Dansylation of bacteriorhodopsin near the retinal attachment site

Harris, Gary J.; Renthal, Robert; Tuley, J.; Robinson, N.

doi:10.1016/0006-291x(79)91968-5

Cited by 21 publications

(5 citation statements)

References 14 publications

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“…If the acceptor is itself fluorescent, then emission will be observed from the acceptor when the donor is excited (sensitized emission). We previously found that, near neutral pH, dansyl chloride exclusively modifies Lys 41 of bacteriorhodopsin ( , ). The Förster critical distance for energy transfer from tryptophan to dansyl is 21 Å (distance at which 50% of the emission is transferred to the acceptor).…”

Section: Discussionmentioning

confidence: 99%

“…Fluorescent Labeling. Purple membrane was specifically modified at Lys 41 with dansyl chloride as previously described (30,32). Dansylbacterioopsin was purified by dissolving lyophilized dansyl-purple membrane in 90% formic acid, diluting to 30% with ethanol (33), and chromatographing on LH-60 in CHCl 3 /methanol (1:1) containing 0.1 M ammonium acetate (5).…”

Section: Methodsmentioning

confidence: 99%

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Estimation of Helix−Helix Association Free Energy from Partial Unfolding of Bacterioopsin

Nannepaga¹,

Gawalapu²,

Velasquez³

et al. 2003

Biochemistry

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To obtain thermodynamic information about interactions between transmembrane helices in integral membrane proteins, partial unfolding of bacterioopsin in ethanol/water mixtures was studied by Förster-type resonance energy transfer (FRET) from tryptophan to a dansyl group on Lys 41. Tryptophan to dansyl FRET was detected by measuring sensitized emission at 490-500 nm from 285 nm excitation. FRET was observed in dansylbacterioopsin in apomembranes and in detergent micelles but not in 90% ethanol/water or in the chymotrypsin fragment C2 (residues 1-71). The main fluorescence donors are Trp 86 and Trp 182. Increase of FRET from C2 with added chymotrypsin fragment C1 (residues 72-248) provides an estimate of the C1-C2 association constant as 7.7 x 10(6) M(-1). With increasing ethanol concentration, the FRET signal from dansylbacterioopsin in detergent micelles disappeared with a sharp transition above 60% ethanol. No transition occurred in Trp fluorescence from bacterioopsin lacking the dansyl acceptor, nor did dansyl model compounds undergo a similar transition. Light scattering measurements show that the detergent micelles dissipate below 50% ethanol. Thus the observed transition is likely to be a partial unfolding of bacterioopsin. Assuming a two-state unfolding model, the free energy of unfolding was obtained by extrapolation as 9.0 kcal/mol. The slope of the transition (m-value) was -0.8 kcal mol(-1) M(-1). The unfolding process probably involves dissociation of several helices. The rate of association was measured by stopped-flow fluorometry. Two first-order kinetic processes were observed, having approximately equal weights, with rate constants of 2.32 s (-1) and 0.185 s(-1).

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Estimation of Helix−Helix Association Free Energy from Partial Unfolding of Bacterioopsin

Nannepaga¹,

Gawalapu²,

Velasquez³

et al. 2003

Biochemistry

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“…Fluorescent Labeling. Purple membrane was reacted with dansyl chloride as previously described (9,11,12). Purple membrane isolated from H. salinarum containing single cysteine bacteriorhodopsin (BR) mutants was labeled with didansyl cystine as follows.…”

Section: Methodsmentioning

confidence: 99%

Transmembrane Helix−Helix Association: Relative Stabilities at Low pH

Valluru¹,

Silva²,

Dhage³

et al. 2006

Biochemistry

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We have previously studied the unfolding equilibrium of bacterio-opsin in a single phase solvent, using Förster mechanism fluorescence resonance energy transfer (FRET) as a probe, from tryptophan donors to a dansyl acceptor. We observed an apparent unfolding transition in bacterio-opsin perturbed by increasing ethanol concentrations [Nannepaga et al.(2004) Biochemistry 43, 50-59]. We have further investigated this transition and find the unfolding is pH-dependent. We have now measured the apparent pK of acid-induced unfolding of bacterio-opsin in 90% ethanol. When the acceptor is on helix B (Lys 41), the apparent pK for unfolding is 4.75; on the EF connecting loop (Cys 163), 5.15; and on helix G (Cys 222), 5.75. Five-helix proteolytic fragments are less stable. The apparent unfolding pKs are, for residues 72-248 (Cys 163), 5.46; and 1-166 (Lys 41), 7.36. When interpreted in terms of a simple equilibrium model for unfolding, the apparent pKs give relative free energies of unfolding, in the range of −0.54 to −3.5 kcal/mol. The results suggest that the C-terminal helix of bacterio-opsin is less stably folded than the N-terminal helices. We analyzed the pair-wise helixhelix interaction surfaces of bacteriorhodopsin and three other 7-transmembrane helix proteins, based on crystal structures. The results show that the interaction surfaces are smoother and the helix axis separations are closer in the amino-terminal two-thirds of the proteins compared with the carboxyl terminal one-third. However, the F helix is important in stabilizing the folded structure, as shown by the instability of the 1-166 fragment. Considering the high resolution crystal structure of bacteriorhodopsin, there are no obvious helix-helix interactions involving protein side chains which would be destabilized by protonation at the estimated pH of the unfolding transitions. However, a number of helix-bridging water molecules could become protonated, thereby weakening the helixhelix interactions.In comparison with water-soluble proteins, integral membrane protein folding mechanisms are poorly understood. Results of several folding studies, both in vivo (1) and in vitro (2-6), have been published on membrane proteins containing transmembrane helices. In some in vitro experiments, the membrane proteins were solubilized in detergent micelles, and unfolding was induced by an anionic amphiphile perturbant, docecyl sulfate. In order to obtain free energies of unfolding, it is necessary to assume that the free energy changes are linear with perturbant concentration (7). Due to non-ideal mixing when charged amphiphiles are added to neutral micelles, the assumption of linearity does not necessarily hold (8). An alternative approach would be to examine unfolding in a single phase solvent system. We have been studying the † Supported by a grant from the National Institutes of Health (GM 08194) *To whom to address correspondence at Dept. unfolding equilibrium of bacterio-opsin (BO) 1 in ethanol/water mixtures. We found an apparent unfolding transition in...

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“…To our knowledge, there are no prior reports of in vitro FRET studies on membrane protein folding. Instead, a number of membrane proteins have been labeled with dyes on solvent-exposed residues to probe conformational changes and global interactions; nearly all of these examples utilized proteins in native membrane environments or solubilized in detergent [42,51–53]. As shown by SDS-PAGE analysis, OmpA that was labeled with dns inserts and folds into membranes with yields that are comparable to unlabeled OmpA.…”

Section: Discussionmentioning

confidence: 99%

Förster resonance energy transfer as a probe of membrane protein folding

Kang¹,

López-Peña²,

Oklejas³

et al. 2012

Biochimica et Biophysica Acta (BBA) - Biomembranes

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The folding reaction of a β-barrel membrane protein, Outer Membrane Protein A (OmpA), is probed with Förster resonance energy transfer (FRET) experiments. Four mutants of OmpA were generated in which the donor fluorophore, tryptophan, and acceptor molecule, 1,5-IAEDANS, are placed in various locations on the protein to report the evolution of distances across the bilayer and across the protein pore during a folding event. Analysis of the FRET efficiencies reveals three timescales for tertiary structure changes associated with insertion and folding into a synthetic bilayer. A narrow pore forms during the initial stage of insertion, followed by bilayer traversal. Finally, a long-time component is attributed to equilibration and relaxation, and may involve global changes such as pore expansion and strand extension. These results augment the existing models that describe concerted insertion and folding events, and highlight the ability of FRET to provide insight into the complex mechanisms of membrane protein folding.

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Dansylation of bacteriorhodopsin near the retinal attachment site

Cited by 21 publications

References 14 publications

Estimation of Helix−Helix Association Free Energy from Partial Unfolding of Bacterioopsin

Estimation of Helix−Helix Association Free Energy from Partial Unfolding of Bacterioopsin

Transmembrane Helix−Helix Association: Relative Stabilities at Low pH

Förster resonance energy transfer as a probe of membrane protein folding

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