Direct observation of differential UV photolytic degradation among the tryptophan residues of gramicidin A in sodium dodecyl sulfate micelles

McKim, Steve; Hinton, James F.

doi:10.1016/0005-2736(93)90421-u

Cited by 10 publications

(5 citation statements)

References 32 publications

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“…This confirms that the tilting of the lipids upon polymerization does not squeeze gramicidin out of the lipid bilayer nor does our low UV irradiation dose inactivate gramicidin. McKim and Hinton have shown that the tryptophan residues in gramicidin undergo UV photolytic degradation over 24 h during exposure to a 75-W UV lamp . The lamp intensity in our experiments is smaller than that used by McKim and Hinton, and our UV exposure time is only two minutes.…”

Section: Resultsmentioning

confidence: 77%

Photopolymerization of Mixed Monolayers and Black Lipid Membranes Containing Gramicidin A and Diacetylenic Phospholipids

Daly¹,

Heffernan²,

Barger³

et al. 2005

Langmuir

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We formed monolayers and black lipid membranes (BLMs) of photopolymerizable lipids mixed with the channel-forming protein gramicidin A to evaluate their miscibility and the potential for improved stability of the BLM scaffold through polymerization. Analyses of surface pressure vs area isotherms indicated that gramicidin A dispersed with three different synthetic, polymerizable, diacetylene-containing phospholipids, 1,2-di-10,12-tricosadiynoyl-sn-glycero-3-phosphocholine (DTPC), 1,2-di-10,12-tricosadiynoyl-sn-glycero-3-phosphoethanolamine (DTPE), and 1-palmitoyl-2,10,12-tricosadiynoyl-sn-glycero-3-phosphoethanolamine (PTPE) to form mixed monolayers at the air-water interface on a Langmuir-Blodgett (LB) trough. Conductance measurements across a diacetylenic lipid-containing BLM confirmed dispersion of the gramicidin channel with the lipid layer and demonstrated gramicidin ion-channel activity before and after UV exposure. Polymerization kinetics of the diacetylenic films were monitored by film pressure changes at constant LB trough area and by UV-vis absorption spectroscopy of polymerized monolayers deposited onto quartz. An initial increase in film pressure of both the pure diacetylene lipid monolayers and mixed films upon exposure to UV light indicated a change in the film structure. Over the time scale of the pressure increase, an absorbance peak indicative of polymerization evolved, suggesting that the structural change in the lipid monolayer was due to polymerization. Film pressure and absorbance kinetics also revealed degradation of the polymerized chains at long exposure times, indicating an optimum time of UV irradiation for maximized polymerization in the lipid layer. Accordingly, exposure of polymerizable lipid-containing black lipid membranes to short increments of UV light led to an increase in the bilayer lifetime.

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Section: Resultsmentioning

confidence: 77%

Photopolymerization of Mixed Monolayers and Black Lipid Membranes Containing Gramicidin A and Diacetylenic Phospholipids

Daly¹,

Heffernan²,

Barger³

et al. 2005

Langmuir

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“…The photochemistry of tryptophan in a protein may differ from that in solution because the photoexcited residues may react with surrounding protein residues (Creed, 1984a). The quantum yields and the distribution of photoproducts may even vary for different tryptophans in the same protein (Tallmadge and Borkman, 1990;McKim and Hinton, 1993). Since previous studies of tryptophan photochemistry were performed with different wavelengths and solvents than used here, we measured the photochemical conversion of tryptophan to its photoproducts using the same light source and solutions employed in our channel modification experiments.…”

Section: Uv Photolysis Of Tryptophan In Solutionmentioning

confidence: 99%

Modification of Cyclic Nucleotide–Gated Ion Channels by Ultraviolet Light

Middendorf

Aldrich²,

Baylor

2000

The Journal of General Physiology

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We irradiated cyclic nucleotide–gated ion channels in situ with ultraviolet light to probe the role of aromatic residues in ion channel function. UV light reduced the current through excised membrane patches from Xenopus oocytes expressing the α subunit of bovine retinal cyclic nucleotide–gated channels irreversibly, a result consistent with permanent covalent modification of channel amino acids by UV light. The magnitude of the current reduction depended only on the total photon dose delivered to the patches, and not on the intensity of the exciting light, indicating that the functionally important photochemical modification(s) occurred from an excited state reached by a one-photon absorption process. The wavelength dependence of the channels' UV light sensitivity (the action spectrum) was quantitatively consistent with the absorption spectrum of tryptophan, with a small component at long wavelengths, possibly due to cystine absorption. This spectral analysis suggests that UV light reduced the currents at most wavelengths studied by modifying one or more “target” tryptophans in the channels. Comparison of the channels' action spectrum to the absorption spectrum of tryptophan in various solvents suggests that the UV light targets are in a water-like chemical environment. Experiments on mutant channels indicated that the UV light sensitivity of wild-type channels was not conferred exclusively by any one of the 10 tryptophan residues in a subunit. The similarity in the dose dependences of channel current reduction and tryptophan photolysis in solution suggests that photochemical modification of a small number of tryptophan targets in the channels is sufficient to decrease the currents.

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“…The tryptophan sidechains of gramicidin A in micellar solutions have previously been shown to be highly susceptible to UV light and air [15]. The 3-hydroperoxytryptophan intermediates expected from Trp photooxidation should be capable of forming ditryptophan crosslinks by a mechanism similar to that of the two-step chemical method (Figure 7) [16].…”

Section: Discussionmentioning

confidence: 99%

“…The resin was resubjected to cleavage conditions for 96 h to provide an additional 1.650 g (77% overall) of white solid. The peptide was purified by preparative HPLC in 50 mg batches (85% MeOH-50 mM AcOH/Et 3 N, pH 6, isocratic 21 min then 5 min ramp to 100% MeOH, 15 13…”

Section: Hco-val-gly-ala-d Leu-ala-d Val-val-d Val-trp-d Leu-phe-d Lementioning

confidence: 99%

Synthesis and study of a gramicidin B mutant possessing a ditryptophan crosslink

Bodnar¹,

Gilbert²,

Yoburn

et al. 2002

Journal of Peptide Science

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Recent studies of peptide dimers linked by Trp-Trp (ditryptophan) crosslinks suggest that the crosslinks can reinforce antiparallel beta-structure. Depending on environment, gramicidins A, B and C form either helical ion channels with parallel beta-structure or non-functional pores with antiparallel beta-structure. In the channel conformation of the gramicidins Trp9 and Trp15 are close in space, but in the pore conformation Trp9 and Trp15 are far apart. We hypothesized that a ditryptophan crosslink between Trp9 and Trp15 could pre-organize gramicidin in an active conformation. To test the potential for preorganization, an intramolecular ditryptophan crosslink was formed between Trp9 and Trp15 in a W13F mutant of gramicidin B. Photooxidative conditions were shown to generate ditryptophan crosslinks in low yields. While not preparatively useful, photooxidative tryptophan crosslinking may have implications for protein aging processes like cataract formation. The ditryptophan crosslink in the gramicidin B mutant substantially lowered the antibiotic activity of the gramicidin B mutant, unlike the ditryptophan crosslink in the antibiotic X-indolicidin. The biaryl chromophore generated diagnostic Cotton effects in the CD spectrum that revealed the absolute stereochemistry of the biaryl chromophore, but the biaryl chromophore obscured diagnostic features below 220 nm. However, changes in peptide conformation were reflected in changes in the biaryl region of the CD spectrum above 240 nm.

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Direct observation of differential UV photolytic degradation among the tryptophan residues of gramicidin A in sodium dodecyl sulfate micelles

Cited by 10 publications

References 32 publications

Photopolymerization of Mixed Monolayers and Black Lipid Membranes Containing Gramicidin A and Diacetylenic Phospholipids

Photopolymerization of Mixed Monolayers and Black Lipid Membranes Containing Gramicidin A and Diacetylenic Phospholipids

Modification of Cyclic Nucleotide–Gated Ion Channels by Ultraviolet Light

Synthesis and study of a gramicidin B mutant possessing a ditryptophan crosslink

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