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Author

Manasi Ghose

Bio: Manasi Ghose is an academic researcher from Bose Institute. The author has contributed to research in topic(s): Quenching (fluorescence) & Fluorescence. The author has an hindex of 1, co-authored 1 publication(s) receiving 11 citation(s).

Papers
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Manasi Ghose1, Surekha Mandal1, Debjani Roy1, R. K. Mandal1, Gautam Basu1 
TL;DR: A remarkable blue shift in fluorescence upon bimolecular quenching in the single‐tryptophan thermostable protein Bj2S, the 2S seed albumin from Brassica juncea, at ambient temperature and viscosity is reported.
Abstract: Although dielectric relaxation can significantly affect the intrinsic fluorescence properties of a protein, usually it is fast compared to fluorescence timescales and needs to be slowed down by adding viscogens or lowering temperature before its impact on fluorescence can be studied. We report here a remarkable blue shift in fluorescence upon bimolecular quenching in the single-tryptophan thermostable protein Bj2S, the 2S seed albumin from Brassica juncea, at ambient temperature and viscosity. The magnitude of the blue shift (∼5 nm at 50% quenching by acrylamide) is striking in a single-tryptophan protein and is attributed to a slowly relaxing dielectric environment in Bj2S from red edge excitation, steady-state polarization and time-resolved fluorescence experiments. Our results have important implications on interpretation of fluorescence of proteins with highly constrained backbones and in designing model systems for studying slow protein solvation dynamics using Trp fluorescence as the reporter probe.

11 citations


Cited by
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TL;DR: This methodology represents a novel approach for the dynamic measurement of homo-FRET in live cells that will be of utility in the biological sciences to detect oligomerization and concentration dependent interactions between identically labeled molecules.
Abstract: Steady-state fluorescence anisotropy measurements can be used to detect fluorescence resonance energy transfer (FRET) between identical fluorophores (homo-FRET). However, the contribution of homo-FRET to the steady-state anisotropy must be discerned from those due to the orientational distribution and rotational diffusion, which so far has required photobleaching controls, largely precluding dynamic measurements in live cells. We describe a variation of steady-state anisotropy microscopy in which the contribution of homo-FRET is dynamically isolated from the total anisotropy by exploiting the loss of energy transfer that occurs at red-edge excitation. Excitation of enhanced green fluorescent protein (EGFP) at the red-edge of its absorption band shows the shift in the emission spectrum compared to main-band excitation that is characteristic for photo-selection of static low energy S(0)-S(1) transitions that fail to exhibit FRET. An experimental setup for steady-state fluorescent anisotropy microscopy is described that can be used to acquire anisotropy images in live cells at main-band and red-edge excitation of EGFP. We demonstrate in live cells homo-FRET suppression of protein fusion constructs that consist of two and three EGFP molecules connected by short linkers. This methodology represents a novel approach for the dynamic measurement of homo-FRET in live cells that will be of utility in the biological sciences to detect oligomerization and concentration dependent interactions between identically labeled molecules.

82 citations

Book ChapterDOI

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01 Jan 2005
TL;DR: Red edge excitation shift (REES) as discussed by the authors is a phenomenon that occurs when the solvent shell around a polar fluorophore relaxes and shifts toward the red edge of the absorption band.
Abstract: A shift in the wavelength of maximum fluorescence emission toward higher wavelengths, caused by a corresponding 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 viscous solutions or condensed phases where the dipolar relaxation time for the solvent shell around a fluorophore is comparable to or longer than its fluorescence lifetime. REES arises from slow rates of solvent relaxation (reorientation) around an excited state fluorophore which depends on the motional restriction imposed on the solvent molecules in the immediate vicinity of the fluorophore. Utilizing this approach, it becomes possible to probe the mobility parameters of the environment itself (which is represented by the relaxing solvent molecules) using the fluorophore merely as a reporter group. Further, since the ubiquitous solvent for biological systems is water, the information obtained in such cases will come from the otherwise ‘optically silent’ water molecules. This makes REES extremely useful since hydration plays a crucial modulatory role in the formation and maintenance of organized molecular assemblies such as folded proteins in aqueous solutions and biological membranes. The application of REES as a powerful tool to monitor the organization and dynamics of a variety of soluble, cytoskeletal, and membrane-bound proteins is discussed

55 citations

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TL;DR: A novel trypsin inhibitor from Indian mustard Brassica juncea that is unique in being the precursor of a 2S seed storage protein that can be used in transforming seed crops for protection to their vegetative parts and early seed stages, when insect damage is maximal.
Abstract: A number of trypsin inhibitor (TI) genes have been used to generate insect-resistant plants. Here we report a novel trypsin inhibitor from Indian mustard Brassica juncea (BjTI) that is unique in being the precursor of a 2S seed storage protein. The inhibitory activity is lost upon processing. The predicted amino acid sequence of the precursor based on the B. juncea 2S albumin (Bj2S) gene cloned and sequenced in this laboratory (Bj2Sc; GenBank(TM) accession number ) showed a soybean-TI active site-like motif GPFRI at the expected processing site. The BjTI was found to be a thermostable Kunitz type TI that inhibits trypsin at a molar ratio of 1:1. The 20-kDa BjTI was purified from midmature seeds and found to be processed in vitro to 9- and 4-kDa subunits upon incubation with seed extract. The Bj2Sc sequence was expressed in Escherichia coli pET systems as the inhibitor precursor. The radiolabeled gene product was expressed in vitro in a coupled transcription-translation system and showed the expected processing into subunits. Two in vitro expressed pre-2S proteins, mutated at Gly and Asp residues, were processed normally to mature subunits, showing thereby no absolute requirement of Gly and Asp residues for processing. Finally, the 2S gene was introduced into tobacco and tomato plants. Third generation transgenics expressing BjTI at 0.28-0.83% of soluble leaf proteins showed remarkable resistance against the tobacco cutworm, Spodoptera litura. This novel TI can be used in transforming seed crops for protection to their vegetative parts and early seed stages, when insect damage is maximal; as the seeds mature, the TI will be naturally processed to the inactive storage protein that is safe for consumption.

52 citations

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TL;DR: There is strong experimental evidence that, in terms of absorption, the conditions in the hydrophobic interior of this protein are very close to those in vacuum, and the absorption of GFP is primarily determined by intrinsic chromophore properties.
Abstract: We have studied the gas-phase absorption properties of the green fluorescent protein (GFP) chromophore in its neutral (protonated) charge state in a heavy-ion storage ring. To accomplish this we synthesized a new molecular chromophore with a charged NH(3) group attached to a neutral model chromophore of GFP. The gas-phase absorption cross section of this chromophore molecule as a function of the wavelength is compared to the well-known absorption profile of GFP. The chromophore has a maximum absorption at 415 +/- 5 nm. When corrected for the presence of the charged group attached to the GFP model chromophore, the unperturbed neutral chromophore is predicted to have an absorption maximum at 399 nm in vacuum. This is very close to the corresponding absorption peak of the protein at 397 nm. Together with previous data obtained with an anionic GFP model chromophore, the present data show that the absorption of GFP is primarily determined by intrinsic chromophore properties. In other words, there is strong experimental evidence that, in terms of absorption, the conditions in the hydrophobic interior of this protein are very close to those in vacuum.

51 citations

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TL;DR: In silico alignment of sequences of napin has revealed that the internal repeats spanning residues 31 to 60 and 73 to 109 are conserved in all Brassica species, which may contribute to the greater stability of nap in nature.
Abstract: The napin from Brassica juncea, oriental mustard, is highly thermostable, proteolysis resistant and allergenic in nature. It consists of two subunits – one small (29 amino acid residues) and one large (86 amino acids residues) – held together by disulfide bonds. The thermal unfolding of napin has been followed by differential scanning calorimetry (DSC) and circular dichroism (CD) measurements. The thermal unfolding is characterized by a three state transition with $T_{M1}$ and $T_{M2}$ at 323.5 K and 335.8 K, respectively; $\Delta C_{P1}$ and $\Delta C_{P2}$ are $2.05 kcal mol^{-1} K^{-1}$ and $1.40 kcal mol^{−1} K^{−1}$, respectively. In the temperature range 310–318 K, the molecule undergoes dimerisation. Isothermal equilibrium unfolding by guanidinium hydrochloride also follows a three state transition, N⇆I⇆U with $\Delta G_{1H2O}$ and $\Delta G_{2H2O}$ values of $5.2 kcal mol^{−1}$ and $5.1 kcal mol^{−1}$ at 300 K, respectively. Excess heat capacity values obtained, are similar to those obtained from DSC measurements. There is an increase in hydrodynamic radius from $20 \AA$ to $35.0 \AA$ due to unfolding by guanidinium hydrochloride. In silico alignment of sequences of napin has revealed that the internal repeats (40%) spanning residues 31 to 60 and 73 to 109 are conserved in all Brassica species. The internal repeats may contribute to the greater stability of napin. A thorough understanding of the structure and stability of these proteins is essential before they can be exploited for genetic improvements for nutrition.

21 citations