Fluorophore environments in membrane-bound probes: A red edge excitation shift study

Chattopadhyay, Amitabha; Mukherjee, Sushmita

doi:10.1021/bi00065a037

Cited by 138 publications

(177 citation statements)

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“…Since the biological membrane offers considerable motional restriction to a fluorophore embedded in it, the slightly larger lifetime components observed at the longer emission wavelength (539 nm) for a given NBD-cholesterol concentration is not surprising. In fact, such an increase in fluorescence lifetime with increasing emission wavelength has previously been reported for fluorophores in environments of restricted mobility (Ware et al, 1971;Matayoshi & Kleinfeld, 1981;Lakowicz et al, 1983;Demchenko & Shcherbatska, 1985;Chattopadhyay & Mukherjee, 1993;. The results shown in Table 1 indicate that, whereas the two lifetimes remain more or less constant over the NBD-cholesterol concentration range investigated when the fluorescence decays are monitored at 522 nm, they exhibit a distinct increase with concentration at 539 nm.…”

Section: Resultssupporting

confidence: 66%

Membrane Organization at Low Cholesterol Concentrations: A Study Using 7-Nitrobenz-2-oxa-1,3-diazol-4-yl-Labeled Cholesterol

Mukherjee

Chattopadhyay

1996

Biochemistry

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Cholesterol is most often found distributed nonrandomly in the plane of the bilayer, giving rise to cholesterol-rich and -poor domains. Many of these domains are thought to be crucial for the maintenance of membrane structure and function. However, such well-characterized domains generally occur in the membranes that contain relatively large amounts of cholesterol. Cholesterol organization in membranes containing very low amounts of cholesterol has not been investigated extensively. Recent evidence from differential-scanning calorimetric studies suggests that cholesterol may not form uniform monodisperse solutions, as assumed earlier, in the membranes even at very low concentrations. Fluorescent cholesterol analogues, when chosen carefully, offer a powerful approach for studying the distribution and organization of cholesterol in membranes at low concentrations. In this paper, we have studied the organization of cholesterol in membranes at very low concentrations (up to 5 mol % of the total lipid) using a fluorescent cholesterol analogue (NBD-cholesterol) which is labeled with the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group at the flexible acyl chain, without any alteration in the structural features necessary for proper membrane incorporation. Our results show that NBD-cholesterol exhibits local organization even at very low concentrations. This is consistent with the recently suggested model of cholesterol organization in membranes at low concentrations, involving the formation of transbilayer, tail-to-tail dimers The fluid mosaic model for biological membranes defines the membrane as an oriented, two-dimensional, viscous solution of proteins and lipids in instantaneous thermodynamic equilibrium (Singer & Nicolson, 1972). Although the basic tenets of this model have stood the test of time, one of its implicit assumptions, that the lipid molecules merely provide the milieu for the membrane proteins and that they are distributed uniformly throughout the bilayer, has been found to be not quite true. In fact, current evidence points out that both transverse and lateral regionalization, which can be described in terms of micro-and macrodomains, are common features of many biological membranes (Curtain et al., 1988;Edidin, 1992;Tocanne, 1992;Glaser, 1993;Jacobson et al., 1995).Cholesterol is one of the membrane components that is found, more often than not, distributed nonrandomly in structural and kinetic domains or pools in both biological and model membranes (Yeagle, 1985;Gennis, 1989;Schroeder et al., 1991;Liscum & Underwood, 1995). The reason for this lies in the rather unusual chemical structure of cholesterol that can by no means be considered a representative component of the "bulk" membrane (see Figure 1). The unusual shape of cholesterol also causes a phase separation in membranes at relatively high concentrations, resulting in the formation of cholesterol-rich and -poor domains that are structurally and compositionally so distinct that they can be observed histochemically and isolated physical...

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Section: Resultssupporting

confidence: 66%

Membrane Organization at Low Cholesterol Concentrations: A Study Using 7-Nitrobenz-2-oxa-1,3-diazol-4-yl-Labeled Cholesterol

Mukherjee

Chattopadhyay

1996

Biochemistry

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“…65 The lifetime of NBD-PE incorporated into DOPC vesicles has been reported to be ∼7.4 ns. 15 In water, however, NBD lifetime reduces to ∼1.5 ns, which has been attributed to hydogen-bonding interactions between the fluorophore and the solvent 64 which is accompanied by an increase in the rate of nonradiative decay. 44 Our results show that the lifetimes of NBD-PE when incorporated into micelles, in general, are smaller than the lifetime obtained for membrane-bound NBD-PE.…”

Section: Resultsmentioning

confidence: 99%

“…Such a marked shortening of mean fluorescence lifetime at the red edge of the absorption band is indicative of slow solvent reorientation around the excited state fluorophore. 15,17,20 Table 2 shows the lifetimes of micelle-bound NBD-PE as a function of emission wavelength, keeping the excitation wavelength constant at 465 nm. All decays corresponding to different emission wavelengths could be fitted to biexponential functions.…”

Section: Resultsmentioning

confidence: 99%

“…In one of our studies, the wavelength-selective fluorescence characteristics of the widely used membrane probe N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (NBD-PE), when bound to model membranes of dioleoyl-sn-glycero-3-phosphocholine (DOPC), was reported. 15 We have also monitored the tryptophan environments in membrane-bound peptides such as melittin, a powerful hemolytic peptide from the venom of the honey bee Apis mellifera, 16,26 and in the channel forming peptide gramicidin, produced by the bacteria Bacillus breVis. 17 Interestingly, in both these peptides the tryptophan residues have been shown to be extremely important for the function of the peptide.…”

Section: Introductionmentioning

confidence: 99%

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Micellar Organization and Dynamics: A Wavelength-Selective Fluorescence Approach

1997

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Wavelength-selective fluorescence comprises a set of approaches based on the red edge effect in fluorescence spectroscopy, which can be used to monitor directly the environment and dynamics around a fluorophore in a complex biological system. A shift in the wavelength of maximum fluorescence emission toward higher wavelengths, caused by a shift in the excitation wavelength toward the red edge of the absorption band, is termed the red edge excitation shift (REES). This effect is mostly observed with polar fluorophores in motionally restricted media such as very viscous solutions or condensed phases. We have previously shown that REES and related techniques (wavelength-selective fluorescence approach) offer a novel way to monitor organization and dynamics of membrane-bound probes and peptides. In this paper, we report REES of NBD-PE, a phospholipid whose headgroup is covalently labeled with the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) moiety, when incorporated into micelles formed by a variety of detergents (SDS, Triton X-100, CTAB and CHAPS) which differ in their charge and organization. In addition, fluorescence polarization of NBD-PE in these micelles shows both excitation and emission wavelength dependence. The lifetime of NBD-PE was found to be dependent on both excitation and emission wavelengths. These wavelength-dependent lifetimes are correlated to the reorientation of solvent dipoles around the excited-state dipole of the NBD group in micellar environments. Taken together, these observations are indicative of the motional restriction experienced by the fluorophore when bound to micelles. Wavelength-selective fluorescence promises to be a powerful tool for studying micellar organization and dynamics.

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“…This change in dipole moment of the NBD group is significant in the context of solvent reorientation in its excited state. 9 An important parameter in the Lippert equation is the Onsager cavity radius.29 It is operationally defined as the radius of a spherical cavity surrounding the molecule in which the molecule resides and is usually equated to its molecular (van der Waals) radius.3s32 We have calculated the Onsager cavity radius for the NBD moiety from the energy minimized structure of the NBD derivative I using the AM 1 program. The distance between the average coordinates of the oxygen atoms (0-14 and -15) of the nitro group on one end of the molecule and the nitrogen atom of the amino group (N-1 1) on the other end is the longest distance across the molecule where charge separation could occur (see Figure 3).…”

Section: Discussionmentioning

confidence: 99%

Dipole moment change of NBD group upon excitation studied using solvatochromic and quantum chemical approaches: Implications in membrane research

Mukherjee¹,

Chattopadhyay²,

Samanta³

et al. 1994

J. Phys. Chem.

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Lipids that are covalently labeled with the 7-nitrobenz-2-oxa-1,3-diazol-4-y1 (NBD) group are widely used as fluorescent analogues of native lipids in model and biological membranes to study a variety of processes. We have recently shown that one such NBD-labeled lipid, NBD-PE, in which the NBD label is covalently attached to the headgroup of a phosphatidylethanolamine molecule, exhibits the red edge excitation shift (REES) effect when incorporated into vesicles of dioleoyl-sn-glycero-3-phosphocholine (DOPC) [Chattopadhyay, A,; Mukherjee, S . Biochemistry 1993,32,3804]. One of the necessary conditions for a fluorophore to be able to exhibit REES is that the fluorophore must be polar and, more importantly, there should be a change in its dipole moment upon excitation. In this paper, we have determined the actual change in dipole moment of the NBD group upon excitation using the solvatochromic shift approach. Our results show that the dipole moment of the NBD group changes by 3.9 D upon excitation. We have complemented this experimental observation by semiempirical quantum chemical calculations of dipole moment changes of various NBD derivatives. These calculated dipole moment changes (3.5-3.6 D) agree very well with our experimental value. These calculations also point out that the process of charge separation is mainly limited to the NBD ring system and is independent of the length of the alkyl chain. These results are relevant to ongoing and future studies that utilize photophysical properties of the NBD group, especially in microheterogeneous media such as membranes and micelles.

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Fluorophore environments in membrane-bound probes: A red edge excitation shift study

Cited by 138 publications

References 82 publications

Membrane Organization at Low Cholesterol Concentrations: A Study Using 7-Nitrobenz-2-oxa-1,3-diazol-4-yl-Labeled Cholesterol

Membrane Organization at Low Cholesterol Concentrations: A Study Using 7-Nitrobenz-2-oxa-1,3-diazol-4-yl-Labeled Cholesterol

Micellar Organization and Dynamics: A Wavelength-Selective Fluorescence Approach

Dipole moment change of NBD group upon excitation studied using solvatochromic and quantum chemical approaches: Implications in membrane research

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