Monitoring membrane protein conformational heterogeneity by fluorescence lifetime distribution analysis using the maximum entropy method

Due to the inherent difficulty in crystallizing membrane proteins, approaches based on fluorescence spectroscopy have proved useful in elucidating their conformational characteristics. The ion channel peptide gramicidin serves as an excellent prototype for monitoring membrane protein conformation and dynamics due to a number of reasons. We have analyzed conformational heterogeneity in membrane-bound gramicidin using fluorescence lifetime distribution analysis of tryptophan residues by the maximum entropy method (MEM). MEM represents a model-free and robust approach for analyzing fluorescence lifetime distribution. In this paper, we show for the first time, that fluorescence lifetime distribution analysis using MEM could be a convenient approach to monitor conformational heterogeneity in membrane-bound gramicidin in particular and membrane proteins in general. Lifetime distribution analysis by MEM therefore provides a novel window to monitor conformational transitions in membrane proteins

Enhancement of rates of H+, Na+ and K+ transport across phospholipid vesicular membrane by the combined action of carbonyl cyanide m-chlorophenylhydrazone and valinomycin: temperature-jump studies

AbstractEnhancement of ΔpH relaxation rate by the combined action of valinomycin (VAL) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) has been studied under a variety of concentration conditions in soyabean phospholipid (SBPL) vesicles after creating a pH gradient across the vesicular membrane ΔpH by temperature jump. After taking note of the changes by VAL and CCCP induced membrane disorder (using nigericin and monensin mediated ΔpH decay as probes) the following could be inferred about the mechanism of enhancement of ΔpH decay rate: (i) in solutions containing KCl, therate limiting species have been identified to be (a) Val-K+-CCCP−, at low [Val]0 and [CCCP]0 (with translocation rate constant k2 ∼ 3.2·103 s−1); (b) CCCPH, at high [Val]0 (with translocation rate constant k1 ∼ 2·105 s−1); (c) the neutral valinomycin species Val, at high [CCCP]0. (ii) In solutions containing NaCl, in our concentration range, the rate limiting species are Val-Na+-CCCP−. (iii) The apparent dissociation constant KM* of Val-M+ decreases with pH in SBPL vesicles but is independent of pH in vesicles prepared from PC + 6% PA. (iv) The differences in the ionic strength dependencies of kinetic data shows that the environments of Na+ and K+ binding sites on VAL are different. (v) In vesicle solutions containing 100 mM MCl, the cation selectivity of VAL (towards K+ in preference to Na+) is reduced when CCCP− is already bound to it in the membrane. The CCCP− dissociation constant of Val-M+-CCCP− is smaller with M+= Na+ (∼ 0.22 mM at 100 mM NaCl) when compared to that with M+ = K+ (∼ 2 mM at 100 mM KCl). Attributing these differences to the differences in electrostatic interaction between CCCP− and M+ in Val-M+-CCCP−, we can say that CCCP− binds closer to the Na+ binding site than to the K+ binding site on VAL

Two mechanisms of nonactin mediated K⁺ ion transport across phospholipid vesicular membranes

283-293From kinetic data it has been possible to show that when a weak acid such as carbonyl cyanide m-chlorophenylhydrazone (CCCP) is also present in phospholipid vesicles the ionophore nonactin (NON) transports K+ ions across the membrane by two mechanisms: (I) As the charged species NON-K+ and (II) as the electroneutral complex NON-K+- CCCP-. In mechanism I, the anion CCCP- is also translocated across the membrane as charged species such that the net charge translocated is zero. In the earlier experiments using valinomycin (VAL) and CCCP (Prabhananda B S & Kombrabail M H (1995) Biochim, Biophys, Acta 1235, 323-335) the existence of a mechanism similar to mechanism I could not be detected. Even with NON (instead of VAL), at the concentrations of our experiments mechanism II is dominant. The relative dominance of mechanism II could be decreased either (i), by lowering the ionophore concentration or (ii), by "catalysing" the transport of CCCP- from the "polar region" to the "nonpolar region" of the membrane by adding VAL at small concentrations. The kinetics of K+ transport used in arriving at such conclusions were inferred from the rate of decay of H+ concentration difference (ΔpH) across phospholipid vesicular membrane after creating ΔpH by temperature jump. A new procedure has been used to estimate the translocation rate constant k+ of NON-K+ in soybean phospholipid vesicles (~1.5 × 103 s-1) by identifying the NON-K+ translocation limited contribution to 1/τ by the combined action of NON, CCCP and tetraphenylphosphonium ions (TPP+). The apparent K+ dissociation constant of NON-K+ (~ 0.14 M), dissociation constant of NON-K+- CCCP - in the membrane (~30mM), and translocation rate constant of NON-K+- CCCP - (k0~ 4.2×103 s-1) have been determined in soybean phospholipid vesicle solutions containing 100 mM KCl

Metal ion specificity in anaesthetic induced increase in the rate of monensin and nigericin mediated H⁺/ M⁺ exchange across phospholipid vesicular membranes

415-421From a study or the decay or the pH difference across vesicular membranes (Δ pH) it has been possible to show that H+ and alkali metal ion (M+) concentration gradients across bilayer membranes  (which are responsible for driving important biochemical processes) can be selectively perturbed by anaesthetics such as chloroform and benzyl alcohol by combining them with a suitable exchange ionophore. On adding the anaesthetic to the membrane in an environment containing metal ions M+=K+. the rate or Δ pH decay by H+/M+ exchange increases by a larger factor or by a smaller factor (when compared to that in a membrane environment with M+=Na+) depending on whether the exchange ionophore chosen is monensin or nigeriein. A rational explanation of this "metal ion specificity" can be given using the exchange ionophore mediated ion transport scheme in which the equilibrations at the "interfaces" are fast compared to the "translocation equilibration" between the species in the two layers of the membrane. The following three factors are responsible for the observed "specificity": On adding the anesthetic (i) translocation rate constants increase. (ii) the concentrations of the M+ bound ionophores increase at the expense of H+ bound ionophores. (iii) Under our experimental conditions the rate determining species are the complexes monensin-K (Mon-K) and nigeriein-H (Nig-H) for M+=K+ whereas they are monensin -H (MonH) and nigeriein-Na (Nig-Na) for M+=Na+ Possible anesthetic induced membrane perturbations contributing to the above mentioned changes in the membrane are (A), the loosening of the membrane structure and (B ), an associated increase in the membrane hydration (and membrane dielectric constant ). An analysis of the consequent changes in the various transport steps shows the following: (a), The anaesthetic induced changes in the translocation rates of electrically charged species are not relevant in the explanation or the observed changes in the Δ pH decay rates. (b), Changes in the rates of fas<span style="font-size: 14.0pt;font-family:HiddenHorzOCR;mso-hansi-font-family:Arial;mso-bidi-font-family: HiddenHorzOCR">t equilibria at the interface contribute to changes in KH and <span style="font-size:14.0pt;font-family:HiddenHorzOCR;mso-hansi-font-family:Arial; mso-bidi-font-family:HiddenHorzOCR">KM (c), A suggestion made in the literature, that a significant interaction between the dipole moment of the monensin-K complex and the membrane slows down its translocation, is not valid. (d), The ability to explain rationally all the Δ p<span style="font-size:14.0pt; font-family:HiddenHorzOCR;mso-hansi-font-family:Arial;mso-bidi-font-family: HiddenHorzOCR">H decay data confirms the validity or the transport scheme used. In our experiments Δ pH across the vesicular membrane was created by pH jump coming from a temperature jump.   </span

Oligomerization of the serotonin<SUB>1A</SUB> receptor in live cells: a time-resolved fluorescence anisotropy approach

The serotonin<SUB>1A</SUB> receptor is a representative member of the G-protein coupled receptor (GPCR) superfamily and serves as an important target in the development of therapeutic agents for neuropsychiatric disorders. Oligomerization of GPCRs is an important contemporary issue since it is believed to be a crucial determinant for cellular signaling. In this work, we monitored the oligomerization status of the serotonin<SUB>1A</SUB> receptor tagged to enhanced yellow fluorescent protein (5-HT<SUB>1A</SUB>R-EYFP) in live cells utilizing time-resolved fluorescence anisotropy decay. We interpret the unresolved fast component of the observed anisotropy decay as fluorescence resonance energy transfer (FRET) between 5-HT<SUB>1A</SUB>R-EYFP molecules (homo-FRET). Homo-FRET enjoys certain advantages over hetero-FRET in the analysis of receptor oligomerization. Our results reveal the presence of constitutive oligomers of the serotonin<SUB>1A</SUB> receptor in live cells. We further show that the oligomerization status of the receptor is independent of ligand stimulation and sphingolipid depletion. Interestingly, acute (but not chronic) cholesterol depletion appears to enhance the oligomerization process. Importantly, our results are independent of receptor expression level, thereby ruling out complications arising due to high expression. These results have potential implications in future therapeutic strategies in pathophysiological conditions in which serotonin<SUB>1A</SUB> receptors are implicated

Site-specific dynamics in TAT triplex DNA as revealed by time-domain fluorescence of 2-aminopurine

Triple helices of DNA are finding increasing level of applications in several areas, including antigene therapy and gene regulation. We have probed site-specific dynamic aspects of TAT triple helices of DNA by using steady-state and time-domain fluorescence of 2-aminopurine (2-AP), a fluorescent analog of adenine. TAT triplexes were formed from repeats of adenine and thymine with 2-AP incorporated at various locations in the polyadenine strand. We find an overall decrease in the level of near-neighbor base-stacking interaction in the TAT triplex when compared to AT duplex as reported by fluorescence decay kinetics of 2-AP. More strikingly, we have observed a stark asymmetry in both the level of base stacking and motional dynamics of the bases in the two ends of TAT triplexes, namely, the 5' end having a higher level of base stacking and segmental dynamics when compared to the 3' end. The possible implications of this asymmetry, which reflects the asymmetry in the strength of Hoogstein base-pairing with the 3' end having stronger Hoogstein pairing when compared to the 5' end, is discussed

Depth-Dependent Heterogeneity in Membranes by Fluorescence Lifetime Distribution Analysis

Biological membranes display considerable anisotropy due to differences in composition, physical characteristics, and packing of membrane components. In this Letter, we have demonstrated the environmental heterogeneity along the bilayer normal in a depth-dependent manner using a number of anthroyloxy fatty acid probes. We employed fluorescence lifetime distribution analysis utilizing the maximum entropy method (MEM) to assess heterogeneity. Our results show that the fluorescence lifetime heterogeneity varies considerably depending on fluorophore location along the membrane normal (depth), and it is the result of the anisotropic environmental heterogeneity along the bilayer normal. Environmental heterogeneity is reduced as the reporter group is moved from the membrane interface to a deeper hydrocarbon region. To the best of our knowledge, our results constitute the first experimental demonstration of anisotropic heterogeneity in bilayers. We conclude that such graded environmental heterogeneity represents an intrinsic characteristics of the membrane bilayer and envisage that it has a role in the conformation and orientation of membrane proteins and their function