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E A Dratz

Publications and source records attributed to E A Dratz.

At least 19 recordsLinked to original sources
PubMed1999

Extraction method for analysis of detergent-solubilized bacteriorhodopsin and hydrophobic peptides by electrospray ionization mass spectrometry.

The analysis of integral membrane proteins or transmembrane peptides by electrospray ionization mass spectrometry (ESI-MS) is difficult since detergents, used to solubilize these hydrophobic proteins and peptides, severely suppress analyte ion formation. This problem has been addressed previously by precipitating the protein, removing the detergent, and resolubilizing the protein in a nonpolar solvent. Here, we demonstrate a method that avoids protein precipitation and resolubilization. Detergent-solubilized bacteriorhodopsin is extracted into a nonpolar solvent phase by adding a chloroform/methanol/water solvent mixture to the aqueous detergent solution. ESI mass spectra of the nonpolar, chloroform-rich phase were dominated by peaks due to bacterioopsin. Bacterioopsin precursors with partially cleaved leader sequences were seen in all mass spectra. Additional peaks were likely due to intact bacteriorhodopsin, i.e., bacterioopsin with the retinal prosthetic group attached, and to bacterioopsin associated with lipid molecules. A separation process that occurred in the fused-silica capillary leading to the electrospray tip was essential for obtaining ESI mass spectra of bacterioopsin. The extraction-into-chloroform procedure also worked well with hydrophobic, transmembrane-type peptides that were insoluble in other electrospray solvents, including 100% formic acid, and the method has application to transmembrane peptides formed from digests of integral membrane proteins.

Bacteriorhodopsins↗
PubMed1999

Actin surface structure revealed by antibody imprints: evaluation of phage-display analysis of anti-actin antibodies.

Phage-display peptide library analysis of an anti-F actin polyclonal antibody identified 12 amino acid residues of actin that appear, in its X-ray crystal structure, to be grouped together in a surface accessible conformational epitope. Phage epitope mapping was carried out by isolating immune complexes containing members of the J404 nonapeptide phage-display library formed in diluted antiserum and isolated on a protein A affinity matrix. Immunoreactive clones were grown as plaques, replica plated onto nitrocellulose, and labeled with anti-actin immune serum. One hundred and forty-four positively staining clones identified in this way were sequenced. Of these, 54 displayed peptides with sequence similarities. When the most abundantly selected sequence, KQTWQQLWD, was produced as a synthetic peptide and derivatized to ovalbumin, the complex was strongly recognized by the antiserum on Western blots and inhibited the binding of the antibody to immobilized F-actin by 60%. A scrambled version of this sequence WQDK WLQTQ, when coupled to ovalbumin, was not recognized by the antiserum and minimally inhibited binding of antiserum to immobilized F-actin by 10%. KQTWQQLWD contained four residues that corresponded, in frame, to a highly conserved six residue region of the chicken beta-actin sequence 351TFQQMW356 (identical residues are shown in bold). Examination of the rabbit skeletal muscle X-ray crystal structure suggested that within a 15 A radius of W356, nine additional residues were arranged on the actin surface in such a way that they could be mimicked by several of the selected phage sequences with root-mean-square deviation fits of 2.1-2.5 A. We conclude that phage-display analysis can provide information about the relative location of amino acids on the surfaces of proteins using antibody imprints of the protein surface structure.

Actins↗
PubMed1998

Sidechain and backbone requirements for anti-invasive activity of laminin peptide 11.

The structure of laminin peptide 11 (CDPGYIGSR-NH2) contains valuable information for the design of mimetic compounds with anti-invasive and anti-metastatic properties. An alanine scan replacement experiment identified Tyr5, Ile6 and Arg9 residues as contributing significantly to anti-invasive activity. Circular dichroism spectra and NMR alphaH chemical shift values both supported the existence of populations of nonrandom coil solution structures for the analogs tested. A D-Ala4 for Gly4 substituted analog completely lost activity, while an L-Ala4 for Gly4 substituted analog retained half the activity of the parent peptide. These results complement our previous findings with D/L alanine substitutions at the Gly7 position, and together they suggest an 'S'-shaped backbone as likely for the active peptide conformation. NMR-constrained molecular modeling supported a direct involvement of the Tyr5 and Ile6 sidechains in conferring bioactivity, and indicated that the Tyr5 sidechain was buried in the Ala2 for Asp2 substitution. Based on the fact that the peptide 11 sequence derives from the disulfide bonded c-loop of an LE-repeat, we synthesized the cyclic CDPGYIGSRC-NH2 peptide. This analog exhibited good anti-invasive and anti-metastatic activity. NMR modeling experiments suggested that the trans-proline cyclic peptide, would favor an 'S'-shaped backbone conformation. Full retro-inverso analogs of peptide 11 were shown to have anti-invasive activity inferior to that of peptide 11. This weak bioactivity was probed using NMR-constrained molecular dynamics, and revealed potential conformations which limited the ability of the required sidechains to mimic the positions of those in the native peptide conformations.

Alanine↗
PubMed1998

Antibody imprint of a membrane protein surface. Phagocyte flavocytochrome b.

Structural features of the integral membrane protein flavocytochrome b (Cyt b) were discovered using an antibody "imprint" of the Cyt b surface. Amino acid sequences were selected from a random nonapeptide phage-display library by their affinity for the monoclonal antibody 44.1 binding site, which recognizes the native conformation of the p22 subunit of Cyt b. Transferred nuclear Overhauser effect spectroscopy and rotating frame Overhauser effect spectroscopy NMR were used to study the antibody-bound conformation of a synthetic peptide derived from phage-displayed sequences. The NMR data supported the phage-display analysis suggesting the existence of a complex epitope and allowed the modeling of the close spatial proximity of the epitope components 29TAGRF33 and 183PQVNPI188 from discontinuous regions of p22. Although these regions are separated by two putative membrane-spanning domains and are 150 residues apart in the sequence, they appear to combine to form a complex epitope on the cytosolic surface of the transmembrane protein. NMR constraints, measured from the antibody-bound conformation of a composite peptide mimetic of the Cyt b epitope, and one constraint inferred from the phage-display results, were used to demonstrate the close proximity of these two regions. This information provides a low resolution view of the tertiary structure of the native discontinuous epitope on the Cyt b surface. Given additional antibodies, such imprint analysis has the potential for producing structural constraints to help support molecular modeling of this and other low abundance or noncrystallizable proteins.

Amino Acid Sequence↗
PubMed1998

NMR studies on the conformation of the CD4 36-59 peptide bound to HIV-1 gp120.

A peptide containing residues 36-59 of the human CD4 receptor includes most of the residues thought to be involved in binding the HIV surface glycoprotein, gp120. This peptide was synthesized and inhibited the binding of gp120 to soluble CD4. NMR relaxation experiments indicated that the peptide was in fast exchange between the free and gp120-bound states. Transferred NOESY NMR showed a number of long-range NOEs, from the gp120-bound state, between residues 38, 40, 45, 48, and 49 of the peptide. NMR evidence also suggested that the Phe43 in the peptide, which corresponds to a critical residue in CD4 for the binding of gp120, makes intimate contact with gp120. The Tr-NOESY cross-peak intensities provided proton-proton distance constraints on the conformation of the gp120-bound peptide. The distance constraints were used in simulated annealing, and a set of 20 very similar structures was obtained for the central region of the gp120-bound peptide. Residues 42-49 of the peptide formed a loop with the side chain of Phe43 pointing away from the rest of the peptide. This Phe43 ring points away from the protein surface in two structures of the amino-terminal domain of CD4 found by X-ray crystallography. Differences in the conformation of CD4 in the two crystal forms suggest that the 36-59 region might be flexible. The NMR data on the 36-59 CD4 peptide predicts a gp120-bound conformation different from either of the CD4 crystal forms in the absence of gp120.

Amino Acid Sequence↗
PubMed1998

Identification of a ligand binding site in the human neutrophil formyl peptide receptor using a site-specific fluorescent photoaffinity label and mass spectrometry.

A novel fluorescent photoaffinity cross-linking probe, formyl-Met-p-benzoyl-L-phenylalanine-Phe-Tyr-Lys-epsilon-N-fluorescei n (fMBpaFYK-fl), was synthesized and used to identify binding site residues in recombinant human phagocyte chemoattractant formyl peptide receptor (FPR). After photoactivation, fluorescein-labeled membranes from Chinese hamster ovary cells were solubilized in octylglucoside and separated by tandem anion exchange and gel filtration chromatography. A single peak of fluorescence was observed in extracts of FPR-expressing cells that was absent in extracts from wild type controls. Photolabeled Chinese hamster ovary membranes were cleaved with CNBr, and the fluorescent fragments were isolated on an antifluorescein immunoaffinity matrix. Matrix-assisted laser desorption ionization mass spectrometry identified a major species with mass = 1754, consistent with the CNBr fragment of fMBpaFYK-fl cross-linked to Val-Arg-Lys-Ala-Hse (an expected CNBr fragment of FPR, residues 83-87). This peptide was further cleaved with trypsin, repurified by antifluorescein immunoaffinity, and subjected to matrix-assisted laser desorption ionization mass spectrometry. A tryptic fragment with mass = 1582 was observed, which is the mass of fMBpaFYK-fl cross-linked to Val-Arg-Lys (FPR residues 83-85), an expected trypsin cleavage product of Val-Arg-Lys-Ala-Hse. Residues 83-85 lie within the putative second transmembrane-spanning region of FPR near the extracellular surface. A 3D model of FPR is presented, which accounts for intramembrane, site-directed mutagenesis results (Miettinen, H. M., Mills, J., Gripentrog, J., Dratz, E. A., Granger, B. L., and Jesaitis, A. J. (1997) J. Immunol. 159, 4045-4054) and the photochemical cross-linking data.

Affinity Labels↗
PubMed1997

The ligand binding site of the formyl peptide receptor maps in the transmembrane region.

We propose that the N-formyl-Met-Leu-Phe binding site in the human neutrophil formyl peptide receptor (FPR) lies in the predicted transmembrane region. We examined the expression, binding, and G protein coupling of 28 mutated forms of FPR in stably transfected Chinese hamster ovary cells. The amino acids we mutated are: 1) predicted to be oriented toward the interhelical space; 2) analogous to those required for ligand binding in various other G protein-coupled receptors; 3) divergent from lipoxin A4 receptor, a low affinity receptor for formylated peptides; and 4) either highly conserved or divergent in other G protein-coupled receptors. Some mutations resulted in intracellular retention, suggesting that the receptors were misfolded. Most mutated receptors that were transported to the plasmalemma bound f-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein with affinities similar to the wild-type receptor (Kd = 6 nM). However, mutations L78A (helix II), D106N, L109A (helix III), T157A (helix IV), R201A, I204Y, and R205A (helix V), W254A and Y257A (helix VI), and F291A (helix VII) resulted in reduced affinities (Kd = 30-128 nM). Of these mutations, D106N, R201A, and R205A also appeared to affect G protein coupling, suggesting that these residues may also be involved in signal transduction and/or are essential for proper folding of the molecule. Some of the FPR residues that appeared to be involved in binding of formylated peptides were located at sites analogous to those identified in ligand binding to certain other G protein-coupled receptors. It is thus possible that several G protein-coupled receptors have a common placement of ligand-binding amino acids.

Amino Acid Sequence↗
PubMed1997

Interaction of human neutrophil flavocytochrome b with cytosolic proteins: transferred-NOESY NMR studies of a gp91phox C-terminal peptide bound to p47phox.

During activation of the neutrophil NADPH oxidase, cytosolic p47(phox) is translocated to the membrane where it associates with flavocytochrome b via multiple binding regions, including a site in the C-terminus of gp91(phox). To investigate this binding site further, we studied the three-dimensional structure of a gp91(phox) C-terminal peptide (551SNSESGPRGVHFIFNKEN568) bound to p47(phox) using transferred nuclear Overhauser effect spectroscopy (Tr-NOESY) NMR. Using MARDIGRAS analysis and simulated annealing, five similar sets of structures of the p47(phox)-bound peptide were obtained, all containing an extended open bend from Ser5 to Phe14 (corresponding to gp91(phox) residues 555-564). The ends of the peptide were poorly defined, however, suggesting they were more flexible. Therefore further refinement was performed on the Ser5-Phe14 region of the peptide after omitting the ends of the peptide from consideration. In this case, two similar structures were obtained. Both structures again exhibited extended open-bend conformations. In addition, the amino acid side chains that showed evidence of immobilization on binding to p47(phox) correlated directly with those that were found previously to be essential for biological activity. Thus during NADPH oxidase assembly, the C-terminus of gp91(phox) binds to 47(phox) in an extended conformation between gp91(phox) residues 555 and 564, with immobilization of all of the amino acid side chains in the 558RGVHFIF564 region except for His561.

Amino Acid Sequence↗
PubMed1997

Identification of transmembrane tryptic peptides of rhodopsin using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The application of mass spectrometry for determining the topography of integral membrane proteins has focused primarily on the mass determination of fragments that do not reside in the lipid bilayer. In this work, we present the accurate mass determination of transmembrane tryptic peptides of bovine rhodopsin using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The ability to determine the accurate mass of hydrophobic transmembrane peptides will facilitate the mapping of ligand binding sites in membrane receptors. It will also augment the determination of membrane spanning regions from integral membrane proteins digested in lipid bilayers. Affinity-purified rhodopsin in detergent and rhodopsin in retinal rod membranes were digested with trypsin. Tryptic peptides were separated using reverse-phase, high-performance liquid chromatography at 55 degrees C with the detergent octyl-beta-glucoside in the mobile phase. Four of the six transmembrane tryptic peptides of rhodopsin were identified, ranging in mass from 3,260 Da to 6,528 Da. The identities of the peptides were confirmed by Edman microsequencing. In addition, heterogeneity in the glycosylation of the N-terminal tryptic peptide of rhodopsin was identified by MALDI MS, without modifying the carbohydrate prior to analysis.

Amino Acid Sequence↗
PubMed1995

Studies of the structure of the metastasis-associated 67 kDa laminin binding protein: fatty acid acylation and evidence supporting dimerization of the 32 kDa gene product to form the mature protein.

The level of expression of the 67 kDa high-affinity laminin binding protein (LBP) correlates with the progression of many solid tumors. The cDNA clone for the 67 kDa LBP is sufficient to encode a polypeptide of only 32 kDa, and there is no readily identifiable mechanism for membrane association. We have overexpressed the transfected 67 kDa hamster LBP in quantities that have enabled us to analyze the membrane-bound form of the protein. Treatment of the purified LBP with methyl transesterification reagents, followed by GC-MS, identified the covalently bound fatty acids palmitate, stearate, and oleate. The fatty acid modification may provide a mechanism for membrane association. Molecular mass determination by MALDI-TOF MS demonstrated the true molecular mass of the protein to be 66.7 kDa, compatible with the SDS-PAGE observation of 67 kDa. Treatment of the LBP with neuraminidase, O-glycanase, or Endo-F glycosidase has no detectable effect on the apparent molecular mass of the protein, and the MALDI-TOF MS did not show evidence of mass heterogeneities typically observed with glycosylated proteins. Reduction with dithiothreitol or beta-mercaptoethanol had no effect on the apparent molecular mass on SDS-PAGE or on the relative quantities of molecular mass species on MALDI-TOF MS. The experimentally determined amino acid composition, however, was found to be consistent with the 67 kDa form being a homodimer of the 32 kDa precursor. Preliminary experiments also suggest that the high-affinity laminin binding characteristic of the protein may be modulated by an, as yet, unidentified membrane accessory molecule.

Acylation↗
PubMed1995

Extensive contact between Gi2 and N-formyl peptide receptor of human neutrophils: mapping of binding sites using receptor-mimetic peptides.

The N-formyl peptide receptor (FPR) of human neutrophils is a member of the G protein-coupled receptor (GPCR) superfamily. Sites on agonist-occupied FPR involved in binding the Gi2 protein were investigated by competition with synthetic receptor-mimetic peptides. Twenty-three synthetic FPR-mimetic and control peptides were tested for their ability to disrupt functionally active complexes of FPR and Gi2 in octyl glucoside, assayed by changes in sedimentation rates of FPR in detergent-containing sucrose gradients. GPCRs are thought to contain seven transmembrane segments with three cytoplasmic connecting loops and a cytoplasmic tail. Only certain peptides from regions in or adjoining each of the four predicted cytoplasmic domains of the 350 amino acid FPR, including the first cytoplasmic loop, were able to disrupt the reconstituted FPR-Gi2 complex. The IC50s of the peptides that were able to fully disrupt the FPR-Gi2 complex ranged from 20 microM (C2W 122-134) to 1.4 mM (C3A 230-246), a range similar to peptide inhibition of other G protein-coupled receptor-G protein interactions. Detergent concentrations above and below the critical micelle concentration had no effect on the activity of even the most hydrophobic peptide, C3B, and there was no apparent correlation of activity with hydrophobic moment, hydrophilic index, or net charge of the peptides. Control peptides from irrelevant proteins with similar physical properties and FPR extracellular domains did not dissociate the reconstituted FPR-Gi2 complex up to 5 mM, the highest concentration tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Immunocytochemical detection of lipid peroxidation in phagosomes of human neutrophils: correlation with expression of flavocytochrome b.

Oxidants generated by the NADPH oxidase of activated neutrophils can react with a number of tissue targets to form toxic metabolites such as 4-hydroxynonenal (4-HNE). 4-HNE is a lipid peroxidation product generated by free radical attack on omega-6 polyunsaturated fatty acids and is a marker for membrane lipid peroxidation. In this study, we examined the accumulation of 4-HNE-protein adducts in phagosomes of neutrophils obtained from a male patient with homozygous X-linked, flavocytochrome b-deficient chronic granulomatous disease (CGD), his heterozygous mother, and his normal father. Specific polyclonal antibodies recognizing 4-HNE-protein adducts and gp91-phox (flavocytochrome b large subunit) were prepared and used to immunocytochemically detect these antigens in cryofixed, molecular distillation-dried neutrophils. No 4-HNE-protein adducts were detected in flavocytochrome b-deficit cells from the homozygous patient or from the heterozygous CGD carrier. However, in gp91-phox-positive cells from both the normal and heterozygous CGD carrier, significant 4-HNE-protein adduct labeling was observed, primarily in the phagosomes. When data from single- and double-labeled cells were combined, the frequency distribution of the labels in phagosomes supported this observation, showing that neutrophils from the heterozygous CGD carrier were 71% 4-HNE-protein adduct-positive and 56% gp91-phox-positive, while cells from the normal father were > 97% positive for both 4-HNE-protein adducts and gp91-phox. These results confirmed the nitroblue tetrazolium tests of 100%, 60 +/- 2%, and 0% positive for the father's, mother's, and son's cells, respectively, and demonstrated that 4-HNE-protein adduct antibodies are useful and accurate probes of the occurrence of lipid peroxidation in vivo. We conclude that 4-HNE and resulting 4-HNE-protein adducts are generated as a result of NADPH oxidase activity in the phagosomes of human neutrophils and that these lipid peroxidation products may contribute to microbial killing and/or damage of neutrophil phagolysosomal proteins.

Aldehydes↗
PubMed1994

Characterization of complex formation between Gi2 and octyl glucoside solubilized neutrophil N-formyl peptide chemoattractant receptor by sedimentation velocity.

The reversible formation of complexes between N-formyl peptide chemoattractant receptor (FPR) and Gi2 protein was analyzed by velocity sedimentation in linear sucrose density gradients. FPR complexed with heterotrimeric Gi2, sediments at different rate than uncomplexed FPR and the two forms have apparent sedimentation coefficients of 7S and 4S, respectively. The biochemical variables important for the reconstitution of the 7S complex from the 4S receptor and Gi2 were studied. The formation of 7S was saturable with Gi2 and addition of excess Gi did not cause oligomerization. The reconstituted 7S complex was stable under a variety of conditions including octyl glucoside concentrations below and above the critical micellar concentration. The optimum pH for the reconstitution is between 7 and 9, where the 4S and 7S species sedimented reproducibly, at distinct positions in the gradient. Below pH 6 both the 4S and the 7S species appear to undergo denaturation and form precipitates. Magnesium ions have no significant effect on the sedimentation of either forms of FPR. Reconstitution was stable up to a NaCl concentration of 0.2 M. At 1 M NaCl reconstitution was inhibited and at 3 M salt FPR aggregated. Since guanine nucleotides GTP, GTP gamma S, GDP beta S selectively dissociated the 7S complex in a concentration-dependent manner and adenine nucleotides had no effect, we conclude that the FPR-Gi2 system displays a vacant guanyl nucleotide binding site, the hallmark of a functional guanine nucleotide exchange complex. Moreover, our results indicate that the reconstitution of FPR-Gi2 complexes is reproducible at physiologically relevant conditions, shows selectivity, specificity, and biochemically functional properties consistent with a specific and functional interaction between solubilized FPR and G protein.

Animals↗
PubMed1994

Human adipose alpha-tocopherol and gamma-tocopherol kinetics during and after 1 y of alpha-tocopherol supplementation.

Alpha-tocopherol and gamma-tocopherol were monitored in human adipose by using needle biopsies in four subjects during a 1-y supplementation trial with 800 mg all-rac-alpha-tocopherol/d, and for 1 additional year after cessation of supplement. Some increase in adipose alpha-tocopherol (per milligram adipose cholesterol) and a more consistent decrease in gamma-tocopherol were observed during the supplementation period. The alpha-tocopherol/gamma-tocopherol ratio rose consistently during supplementation and fell only gradually after the supplement was stopped. We estimate that > or = 2 y are required for the alpha-tocopherol/gamma-tocopherol ratio to reach a new steady state after a change in alpha-tocopherol intake. In a cross-sectional measurement in five subjects who reported long-term use of alpha-tocopherol supplements (> or = 250 mg/d), and in five other subjects who reported no supplement use, the adipose alpha-tocopherol/gamma-tocopherol ratio clearly discriminated between the two groups (P < 0.002). This ratio may be of value in ranking individuals according to long-term alpha-tocopherol intake.

Adipose Tissue↗
PubMed1993

NMR structure of a receptor-bound G-protein peptide.

Heterotrimeric GTP-binding proteins (G proteins) regulate cellular activity by coupling to hormone or sensory receptors. Stimulated receptors catalyse the release of GDP from G protein alpha-subunits and GTP bound to the empty alpha-subunits provides signals that control effectors such as adenylyl cyclases, phosphodiesterases, phospholipases and ion channels. Three cytoplasmic loops of the activated receptor are thought to interact with three sites on the heterotrimeric G protein to provide high-affinity interaction and catalyse G-protein activation. The carboxyl terminus of the alpha-subunit is particularly important for interaction with the receptor. Here we study the structure of part of the active interface between the photon receptor rhodopsin and the G protein transducin, or Gt, using nuclear magnetic resonance. An 11-amino-acid peptide from the C terminus of the alpha-subunit of Gt (alpha t (340-350)) binds to rhodopsin and mimics the G protein in stabilizing its active form, metarhodopsin II. The peptide alpha t (340-350) binds to both excited and unexcited rhodopsin and conformational differences between the two bound forms suggest a mechanism for activation of G proteins by agonist-stimulated receptors. Insight into receptor-catalysed GDP release will have broad application because the GTP/GDP exchange and the intrinsic GTPase activity of GTP-binding proteins constitute a widespread regulatory mechanism.

Amino Acid Sequence↗
PubMed1993

A carboxyl-terminal tail peptide of neutrophil chemotactic receptor disrupts its physical complex with G protein.

The binding of G protein to the N-formyl peptide receptor of human neutrophils was investigated with site-specific synthetic peptides. Peptide CT336(322) (322RALTEDSTQTSDTAT336) from the carboxyl-terminal tail region of the receptor competed with the receptor for binding to bovine Gi protein. The peptide competition was assayed by dissociation of a GTP-sensitive, rapidly sedimenting (7S) form of receptor-G protein complex as analyzed by velocity sedimentation on linear sucrose density gradients. An IC50 of 590 microM was determined for CT336(322) peptide. A control peptide, with the reverse sequence, rCT322(336) (336TATDSTQTSDETLAR322), did not perturb the sedimentation of the reconstituted receptor-G protein complex up to the highest tested concentration, 3 mM. Other peptides tested, corresponding to central portions of the predicted intracellular loop regions CII140(127) (127VLHPVWTQNHRTVS140) and CIII239(227) (227KIHKQGLIKSSRP239) of the receptor, failed to dissociate the reconstituted receptor-G protein complex. Control peptides from the extracellular region EII184(170) (170KTGTVACTFNFSPWT184) and an unrelated sequence matching a portion of neutrophil cytochrome b, CYT306(296) (296KVVITKVVTHPFKTIE306), were also ineffective. Our results suggest that the cytoplasmic tail of the formyl chemotactic peptide receptor is involved in its coupling to the signal-transducing G protein.

Amino Acid Sequence↗
PubMed1992

NMR constrained solution structures for laminin peptide 11. Analogs define structural requirements for inhibition of tumor cell invasion of basement membrane matrix.

Peptide 11, CDPGYIGSR-NH2, is a segment of laminin which blocks tumor cell invasion. A high affinity laminin receptor in tumor cells is thought to be blocked by the carboxyl-terminal YIGSR, and conformational energy calculations suggest that the glycine in YIGSR allows an important conformational bend. We replaced the YIGSR glycine residue in peptide 11 with either D-alanine or L-alanine to allow or disfavor the proposed glycine bend. We found the Gly7-->D-Ala7 analog to be equal to peptide 11 in inhibiting tumor cell invasion of basement membrane matrix. The Gly7-->L-Ala7 analog was much less capable of invasion inhibition. Two-dimensional 1H-1H NMR was used to study the solution conformations of the peptide 11 analogs. NOESY experiments revealed close NH-NH contacts in peptide 11 and the D-Ala7 analog, but not in the L-Ala7 analog. Molecular dynamics generated low energy structures with excellent NOE agreement for peptide 11 and its analogs. Both peptide 11 and the D-Ala7 analog, but not the less active L-Ala7 analog, were predicted to have similar bends around Gly7 or D-Ala7. These results suggest that a bend in the YIGSR region of peptide 11 may be important for the binding of laminin to its metastasis-associated receptor.

Amino Acid Sequence↗