR spectroscopy and molecular modeling. The results obtain from this studies will identify the specific structure of the amino acids which are within the G1 and G1TE sequence necessary for binding activity, and will help for further drug design in the area of anticancer research.
The development of the bilayered micelle, or "bicelle," has permitted investigations into the structure as well as orientation of channel forming peptides with respect to the membrane. These bicelles, composed entirely of phospholipids, spontaneously orient in a magnetic field at high long chain:short chain phospholipid ratios while forming an isotropic phase suitable for high resolution NMR studies at low long chain:short chain phospholipid ratios. Mastoparan X (MPX), from wasp venom, serves as a model peptide for studying the structural properties of such peptides. Many short peptidic toxins form ion-channels with only 14 amino acids while an alpha-helix of approximately 25 residues is normally required to span a membrane. We have used high resolution NMR to show that MPX in bicellar solution assumes an amphipathic alpha-helical structure which could be resting in the membrane surface or inserted into the membrane to form a channel. Subsequent 2H NMR experiments with MPX labeled at A7 allowed determination of the orientation. We also performed binding affinity studies to examine the dependence of MPX binding on ionic strength and bicelle composition.
Amide hydrogen exchange (HX) coupled with high resolution NMR has provided a powerful platform for monitoring non-covalent interactions in proteins. Proton exchange rate constants measured directly or indirectly can serve as a sensitive probe for observing intra-protein interactions and defining protein-protein contact sites with single residue resolution. Due to the rapid rate of amide HX in solvent exposed residues, most studies have concentrated on structured proteins. The present study describes a technique to indirectly measure the amide HX rate constants of the 15 residue antigenic peptide RIQRGPGRAFVTIGK, derived from gp120 protein of the HIV-1 virus, while associated with its cognate antibody. A reduction in the amide HX rate constants relative to the free rate constants define the residues on the peptide involved in contact with the antibody. The peptide undergoes exchange with deuterated solvent while associated with antibody coupled to a hydrophilic and highly crosslinked bis-acrylamide/copolymer. After a period of deuterium exchange, the peptide is eluted from the column in <50 seconds, rapidly frozen, lyophilized and redissolved in an organic based quenching solvent1 for 1H TOCSY acquisition. The results to be presented discuss the minimization of artifactual exchange and compare mapped contact sites to previous PEPSCAN2 results for this complex. 1. Zhang, Y. Z., Paterson Y., and Roder H., Protein Sci., 4:804 (1995). 2. Langedijk, J. P. M., Back, N. K. T., Durda, P. J., Goudsmit, J., and R. H. Meloen, Journal of General Virology 72:2519 (1991).
Trichorzianin TAVII is a nonadecapeptide member of the biologically active peptaibol series. These peptides are of interest because of their voltage-dependent channel forming property and their membrane activity. The sequence of this peptide is the following : Ac0 U1 A2 A3 U4 J5 Q6 U7 U8 U9 S10 L11 U12 P13 V14 U15 I16 Q17 Q18 Fol19 In order to provide insights into the lipid-peptide interaction promoting the voltage gating, we have carried out the conformational study of TAVII in the presence of perdeuterated sodium dodecyl sulfate (SDS) micelles. 1H sequential assignments were performed with use of 2D-homo and -heteronuclear NMR techniques including DQF-COSY, HOHAHA, NOESY, 1H-13C HSQC and HMBC. Conformational parameters, 3JNHCalphaH coupling constant values, temperature coefficients of amide protons, Chemical Shift Index values of alpha-protons (CSI) and quantitative nuclear Overhauser enhancement (nOe), gave detailed structural information. A set of 231 inter-proton distances derived from NOESY experiments and six Phi dihedral angle restraints were used to generate, by the use of restrained molecular dynamics and energy minimization calculations, 98 three dimensional structures consistent with NMR data. The conformation of trichorzianin TAVII associated with SDS micelles, as determined by these methods, appears to be characterized by two right-handed helix segments linked by a beta-turn which leads to an angle about 100 between the two helix axes ; residues 18 and 19 at the end of the C-terminal helix exhibit multiple conformations.
The interaction of a CXC chemokine and its receptor was analyzed by titration experiments using 1H-15N HSQC spectra. A unique feature of rat CXC chemokine CINC/Gro (a cytokine induced neutrophil chemoattractant) compared to other cytokines like IL-8, is that its monomer and dimer states could be diversely populated by changing the solution condition, and could then be separately observed. We synthesized fragment peptides of the CINC/Gro receptor, a G-protein-coupled 7TM type receptor, corresponding to the extracellular N-terminal region: loop 1 (loop between helix 2 and helix 3), loop 2 (loop 2 between helix 4 and helix 5) and loop 3 (loop between helix 6 and 7), as well as the disulfide-bridged heterodimer of loop 1 and loop 2 (loop 1-loop 2). Under solution conditions enabling the observation of both the monomer and the dimer states of uniformly 15N-labeled CINC/Gro, the chemical shift perturbation induced by the added receptor peptides was monitored by 1H-15N HSQC spectra. Data showed that loop 2 and loop 3 peptide did not effect a chemical shift, and loop1 peptide was not dissolvable. The N-terminal region was found to bind to both the monomer and the dimer state of CINC/Gro. On the other hand, the loop 1-loop 2 peptide only affected the resonance of the monomeric CINC/Gro and perturbations were localized at the dimer interface, suggesting that only CINC/Gro monomer bind to the receptor.
In order to understand the effect that 2'-pendant groups have on the dynamics of base pairs in oligonucleotide hybrids, a series of base pair breathing rate studies have been conducted using 1H-NMR spectroscopy. For these studies, a series of oligonucleotides with the sequence 5'-C-G-C-T-G-A-C-C-3' was synthesized and hybridized to RNA complement. The base pair opening rates were calculated from imino exchange rates, which were determined via selective inversion recovery experiments in a titration with the exchange catalyst ammonia (in ammonium chloride buffer). The exchange rates of the imino protons in the hybrids extrapolate to give base pair lifetimes that differ over an order of magnitude, depending upon the modification of the ribose. Three forms of modification at the 2' position were tested in this study: DNA, 2'-O-methyl RNA, and 2'-O-(2-methoxy)ethyl RNA. Based upon the results of our experiments with the 2'-O-(2-methoxy)ethyl compound, we also investigated whether a modification to the phosphodiester backbone would also influence base pair dynamics. To that end, a phosphorothioate oligodeoxynucleotide was tested for comparison to standard phosphodiester DNA. Comparisons were made between RNA and 2'-O-methyl compounds as oligonucleotide complements for imino exchange studies in order to determine the suitability of 2'-O-methyl as a substitute for RNA in these studies. The imino proton resonances were assigned using proton TOCSY and NOESY experiments. The results of these experiments suggest that the 2'-O-(2-methoxy)ethyl group has a large effect in stabilizing the base pair.
Hydrophobic interactions are a major driving force for the formation of non-covalent aggregates of peptides and proteins in aqueous solutions. One strategy for reducing or eliminating aggregation employs the addition of simple reagents that prevent self-association by binding at the site of the hydrophobic interaction. NMR chemical shift, relaxation parameters, correlation times and diffusion coefficients have been used to probe interactions between several small molecules and a fragment of the Alzheimer's associated peptide, beta(12-28), VHHQKLVFFAEDVGSNK. The interaction of several chemical agents with different hydrophobic structural features and the central hydrophobic region of the beta(12-28) peptide have been examined. Chemical agents investigated include beta-cyclodextrin, trimethylsilylpropionic acid and fluoro-phenylalanine. Because aggregation is a concentration dependent process, binding of additives to the peptide is examined at high and low concentrations.
PFG-NMR measurements for the determination of diffusion coefficients are a well-established method for probing the molecular size and hydrodynamic radii of molecules. We have investigated the use of the BPPLED pulse sequence to evaluate the relative hydrodynamic radii of cis and trans prolyl isomers of both a simple tripeptide (Phe-Pro-Ala) and a more complex peptide hormone (Arginine Vasopressin). Proline isomerization is often the rate-limiting step in protein folding or refolding in many denatured proteins in vitro. Differences in hydrodynamic radii of proline-containing peptides may be linked to the conformational recognition system of certain hormone receptors. Therefore, it is important to be able to study these interconverting isomers separately. NMR is the best physical method available to study this phenomenon because the cis and trans isomers are usually in slow exchange on the NMR time scale. Obtaining quantitative diffusion coefficients from the PFG-NMR spectra can be challenging due to the low content of cis isomer in most peptides and the limitations imposed by spectral overlap. However, the results of these PFG-NMR experiments suggest that statistically different diffusion coefficients for prolyl cis and trans isomers can be measured for both peptides under investigation using this method.
In the intricacy of the three-dimensional (3-D) arrangement of biologically active molecules lie unique, distinctive structural features that are subtly related to function. Accurate structural determination is therefore crucial to structure-function relationships. However, there are no straightforward methods to study membrane bound species with solid-like properties. Solid state NMR has been widely applied to membrane inserted polypeptides such as gramicidin. Gramicidin A, a 15-amino acid polypeptide forms dimeric cation-selective channels in lipid bilayers and is often used as a model for ion channels. Amino acid side chains are believed to mediate key protein-lipid interactions and thereby to play important roles in function. The aromatic, amphipathic side chain of tryptophan (Trp) is a very versatile amino acid in membrane proteins and the focus of our study has been on the structural role of the four tryptophans of gramicidin A. For this purpose, we have substituted aromatic, non-polar phenylalanine side chains for all four tryptophans. Double-labeled 13C and 15N gramicidin A (Formyl valine1-13C-1, alanine5-15N) and its analog, gramicidin M (Alanine5-13C-1, phenylalanine11-15N) have been incorporated separately into hydrated lipid bilayers and studied above the phase transition of the lipids. In this environment, motional averaging scales down the 13C-15N dipolar interactions. In this presentation, we used a newly developed simultaneous frequency and amplitude modulation (SFAM) which is capable of detecting weak interactions in solid state magic angle spinning (MAS) NMR to determine distance constraints in gramicidin dimers. The observed distances show that gramicidin A forms an amino terminus to amino terminus dimer in our samples and gramicidin M forms a double-stranded antiparallel helix. The sensitivity and limits of the technique as well as the structural role of tryptophan in gramicidin A will be discussed.