Spectroscopic Evaluation of Excited State Depopulation in Red Fluorescent Proteins Developed Using Fluorescence Lifetime Selections

Fluorescent Proteins are intriguing molecules that are ubiquitous tools for biological research. This thesis investigates the relationship between engineering improved fluorescent proteins and fundamental physical chemistry principles that drive the molecular design of brighter and more photostable variants of existing fluorescent proteins. This work primarily focuses on designing brighter variants of the red-fluorescent proteins, FusionRed and mCherry, and the consequent physical investigations that lead to increments in brightness. To achieve this, droplet-based and single stream microfluidic sorters that discriminate members of a cell-based mutagenesis library of variants on their excited state lifetime and fluorescence intensity were developed and utilized. These lifetime-based selections led to variants with greater than 3-fold higher values of fluorescence quantum yield in comparison to their progenitors. Finally, the mechanisms and pathways that lead to excited state depopulation after photoexcitation, which govern observables like fluorescence quantum yield, fluorescence brightness, reversible and permanent photobleaching, and blinking at the single molecule level were investigated. This work highlights the progress and the many knowledge gaps in understanding the interwoven relationships of observed photophysics in fluorescent proteins, and suggests ways in which one might further examine the physical basis of brightness and photostability in these molecules

Giant Dipole Resonance Width in near-Sn Nuclei at Low Temperature and High Angular Momentum

High energy gamma-rays in coincidence with low energy yrast gamma-rays have been measured from 113Sb, at excitation energies of 109 and 122 MeV, formed by bombarding 20Ne on 93Nb at projectile energies of 145 and 160 MeV respectively to study the role of angular momentum (J) and temperature (T) over Giant Dipole Resonance (GDR) width. The maximum populated angular momenta for fusion were 67hbar and 73hbar respectively for the above-mentioned beam energies. The high energy photons were detected using a Large Area Modular BaF2 Detector Array (LAMBDA) along with a 24-element multiplicity filter. After pre-equilibrium corrections, the excitation energy E* was averaged over the decay steps of the compound nucleus (CN). The average values of temperature, angular momentum, CN mass etc. have been calculated by the statistical model code CASCADE. Using those average values, results show the systematic increase of GDR width with T which is consistent with Kusnezov parametrization and the Thermal Shape Fluctuation Model. The rise of GDR width with temperature also supports the assumptions of adiabatic coupling in the Thermal Shape Fluctuation Model. But the GDR widths and corresponding reduced plots with J are not consistent with the theoretical model at high spins.Comment: 19 pages, 10 figures, Submitted to Physics Review

Onset of deformation at N=112N = 112 in Bi nuclei

The high spin states in $^{195}$Bi has been studied by $\gamma$-ray spectroscopic method using the $^{181}$Ta($^{20}$Ne, 6n) fusion evaporation reaction at 130 MeV. The $\gamma\gamma$ coincidence data were taken using an array of 8 clover HPGe detectors. The spin and parity assignments of the excited states have been made from the measured directional correlation from oriented states (DCO) ratios and integrated polarization asymmetry (IPDCO) ratios. The results show, for the first time, the evidence of a rotational like band based on a 13/2$^+$ band head in this nucleus, indicating the onset of deformation at neutron number $N = 112$ for the Bismuth isotopes. The results obtained were found to be consistent with the prediction of the total Routhian surface calculations using Woods Saxon potential. The same calculations also predict a change in shape from oblate to triaxial in $^{195}$Bi at high rotational frequency

Technology Transfer and the Intellectual Property Issues Emerging from It – An Analysis from a Developing Country Perspective

260-274The exploitation of technological knowledge is central to the development process. Less developed economies typically obtain this knowledge from more advanced ones rather than by creating it themselves. Transfer of technology is the transfer of a patent with respect to a particular product, which is patented. The product of technology is mixed up with the technology itself. No effort is made to make a distinction between the two. This paper attempts to clarify the meaning of technology transfer and what it actually transfers and elaborates on the modalities of technology transfer agreement and the various types of the same. It also deals with the situation where there is a loss of confidentiality and elaborates on its effect on the liabilities of the contracting parties in various jurisdictions. The article also attempts to deal with the various intellectual property issues involved in technology transfer and attempts to analyse things from a developing country perspective

Influence of fluorescence lifetime selections and conformational flexibility on brightness of FusionRed variants

Fluorescent Proteins (FPs) for bioimaging are typically developed by screening mutant libraries for clones with improved photophysical properties. This approach has resulted in FPs with high brightness, but the mechanistic origins of the improvements are often unclear. We focused on improving the molecular brightness in the FusionRed family of FPs with fluorescence lifetime selections on targeted libraries, with the aim of reducing non-radiative decay rates. Our new variants show fluorescence quantum yields up to 75% and lifetimes >3.5 ns. We present a comprehensive analysis of these new FPs, including trends in spectral shifts, photophysical data, photostability, and cellular brightness resulting from codon optimization. We also performed all-atom molecular dynamics simulations to investigate the impact of sidechain mutations. The trajectories reveal that individual mutations reduce the flexibility of the chromophore and sidechains, leading to an overall reduction in non-radiative rates

Constraining scalar-Gauss-Bonnet inflation by reheating, unitarity, and Planck data

We revisit the inflationary dynamics in detail for theories with Gauss-Bonnet gravity coupled to scalar functions, in light of the Planck data. Considering the chaotic inflationary scenario, we constrain the parameters of two models involving inflaton-Gauss-Bonnet coupling by current Planck data. For nonzero inflaton-Gauss-Bonnet coupling β , an inflationary analysis provides us a big cosmologically viable region in the space of ( m , β ), where m is the mass of the inflaton. However, we study further on constraining β arising from reheating considerations and unitarity of tree-level amplitude involving 2-graviton → 2 -graviton ( h h → h h ) scattering. Our analysis, particularly on reheating significantly reduces the parameter space of ( m , β ) for all models. The quadratic Gauss-Bonnet coupling parameter turns out to be more strongly constrained than that of the linear coupling. For the linear Gauss-Bonnet coupling function, we obtain β ≲ 1 0 3 , with the condition β ( m / M P ) 2 ≃ 10 − 4 . However, the study of the Higgs inflation scenario in the presence of a Gauss-Bonnet term turns out to be completely disfavored.by Srijit Bhattacharjee and Sudipta Sarka

Conformational dynamics of mCherry variants: a link between sidechain motions and fluorescence brightness

We recently developed the red fluorescent protein (RFP) mCherry-XL, which is threefold brighter than its predecessor, mCherry, with a directed evolution approach using fluorescence-lifetime flow cytometry selections. The enhanced brightness is due to a significant decrease in the non-radiative decay rate underlying its increase in fluorescence quantum yield. To examine the dynamic role of the four mutations that distinguish the two RFPs and closely-related variants, we employed microsecond-timescale, all-atom molecular dynamics simulations to sample their ground state conformational landscapes. The simulations revealed the significance of the I197R mutation, which leads to the formation of multiple hydrogen-bonded contacts. The triad of interactions observed between residues K70, E148 and 197R is also seen in mScarlet, another RFP of unrelated origin, but of comparably high brightness. These substitutions in mCherry-XL increased the rigidity of the β-barrel, for example as shown by increased hydrogen-bonding in the chromophore region and decreased root-mean-square backbone deviations. Furthermore, mCherry-XL showed significantly less ns-timescale breathing of the gap between β-7 and β-10 strands. This gap was previously shown to be the most flexible region of mCherry, permitting entry of O2 into the barrel. We also characterize the role played by the sidechain of residue 161 using a combination of MD simulations and in-vitro experimental characterization. We find this position is critical to the steric interactions that perturb the chromophore electronic structure. MD simulations also help us to recognize a network of hydrogen-bonded interactions between the chromophore, the residue 143, 163 and 59, which can potentially impact the electron distribution of the chromophore. Finally, we shed light on the conformational dynamics of the conserved residues R95 and S146, which are hydrogen bonded to the chromophore, and provide physical insights into the observed photophysics. To the best of our knowledge, this is the first study that evaluates the conformational space for a set of closely related FPs generated by directed evolution

Characterizing Dark State Kinetics and Single Molecule Fluorescence of FusionRed and FusionRed-MQ at Low Irradiances

The presence of dark states causes fluorescence intermittency of single molecules due to transitions between “on” and “off” states. Genetically encodable markers such as fluorescent proteins (FPs) exhibit dark states that make several super-resolved single-molecule localization microscopy (SMLM) methods possible. However, studies quantifying the timescales and nature of dark state behavior for commonly used FPs under conditions typical of widefield and total internal reflection fluorescence (TIRF) microscopy remain scarce and pre-date many new SMLM techniques. FusionRed is a relatively bright red FP exhibiting fluorescence intermittency and has thus been identified as a potential candidate for SMLM. We herein characterize the rates for dark-state conversion and the subsequent ground-state recovery of FusionRed and its 2.5-fold brighter descendent FusionRed L175M M42Q (FusionRed-MQ) at low irradiances (1-10 W/cm2), which were previously unexplored experimental conditions. We characterized the kinetics of dark state transitions in these two FPs by using single molecule blinking and ensemble photobleaching experiments bridged with a dark state kinetic model. We find that at low irradiances, the recovery process to the ground state is minimally light-driven and FusionRed-MQ has a 1.3-fold higher ground state recovery time indicating a conformationally restricted dark-state chromophore in comparison to FusionRed. Our studies indicate that the brighter FusionRed-MQ exhibits higher tendency in dark state conversion, thus it is potentially a better candidate for SMLM applications than its progenitor FusionRed

Directed evolution of a bright variant of mCherry: Suppression of non-radiative decay by fluorescence lifetime selections

The approximately linear scaling of fluorescence quantum yield (QY) with fluorescence lifetime (τ) in fluorescent proteins (FPs) has inspired engineering of brighter fluorophores based on screening for increased lifetimes. Several recently developed FPs such as mTurquoise2, mScarlet and FusionRed-MQV which have become useful for live cell imaging are products of lifetime selection strategies. However, the underlying photophysical basis of the improved brightness has not been scrutinized. In this study, we focused on understanding the outcome of lifetime-based directed evolution of mCherry, which is a popular red-FP (RFP). We identified four positions (W143, I161, Q163, and I197) near the FP chromophore that can be mutated to create mCherry-XL (eXtended Lifetime: QY = 0.70; τ =3.9 ns). The threefold higher quantum yield of mCherry-XL is on par with that of the brightest RFP to date, mScarlet. We examined selected variants within the evolution trajectory and found a near-linear scaling of lifetime with quantum yield and consistent blue-shifts of the absorption and emission spectra. We find that the improvement in brightness is primarily due to a decrease in the non-radiative decay of the excited state. In addition, our analysis revealed the decrease in non-radiative rate is not limited to the blue-shift of the energy gap and changes in the excited state reorganization energy. Our findings suggest that non-radiative mechanisms beyond the scope of energy-gap models such the Englman-Jortner are suppressed in this lifetime evolution trajectory