Tryptophan gramicidin

Other workers began to study the structure of gramicidin. Christensen and coworkers12 isolated crystalline tryptophane and leucine from a hydrolysate. They found no evidence for a fatty acid component and established that phenylalanine, proline and hydroxyproline were absent from a hydrolysate. These workers isolated alanine diox-pyridate from a hydrolysate and also established that gramicidin contained a compound with vicinal hydroxy and amino groups. They speculated that this compound might be serine or isoserine and proposed that gramicidin contains two tryptophane, 2 leucine, 2 or 3 alanine and 1 hydroxyamino residues or a multiple of this composition. 

Gordon, Martin and Synge18 utilized their new and elegant technique, chromatography, to establish the amino acid composition of gramicidin. They proposed a 24 unit cyclic peptide consisting of six moles each of leucine, and tryptophane, 5 moles of valine, 3 moles of alanine and 2 moles each of glycine and unknown hydroxyamino compound. 

The tryptophane content of the gramicidin complex was measured by the ultraviolet absorbance47. 

Rittenberg and coworkers used a colorimetric assay for tryptophane to determine tyrothricin in fermentation broth152. Kreuzig described a semi-automated colorimetric assay for gramicidin utilizing the reaction of... 

Fluorescence quantum yield and emission maximum determinations as a function of peptide concentration may also permit the detection of peptide self-aggregation at concentrations below 10-4 M, because the peptide fluorophore is likely to be located in a different environment in the peptide aggregate. For example, the concentration-dependent changes in the tryptophan fluorescence emission maximum of mellitin were monitored to determine the equilibrium dissociation constant and thermodynamic parameters of the monomer-tetramer self-association reaction of this peptide. 25 Similarly, measurement of the changes in the tryptophan fluorescence intensity of gramicidin A as a function of its concentration permitted the determination of an average monomer-dimer equilibrium con-stant. 26 ... 

There are many examples of peptides that cannot form transmembrane channels on their own but can do so through aggregation. The gramicidin antibiotics, produced by bacteria as part of their chemical defence system, are only 1.6 nm or so in length [11], Specific placement of side chains, such as four tryptophan residues towards the C-terminus, ensures that the helix penetrates cell membranes to a particular depth but does not pass through the membrane. 

Gramicidin A, however, is attacked by JV -bromoacetamide (NBA) and N-bromosuccinimide (NBS) (Gross and Witkop, unpublished observation). In 50 % aqueous ethyl alcohol at room temperature 5 % of the peptide bonds (20% of the tryptophyl peptide bonds) are cleaved with NBS. Methyl alcohol must be avoided because it opens the spirodioxindole lactone from oxidized tryptophan to the ester even at room temperature. The cleavage mixture separates on electrophoresis (pH = 2.5, sodium-formate buffer) into four ninhydrin-positive components of which the fastest migrating one was identified as ethanolamine. Dinitrophenylation showed leucine and alanine to be additional NHs-terminals of the released fragments. 

Fluorotryptophan (F-Trp) was one of the first labels used for investigation of peptides and proteins by 19F NMR. Both 5- and 6-fluorotryptophan are commercially available. In this case, orientational constraints and distance measurements are very useful for obtaining information about the side-chain conformation [74, 75], The tryptophan side-chain is often found to anchor transmembrane helices in the lipid head-group region of the membrane and it often plays a functional role in peptides that form channels in the membrane, such as gramicidin A [74, 76] or the influenza virus peptide M2 [75], Fluorotryptophans are easily incorporated into peptides and proteins using biosynthetic and SPPS protocols. 

Grage, S. L., Wang, J., Cross, T. A. and Ulrich, A. S. (2002) Structure analysis of fluorine-labelled tryptophan side-chains in gramicidin A by solid state 19F-NMR. Biophysical Journal, 83, 3336-3350. 

Gramicidin contains no acidic or basic groups. Tyrocidine contains strongly basic groups due to ornithine residues. It can be isolated only as the hydrochloride. The crystalline mixture tyrocidine contains 1-omithine, 1-proUne, 1-valine, 1-leucine, d-phenylalanihe, 1-tryptophane, 1-tyrosine, 1-aspartic acid and 1-glutamic acid (11). 

NMR results for gramicidin A incorporated into oriented DMPC bilayers produced results that were interpreted as being consistent with the structure of gramicidin A incorporated into SDS micelles (i.e. no tryptophan 9/15 stacking). The results of a Raman spectroscopy study of water accessibility to the tryptophan indole NH sites of gramicidin A incorporated into a hposome (dilauroyl-L-a-phosphatidylchoUne) aqueous suspension were interpreted as being consistent with the stacked tryptophan structure. Obviously, the... 

The structure of gramicidin B and gramiddin C incorporated into SDS micelles has been determined. No significant differences in the backbone of the channel are observed. A comparison of the tryptophan region (i.e. 9 to 15) reveals some small differences in residue orientation that may be of importance in ion binding and transport (Fig. 5). With the Gly-15 analogue of... 

Figure 34. A, Circular dichroism spectra of the Gramicidin A transmembrane channel in phospholipid bilayers (curve a) and of hydrogenated Gramicidin A in trifluoroethanol at high concentration ( 100 mg/ml), which is likely a double-stranded j3-helix. See text for discussion. Mean residue ellipticities are given. B, Absorption spectrum of the Gramicidin A transmembrane channel in phospholipid bilayers. The absorption spectrum is dominated by the four tryptophan residues per pentadecapeptide. The extinction coefficient is given on a per residue basis.

Proteins or peptides containing either tyrosine or tryptophan, hut not both, give absorption curves which resemble very closely, both qualitatively and quantitatively, the curves of the appropriate free aromatic amino acid, after allowing for the longwave shift and loss of resolution which results from peptide combination (Section VI, 2). Typical substances which satisfy this condition are insulin (12% tyrosine) and gramicidin (39% tryptophan). (See Table VII and Fig. 11.)... 

Firstly, it is evident that the free aromatic amino acids are not the correct standards from which to determine the contributions to protein absorption. The same can be said of peptides of the aromatic amino acids. Although peptide combination causes some shift in the spectra of the aromatic amino acids it is not sufficient to account for the shift occurring in proteins (Section VI, 2). However it may be an improvement to use the peptides for comparison rather than the free amino acids, for Edwards (1949) found a much better fit to gramicidin curves by using glycyl-tryptophan as a standard. A drawback to the use of peptides is that they are more difficult to purify than the amino acids and some are difficult to synthesize. One should ideally employ a sandwich tripeptide with the aromatic acid in the middle, but no reports of the synthesis of such peptides have come to the authors notice. Secondly, if it were possible to measure precisely the shift in the spectra it should be possible to allow for this in the calculations. For instance if the shift were estimated as AX it would be possible to measure the absorption of the protein at Xi - - AX and Xz -h AX or alternatively to measure the amino acids at Xi — AX and Xj — AX and the protein at Xi and X2. 

Dubos showed that tyrothricin could be separated into two components, gramicidin and tyrocidine. Craig showed that tyrocidine could be resolved into three components, tyrocidines A, B and C. Tyrocidine A has the structure XI and t5nrocidine B differs from XI only in containing an L-tryptophan residue in place of an L-phenylalanine residue . 

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