Sourav Haldar

Centre for Cellular and Molecular Biology, Bhaganagar, Andhra Pradesh, India

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Publications (19)78.53 Total impact

  • [show abstract] [hide abstract]
    ABSTRACT: The linear ion channel peptide gramicidin represents an excellent model for exploring the principles underlying membrane protein structure and function, especially with respect to tryptophan residues. The tryptophan residues in gramicidin channels are crucial for the structure and function of the channel. In order to test the importance of indole hydrogen bonding for the biophysical properties of gramicidin channels, we monitored the effect of N-methylation of gramicidin tryptophans, using a combination of steady state and time-resolved fluorescence approaches along with circular dichroism spectroscopy. We show here that in the absence of hydrogen bonding ability of tryptophans, tetramethyltryptophan gramicidin (TM-gramicidin) is unable to maintain the single stranded, head-to-head dimeric channel conformation in membranes. Our results show that TM-gramicidin displays a red-shifted fluorescence emission maximum, lower red edge excitation shift (REES), and higher fluorescence intensity and lifetime, consistent with its nonchannel conformation. This is in agreement with the measured location (average depth) of the 1-methyltryptophans in TM-gramicidin using the parallax method. These results bring out the usefulness of 1-methyltryptophan as a fluorescent tool to examine the hydrogen bonding ability of tryptophans in proteins and peptides. We conclude that changes in the hydrogen bonding ability of tryptophans, along with coupled changes in peptide backbone structure induce the loss of single stranded β(6.3) helical dimer conformation. These results agree with earlier results from size-exclusion chromatography and single-channel measurements for TM-gramicidin, and confirm the importance of indole hydrogen bonding for the conformation and function of ion channels and membrane proteins.
    Biochimica et Biophysica Acta 10/2013; · 4.66 Impact Factor
  • Amitabha Chattopadhyay, Sourav Haldar
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    ABSTRACT: Proteins are considered the workhorses in the cellular machinery. They are often organized in a highly ordered conformation in the crowded cellular environment. These conformations display characteristic dynamics over a range of time scales. An emerging consensus is that protein function is critically dependent on its dynamics. The subtle interplay between structure and dynamics is a hallmark of protein organization and is essential for its function. Depending on the environmental context, proteins can adopt a range of conformations such as native, molten globule, unfolded (denatured), and misfolded states. Although protein crystallography is a well established technique, it is not always possible to characterize various protein conformations by X-ray crystallography due to transient nature of these states. Even in cases where structural characterization is possible, the information obtained lacks dynamic component, which is needed to understand protein function. In this overall scenario, approaches that reveal information on protein dynamics are much appreciated. Dynamics of confined water has interesting implications in protein folding. Interfacial hydration combines the motion of water molecules with the slow moving protein molecules. The red edge excitation shift (REES) approach becomes relevant in this context. REES is defined as the shift in the wavelength of maximum fluorescence emission toward higher wavelengths, caused by a shift in the excitation wavelength toward the red edge of absorption spectrum. REES arises due to slow rates (relative to fluorescence lifetime) of solvent relaxation (reorientation) around an excited state fluorophore in organized assemblies such as proteins. Consequently, REES depends on the environment-induced motional restriction imposed on the solvent molecules in the immediate vicinity of the fluorophore. In the case of a protein, the confined water in the protein creates a dipolar field that acts as the solvent for a fluorophore in the protein. In this Account, we focus on REES to monitor organization and dynamics of soluble and membrane proteins utilizing intrinsic protein fluorescence. We discuss here the application of REES in various conformations of proteins. While application of REES to proteins in native conformation has been in use for a long time, our work highlights the potential of this approach in case of molten globule and denatured conformations. For example, we have demonstrated the presence of residual structure, that could not be detected using other methods, by REES of denatured spectrin. Given the functional relevance of such residual structures, these results are very far reaching. We discuss here the application of REES to molten globule conformation and to the green fluorescent protein (GFP). The case of GFP is particularly interesting since the dipolar field in this case is provided by the protein matrix itself and not confined water. We envision that future applications of REES in proteins will involve generating a dynamic hydration map of the protein, which would allow us to explore protein function in terms of local dynamics and hydration.
    Accounts of Chemical Research 08/2013; · 20.83 Impact Factor
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    ABSTRACT: Water plays a fundamental role in the folding, structure, dynamics and function of proteins and peptides. The extracellular N-terminal domain of chemokine receptors is crucial in mediating binding affinity, receptor selectivity, and regulating function. The flexible N-terminal domain becomes ordered in membranes and membrane-mimetic assemblies, thereby indicating that the membrane could play an important role in regulating CXC chemokine receptor 1 (CXCR1) function. In view of the role of hydration in lipid-protein interactions in membranes, we explored the organization and dynamics of a 34-mer peptide of CXCR1 N-terminal domain in reverse micelles by utilizing a combination of fluorescence-based approaches and circular dichroism spectroscopy. Our results show that the secondary structure adopted by the CXCR1 N-domain is critically dependent on hydration. The tryptophan residues of the CXCR1 N-domain experience motional restriction and exhibit red edge excitation shift (REES) upon incorporation in reverse micelles. REES and fluorescence lifetime exhibit reduction with increasing reverse micellar hydration. Time-resolved fluorescence anisotropy measurements reveal the effect of hydration on peptide rotational dynamics. Taken together, these results constitute the first report demonstrating modulation in the organization and dynamics of the N-terminal domain of a chemokine receptor in membrane-like environment of varying hydration. We envisage that these results are relevant in the context of hydration in the function of G protein-coupled receptors.
    The Journal of Physical Chemistry B 01/2013; · 3.61 Impact Factor
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    ABSTRACT: Dipole potential is the potential difference within the membrane bilayer, which originates due to the nonrandom arrangement of lipid dipoles and water molecules at the membrane interface. In this work, we have explored the possible correlation between functional modulation of a G protein-coupled receptor (the serotonin(1A) receptor) and membrane dipole potential, under conditions of altered membrane sterol composition. We have previously shown that the ligand binding activity of the hippocampal serotonin(1A) receptor is reduced upon cholesterol depletion and could be restored upon replenishment with cholesterol. Interestingly, when the replenishment was carried out with an immediate biosynthetic precursor of cholesterol (7-DHC), differing with cholesterol merely in a double bond, the ligand binding activity of the receptor was not restored. In order to understand the mechanistic framework of receptor-cholesterol interaction, we carried out dipole potential measurements of hippocampal membranes under these conditions, by the dual wavelength ratiometric approach using an electrochromic probe (di-8-ANEPPS). We show here that dipole potential of hippocampal membranes is reduced upon progressive depletion of cholesterol and is restored upon replenishment with cholesterol, but not with 7-DHC. Our results show that the recovery of ligand binding activity of the serotonin(1A) receptor upon replenishment with cholesterol (but not with 7-DHC) could be related to the differential ability of these closely related sterols to modulate membrane dipole potential. We conclude that subtle changes in membrane dipole potential could be crucial in understanding the complex interplay between membrane lipids and proteins in the cellular milieu.
    Biochimica et Biophysica Acta 11/2012; · 4.66 Impact Factor
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    ABSTRACT: The linear ion channel peptide gramicidin serves as an excellent prototype for monitoring the organization, dynamics, and function of membrane-spanning channels. The tryptophan residues in gramicidin channels are crucial for establishing and maintaining the structure and function of the channel in the membrane bilayer. In order to address the basis of differential importance of tryptophan residues in the gramicidin channel, we monitored the effects of pairwise substitution of two of the four gramicidin tryptophans, the inner pair (Trp-9 and -11) and the outer pair (Trp-13 and -15), using a combination of steady state and time-resolved fluorescence approaches and circular dichroism spectroscopy. We show here that these double tryptophan gramicidin analogues adopt different conformations in membranes, suggesting that the conformational preference of double tryptophan gramicidin analogues is dictated by the positions of the tryptophans in the sequence. These results assume significance in the context of recent observations that the inner pair of tryptophans (Trp-9 and -11) is more important for gramicidin channel formation and channel conductance. These results could be potentially useful in analyzing the effect of tryptophan substitution on the functioning of ion channels and membrane proteins.
    The Journal of Physical Chemistry B 08/2012; 116(36):11056-64. · 3.61 Impact Factor
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    ABSTRACT: Dipole potential is the potential difference within the membrane bilayer, which originates due to the nonrandom arrangement of lipid dipoles and water molecules at the membrane interface. Cholesterol, a representative sterol in higher eukaryotic membranes, is known to increase membrane dipole potential. In this work, we explored the effects of immediate (7-DHC and desmosterol) and evolutionary (ergosterol) precursors of cholesterol on membrane dipole potential, monitored by the dual wavelength ratiometric approach utilizing the probe di-8-ANEPPS. Our results show that the effect of these precursors on membrane dipole potential is very different from that observed with cholesterol, although the structural differences among them are subtle. These results assume relevance, since accumulation of cholesterol precursors due to defective cholesterol biosynthesis has been reported to result in several inherited metabolic disorders such as the Smith-Lemli-Opitz syndrome. Interestingly, cholesterol (and its precursors) has a negligible effect on dipole potential in polyunsaturated membranes. We interpret these results in terms of noncanonical orientation of cholesterol in these membranes. Our results constitute the first report on the effect of biosynthetic and evolutionary precursors of cholesterol on dipole potential, and imply that a subtle change in sterol structure can significantly alter the dipolar field at the membrane interface.
    Biophysical Journal 04/2012; 102(7):1561-9. · 3.67 Impact Factor
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    ABSTRACT: Dipole potential is the potential difference within the membrane bilayer, which originates due to the nonrandom arrangement of lipid dipoles and water molecules at the membrane interface. Cholesterol, a representative sterol in higher eu-karyotic membranes, is known to increase membrane dipole potential. In this work, we explored the effects of immediate (7-DHC and desmosterol) and evolutionary (ergosterol) precursors of cholesterol on membrane dipole potential, monitored by the dual wavelength ratiometric approach utilizing the probe di-8-ANEPPS. Our results show that the effect of these precursors on membrane dipole potential is very different from that observed with cholesterol, although the structural differences among them are subtle. These results assume relevance, since accumulation of cholesterol precursors due to defective cholesterol biosynthesis has been reported to result in several inherited metabolic disorders such as the Smith-Lemli-Opitz syndrome. Inter-estingly, cholesterol (and its precursors) has a negligible effect on dipole potential in polyunsaturated membranes. We interpret these results in terms of noncanonical orientation of cholesterol in these membranes. Our results constitute the first report on the effect of biosynthetic and evolutionary precursors of cholesterol on dipole potential, and imply that a subtle change in sterol struc-ture can significantly alter the dipolar field at the membrane interface.
    Biophysical Journal 03/2012; · 3.67 Impact Factor
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    ABSTRACT: Structural transitions involving shape changes play an important role in cellular physiology. Such transition can be conveniently induced in charged micelles by increasing ionic strength of the medium. Shape changes have recently been shown to result in altered packing and lowering of micellar polarity. As a consequence of reduced polarity, the ionization states of micelle-bound molecules vary in micelles of different shape. The changes in micellar organization and dynamics due to structural transition can be effectively monitored utilizing the red edge excitation shift (REES). These changes are influenced by the position (location) of the probe in the micelle, i.e., the region of the micelle being monitored. Changes in organization and dynamics of probes and peptides upon structural transition are discussed with representative examples. We envisage that the reduction in micellar polarity and tighter packing upon structural transition represent important factors in the incorporation of drugs in micelles (nano-carriers), since micellar polarity plays a crucial role in the incorporation of drugs.
    Chemistry and physics of lipids 09/2011; 165(4):497-504. · 2.15 Impact Factor
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    ABSTRACT: Dynamics of confined water has interesting implications in the organization and function of molecular assemblies such as membranes. A direct consequence of this type of organization is the restriction imposed on the mobility of the constituent structural units. Interestingly, this restriction (confinement) of mobility couples the motion of solvent (water) molecules with the slow moving molecules in the assembly. It is in this context that the red edge excitation shift (REES) represents a sensitive approach to monitor the environment and dynamics around a fluorophore in such organized assemblies. 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 REES. REES relies on slow solvent reorientation in the excited state of a fluorophore that can be used to monitor the environment and dynamics around a fluorophore in a host assembly. In this article, we focus on the application of REES to monitor organization and dynamics of membrane probes and proteins.
    The Journal of Physical Chemistry B 03/2011; 115(19):5693-706. · 3.61 Impact Factor
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    ABSTRACT: The relevance of partially ordered states of proteins (such as the molten globule state) in cellular processes is beginning to be understood. Bovine alpha-lactalbumin (BLA) assumes the molten globule state at acidic pH. We monitored the organization and dynamics of the functionally important tryptophan residues of BLA in native and molten globule states utilizing the wavelength-selective fluorescence approach and fluorescence quenching. Quenching of BLA tryptophan fluorescence using quenchers of varying polarity (acrylamide and trichloroethanol) reveals varying degrees of accessibility of tryptophan residues, characteristic of native and molten globule states. We observed red edge excitation shift (REES) of 6 nm for the tryptophans in native BLA. Interestingly, we show here that BLA tryptophans exhibit REES (3 nm) in the molten globule state. These results constitute one of the early reports of REES in the molten globule state of proteins. Taken together, our results indicate that tryptophan residues in BLA in native as well as molten globule states experience motionally restricted environment and that the regions surrounding at least some of the BLA tryptophans offer considerable restriction to the reorientational motion of the water dipoles around the excited-state tryptophans. These results are supported by wavelength-dependent changes in fluorescence anisotropy and lifetime for BLA tryptophans. These results could provide vital insight into the role of tryptophans in the function of BLA in its molten globule state in particular, and other partially ordered proteins in general.
    Biophysics of Structure and Mechanism 04/2010; 39(10):1453-63. · 2.44 Impact Factor
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    ABSTRACT: The N-terminal domain of chemokine receptors constitutes one of the two critical ligand binding sites, and plays important roles by mediating binding affinity, receptor selectivity, and regulating function. In this work, we monitored the organization and dynamics of a 34-mer peptide of the CXC chemokine receptor 1 (CXCR1) N-terminal domain and its interaction with membranes by utilizing a combination of fluorescence-based approaches and surface pressure measurements. Our results show that the CXCR1 N-domain 34-mer peptide binds vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and upon binding, the tryptophan residues of the peptide experience motional restriction and exhibit red edge excitation shift (REES) of 19nm. These results are further supported by increase in fluorescence anisotropy and mean fluorescence lifetime upon membrane binding. These results constitute one of the first reports demonstrating membrane interaction of the N-terminal domain of CXCR1 and gain relevance in the context of the emerging role of cellular membranes in chemokine signaling.
    Biochimica et Biophysica Acta 03/2010; 1798(6):1056-61. · 4.66 Impact Factor
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    ABSTRACT: Bovine alpha-lactalbumin (BLA) is known to be present in molten globule form in its apo-state (i.e., Ca(2+) depleted state). We explored the organization and dynamics of the functionally important tryptophan residues of BLA in native, molten globule and denatured states utilizing the wavelength-selective fluorescence approach. We observed red edge excitation shift (REES) of 7 nm for the tryptophans in native BLA. Interestingly, we show here that BLA tryptophans exhibit considerable REES (8 nm) in its molten globule state. Taken together, these results indicate that tryptophan residues in BLA in native as well as molten globule states experience motionally restricted environment. We further show that even the denatured form of BLA exhibits a modest REES of 3 nm, indicating that the tryptophans are shielded from bulk solvent, even when denatured, due to the presence of residual structure around tryptophan(s). This is further supported by wavelength-dependent changes in fluorescence anisotropy and lifetime for BLA tryptophans. These novel results constitute one of the first reports of REES in the molten globule state of proteins, and could provide vital insight into the role of tryptophans in the function of BLA in its molten globule state in particular, and other partially ordered proteins in general.
    Biochemical and Biophysical Research Communications 03/2010; 394(4):1082-6. · 2.41 Impact Factor
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    ABSTRACT: Structural transitions involving shape changes play an important role in cellular physiology. Such transition can be induced in charged micelles at a given temperature by increasing ionic strength of the medium. We have monitored the change in organization and dynamics associated with sphere-to-rod transition of SDS micelles utilizing pyrene fluorescence. We report here, utilizing changes in the ratio of pyrene vibronic peak intensities (I(1)/I(3)), the apparent dielectric constant experienced by pyrene in spherical SDS micelles (in absence of salt) to be approximately 32. Interestingly, the apparent micellar dielectric constant exhibits a reduction with increasing NaCl concentration. The dielectric constant in rod-shaped micelles of SDS (in presence of 0.5M NaCl) turns out to be approximately 22. To the best of our knowledge, these results constitute one of the early reports on polarity estimates in rod-shaped micelles. In addition, pyrene excimer/monomer ratio shows increase in SDS micelles with increasing NaCl concentration. We interpret this increase due to an increase in average number of pyrene molecules per micelle associated with the sphere-to-rod structural transition. These results could be significant in micellar drug solubilization and delivery, and in membrane morphology changes.
    Biochemical and Biophysical Research Communications 10/2009; 390(3):728-32. · 2.41 Impact Factor
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    ABSTRACT: 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.
    Journal of Fluorescence 10/2009; 20(1):407-13. · 1.79 Impact Factor
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    Sourav Haldar, Amitabha Chattopadhyay
    Journal of Biosciences 07/2009; 34(2):169-72. · 1.76 Impact Factor
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    ABSTRACT: Fluorescent analogues of cholesterol offer a powerful approach for monitoring cholesterol behavior in membranes because of their high sensitivity, suitable time resolution, and multiplicity of measurable parameters. In this work, we have monitored the orientation and dynamics of a novel fluorescent cholesterol probe, 6-dansylcholestanol (DChol), in membranes of different phase type utilizing sensitive fluorescence techniques including the red-edge excitation shift (REES) approach. Our results show that fluorescence emission maximum, anisotropy, and lifetime of DChol are dependent on the phase of the membrane. Interestingly, DChol exhibits significant red-edge excitation shift (REES) that appear to depend on the phase of the membrane. Analysis of membrane penetration depth by the parallax method shows that the dansyl group of DChol is localized at the interfacial region of the membrane ( approximately 15.6 A from the center of the bilayer). This is in excellent agreement with the previously reported location of cholesterol in fluid-phase membranes. We propose that DChol could be a potentially useful cholesterol analogue in future studies of model and biological membranes.
    The Journal of Physical Chemistry B 03/2009; 113(13):4475-81. · 3.61 Impact Factor
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    Sourav Haldar, Amitabha Chattopadhyay
    Journal of Fluorescence 01/2009; 19(1):1-2. · 1.79 Impact Factor
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    ABSTRACT: Melittin is a cationic hemolytic peptide isolated from the European honey bee, Apis mellifera. In spite of a number of studies, there is no consensus regarding the orientation of melittin in membranes. In this study, we used a melittin analogue that is covalently labeled at its amino terminal (Gly-1) with the environment-sensitive 1-dimethylamino-5-sulfonylnaphthalene (dansyl) group to obtain information regarding the orientation and dynamics of the amino terminal region of membrane-bound melittin. Our results show that the dansyl group in Dns-melittin exhibits red edge excitation shift in vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine, implying its localization in a motionally restricted region of the membrane. This is further supported by wavelength-dependent anisotropy and lifetime changes and time-resolved emission spectra characterized by dynamic Stokes shift, which indicates relatively slow solvent relaxation in the excited state. Membrane penetration depth analysis using the parallax method shows that the dansyl group is localized at a depth of approximately 18 A from the center of the bilayer in membrane-bound Dns-melittin. Further analysis of dansyl and tryptophan depths in Dns-melittin shows that the tilt angle between the helix axis of membrane-bound melittin and the bilayer normal is approximately 70 degrees. Our results therefore suggest that melittin adopts a pseudoparallel orientation in DOPC membranes at low concentration.
    The Journal of Physical Chemistry B 11/2008; 112(44):14075-82. · 3.61 Impact Factor
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    Sourav Haldar, Amitabha Chattopadhyay
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    ABSTRACT: The fluorophore in green fluorescent protein (GFP) is localized in a highly constrained environment, protected from the bulk solvent by the barrel-shaped protein matrix. We have used the wavelength-selective fluorescence approach (red edge excitation shift, REES) to monitor solvent (environment) dynamics around the fluorophore in enhanced green fluorescent protein (EGFP) under various conditions. Our results show that EGFP displays REES in buffer and glycerol, i.e., the fluorescence emission maxima exhibit a progressive shift toward the red edge, as the excitation wavelength is shifted toward the red edge of the absorption spectrum. Interestingly, EGFP displays REES when incorporated in reverse micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT), independent of the hydration state. We interpret the observed REES to the constrained environment experienced by the EGFP fluorophore in the rigid protein matrix, rather than to the dynamics of the bulk solvent. These results are supported by the temperature dependence of REES and characteristic wavelength-dependent changes in fluorescence anisotropy.
    The Journal of Physical Chemistry B 01/2008; 111(51):14436-9. · 3.61 Impact Factor