Photochemical labeling of membrane-associated and channel-forming domains of proteins directed by energy transfer (original) (raw)
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Biochimica et Biophysica Acta (BBA) - Biomembranes, 1997
Ž 4. We have examined conditions optimal for 5-iodonaphthyl-1-azide INA labeling of membrane proteins proximal to known membrane sites. Membrane-bound INA can be indirectly activated by energy transfer from visible chromophores. We demonstrate that the efficiency of this sensitized activation is enhanced by use of triplet-forming chromophores such as eosin and by deoxygenation. Variation of sensitized activation efficiency with INA concentration indicates that the critical distance for eosin-INA energy transfer in solution is 8-14 A. We suggest that photosensitization occurs through triplet exchange and present an improved labeling protocol based on these findings. This protocol was used to examine whether different accessory proteins are associated with isolated and crosslinked Type I Fc receptors on 2H3 rat basophilic e w 125 x leukemia cells. 2H3 cells were incubated with eosin-conjugated IgE and irradiated at 514 nm yielding I INA derivatized peptides at 53, 38, 34, and 29 kDa. Crosslinking IgE with mouse anti-rat IgE prior to irradiation labeled three additional proteins at 60, 54, and 43 kDa. These results demonstrate the utility of sensitized INA labeling in characterizing protein-protein interactions in membranes of intact cells and indicate the importance of considering photophysical factors when selecting sensitizers and reaction conditions. We discuss estimation of the size of the membrane region surrounding a sensitizing chromophore within which INA labeling of membrane proteins occurs.
Analytical Biochemistry, 1999
An apparatus has been developed that allows photoaffinity ligands to be crossed-linked to milligram quantities of membrane proteins with maximum attainable yield following contact times of approximately 1 ms. The apparatus consisted of three parts: a conventional rapid mixing unit, a novel freezequench unit, and a photolabeling unit. The freezequench unit consisted of a rapidly rotating metal disk which was precooled in liquid nitrogen. Correct alignment of the exit jet from the sample mixer allowed up to 2 ml of sample to be frozen in a thin film on the disk. Experiments with colorimetric reactions showed the combined dead time of mixing and freeze-quenching to be submillisecond. Photoincorporation was maximized by prolonged irradiation of the freezequenched sample. Using this apparatus we determine the binding kinetics of the resting state channel inhibitor 3-[ 125 I](trifluoromethyl)-3-(m-iodophenyl) diazirine (TID) to nicotinic acetylcholine receptor-rich membranes from Torpedo. The binding kinetics for the 125 I-labeled ␣ and ␦ subunits were biphasic; about half the binding was complete by 2.4 ms, and the remainder could be resolved and occurred with a pseudo-firstorder rate constant determined at 4 M [ 125 I]TID of 12.0 ؎ 2.3 and 13.6 ؎ 4.0 s ؊1 , respectively. This compares well to the same constant determined for the inhibition of agonist-induced cation flux in Torpedo membranes.
Design and synthesis of new fluorescent photoaffinity labels to study membrane structure
1984
Photoaffinity labelling has been used as a technique to study membrane structure. This technique necessitates design and synthesis of suitable carbene and nitrene precursors referred to as photoaffinity (PA) labels. The PA labels should preferably be hydrophobic in nature, photolyse with light of wavelength greater than 300nm to give reactive intermediates i.e., carbenes which should undergo intermolecular insertion exclusively. The latter reaction, on incorporation of the PA labels in membranes, gives rise to crosslinked products, the analyses of which give useful information on the nature of bio-molecular interaction in membranes.
Photoreactive labeling of M13 coat protein in model membranes by use of a glycolipid probe
Proceedings of the National Academy of Sciences of the United States of America, 1979
Coliphage M13 coat protein in synthetic bilayer membranes was labeled by use of 12,(4-azido-2-nitrophenoxy)stearoyl[I-14Clglucosamine, a photoreactive glycolipid probe that spontaneously inserts into membranes. In this model system, the probe preferentially labeled the proteins over the lipids. Experiments designed to test the probe's restriction to integral membrane proteins revealed that extrinsic proteins as well as external peptide fragments of integral membrane proteins were not accessible to the photogenerated nitrene on the fatty acid chain. Only integral membrane peptides were labeled by the membrane-bound probe. These results indicate that protein labeling can be effected specifically from within the
Fluorescence energy transfer as a tool to locate functional sites in membrane proteins
Biochemical Society Transactions
Biochemical Society Transactions 784 whether interacting components are separated by barriers or entrapped within the same domain. Whether or not particular proteins are randomly dispersed in the membrane raises interesting questions in relation to membrane biosynthesis. It is likely that SPT experiments will contribute much in the coming years to our understanding of membrane structure and function. We are grateful to the Wellcome Trust and SEKC for financial support.
Journal of Biological Chemistry
CAAX motif peptides, which are substrates for isoprenylation, were synthetically derivatized with the light-sensitive benzophenone (Bz) group in order to determine their potential use as catalytic site-directed covalent photocross-linking ligands for one of the enzymes catalyzing protein isoprenylation, farnesyl-protein transferase (FPTase). Bz-peptides could be synthesized with [8H]benzophenone and possessed either one or two benzophenone groups located at or near the peptide's N H z terminus (e.g. the mono-Bz probes Bz-ACVIM and Bz-LPCVVM, and the di-Bz derivatized probe Bz-GY-(Bz)PCVVM, referred to as Bzz-GYPCVVM). Each type of derivatized peptide, behaved as a substrate for farqesylation in vitro without irradiation, while under 366-nm irradiation each demonstrated covalent crosslinking ability as a catalytic site-directed photoaffinity ligand with tissue-purified or enriched but impure fractions from rat and bovine brain FPTase, as well as with a recombinant human FPTase variant, FPTase(pat) expressed in Escherichia coli. Without photoactivation, Bz-ACVIM yielded a & of 37 IIM for the cloned variant of human FPTase. Pseudo first-order photolytic inhibition of FPTase preparations with Bz-peptides, as well as protection from photoinactivation by unmodified -CAAX motif peptides, supported the capacity of these Bz-peptides to serve as co-substrates and their specificity for seeking the catalytic site of the enzyme. SDS-polyacrylamide gel electrophoresis analysis subsequent to photolysis indicated that the mono-Bz-derivatized peptides (e.g.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2001
To identify membrane-associated polypeptides present in Torpedo nicotinic acetylcholine receptor (AChR)-rich membranes, we used hydrophobic photolabeling with [ 3 H]diazofluorene ([ 3 H]DAF) and 1-azidopyrene (1-AP) to tag the membrane proteins which were then identified by amino-terminal sequence analysis of labeled fragments isolated from proteolytic digests by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by reverse-phase high-performance liquid chromatography. In addition to AChR subunits, identified polypeptides include the 95 kDa K-subunit of the (Na +K)-ATPase, the 89 kDa voltage-gated chloride channel (CLC-0), the 105 kDa SITS-binding protein, and 32 and 34 kDa polypeptides identified as Torpedo homologues of the mitochondrial membrane ATP/ADP carrier protein and the voltage-dependent anion channel (VDAC), respectively. Further, individual amino acids that reacted with [ 3 H]DAF and therefore likely to be in contact with lipid were identified in the transmembrane segment M3 of the K-subunit of the (Na +K)-ATPase and in a putative transmembrane L-strand in VDAC. Collectively these results demonstrate that [ 3 H]DAF/1-AP photolabeling provides an effective method for tagging the membrane-associated segments of polypeptides in a way that makes it easy to isolate the labeled polypeptide or polypeptide fragments by fluorescence and then to identify amino acids at the lipid-protein interface by 3 H release.
Photolysis of gramicidin A channels in lipid bilayers
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1983
We have tested the hypothesis that peptide tryptophan groups can control the ionic conductance of transmembrane channels. We report here that single gramicidin A channels change conductance state when the peptide tryptophans are flash photolyzed with ultraviolet light. The current ...
The Mechanism of Photoaffinity Labeling
Proceedings of the National Academy of Sciences, 1973
Photoaffinity labeling is a recently introduced method for covalently binding chemical tags to the active sites of protein molecules, which is potentially capable of very great specificities of labeling. A labeling reagent is used that is converted by photolysis to an extremely reactive intermediate. According to the expected mechanism, the reagent molecules that are specifically and reversibly bound to the active site at the instant of photolysis react irreversibly in the site before they can dissociate from the site. In two such reagent-protein systems studied in this paper, however, it is shown that, while by the usual criteria photoaffinity labeling appears to have occurred, the expected mechanism in fact does not hold. This was discovered in experiments with scavengers present in the mixtures that were photolyzed. The general properties of, and criteria for, photoaffinity labeling reactions are discussed in the light of these findings.