Green fluorescent protein: a molecular lantern that illuminates the cellular interior

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Organization and dynamics of tryptophans in the molten globule state of bovine α -lactalbumin utilizing wavelength-selective fluorescence approach: comparisons with native and denatured states

Bovine a-lactalbumin (BLA) is known to be present in molten globule form in its apo-state (i.e., Ca2+ 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

Organization and dynamics in micellar structural transition monitored by pyrene fluorescence

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 (I1/I3), the apparent dielectric constant experienced by pyrene in spherical SDS micelles (in absence of salt) to be ˜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 0.5 M NaCl) turns out to be ˜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

Effect of ionic strength on the organization and dynamics of membrane-bound melittin

Melittin, a cationic hemolytic peptide, is intrinsically fluorescent due to the presence of a single functionally important tryptophan residue. We have previously shown that the sole tryptophan of melittin is localized in a motionally restricted environment in the membrane interface. We have monitored the effect of ionic strength on the organization and dynamics of membrane-bound melittin utilizing fluorescence and circular dichroism (CD) spectroscopic approaches. Our results show that red edge excitation shift (REES) of melittin bound to membranes is sensitive to the change in ionic strength of the medium. This could be attributed to a change in the immediate environment around melittin tryptophan with increasing ionic strength due to differential solvation of ions. Interestingly, the rotational mobility of melittin does not appear to be affected with change in ionic strength. In addition, fluorescence parameters such as lifetime and acrylamide quenching of melittin indicate an increase in water penetration in the membrane interface upon increasing ionic strength. Our results suggest that the solvent dynamics and water penetration in the interfacial region of the membranes are significantly affected at physiologically relevant ionic strength. These results assume significance in the overall context of the influence of ionic strength in the organization and dynamics of membrane proteins and membrane-active peptides

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

Organization of higher-order oligomers of the serotonin<SUB>1A</SUB> receptor explored utilizing homo-FRET in live cells

The serotonin1A receptor is a representative member of the GPCR superfamily and serves as an important drug target. The possible role of GPCR oligomerization in receptor function is an active area of research. We monitored the oligomerization state of serotonin1A receptors using homo-FRET and fluorescence lifetime measurements. Homo-FRET is estimated by a reduction in fluorescence anisotropy and provides a superior approach for exploring oligomerization. In addition, homo-FRET offers the possibility of detecting higher-order oligomers. On the basis of an observed increase in fluorescence anisotropy upon progressive photobleaching and analysis of the difference between the extrapolated anisotropy and the predicted anisotropy of an immobile monomer, we propose the presence of constitutive oligomers of the serotonin1A receptor. To the best of our knowledge, these results constitute the first report of higher-order oligomers for the serotonin1A receptor. We further show that cholesterol depletion and antagonist treatment result in a reduced population of higher-order oligomers. In contrast, agonist stimulation and destabilization of the actin cytoskeleton lead to an increased contribution from higher oligomers. These results provide novel insight into the oligomerization status of the serotonin1A receptor that could enhance the ability to design better therapeutic strategies to combat diseases related to malfunctioning of GPCRs

Differential Effect of Cholesterol and Its Biosynthetic Precursors on Membrane Dipole Potential

AbstractDipole 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

Quantum Query-To-Communication Simulation Needs a Logarithmic Overhead

Buhrman, Cleve and Wigderson (STOC'98) observed that for every Boolean function $f : \{-1, 1\}^n \to \{-1, 1\}$ and $\bullet : \{-1, 1\}^2 \to \{-1, 1\}$ the two-party bounded-error quantum communication complexity of $(f \circ \bullet)$ is $O(Q(f) \log n)$, where $Q(f)$ is the bounded-error quantum query complexity of $f$. Note that the bounded-error randomized communication complexity of $(f \circ \bullet)$ is bounded by $O(R(f))$, where $R(f)$ denotes the bounded-error randomized query complexity of $f$. Thus, the BCW simulation has an extra $O(\log n)$ factor appearing that is absent in classical simulation. A natural question is if this factor can be avoided. H{\o}yer and de Wolf (STACS'02) showed that for the Set-Disjointness function, this can be reduced to $c^{\log^* n}$ for some constant $c$, and subsequently Aaronson and Ambainis (FOCS'03) showed that this factor can be made a constant. That is, the quantum communication complexity of the Set-Disjointness function (which is $\mathsf{NOR}_n \circ \wedge$) is $O(Q(\mathsf{NOR}_n))$. Perhaps somewhat surprisingly, we show that when $\bullet = \oplus$, then the extra $\log n$ factor in the BCW simulation is unavoidable. In other words, we exhibit a total function $F : \{-1, 1\}^n \to \{-1, 1\}$ such that $Q^{cc}(F \circ \oplus) = \Theta(Q(F) \log n)$. To the best of our knowledge, it was not even known prior to this work whether there existed a total function $F$ and 2-bit function $\bullet$, such that $Q^{cc}(F \circ \bullet) = \omega(Q(F))$

Assessment of Lockdown Effect in Some States and Overall India: A Predictive Mathematical Study on COVID-19 Outbreak

In the absence of neither an effective treatment or vaccine and with an incomplete understanding of the epidemiological cycle, Govt. has implemented a nationwide lockdown to reduce COVID-19 transmission in India. To study the effect of social distancing measure, we considered a new mathematical model on COVID-19 that incorporates lockdown effect. By validating our model to the data on notified cases from five different states and overall India, we estimated several epidemiologically important parameters as well as the basic reproduction number ($R_{0}$). Combining the mechanistic mathematical model with different statistical forecast models, we projected notified cases in the six locations for the period May 17, 2020, till May 31, 2020. A global sensitivity analysis is carried out to determine the correlation of two epidemiologically measurable parameters on the lockdown effect and also on $R_{0}$. Our result suggests that lockdown will be effective in those locations where a higher percentage of symptomatic infection exists in the population. Furthermore, a large scale COVID-19 mass testing is required to reduce community infection. Ensemble model forecast suggested a high rise in the COVID-19 notified cases in most of the locations in the coming days. Furthermore, the trend of the effective reproduction number ($R_{t}$) during the projection period indicates if the lockdown measures are completely removed after May 17, 2020, a high spike in notified cases may be seen in those locations. Finally, combining our results, we provided an effective lockdown policy to reduce future COVID-19 transmission in India.Comment: 43 pages, 17 figure