Biology subjects

Manna, P.

Publications and source records attributed to Manna, P..

6 recordsLinked to original sources

bioRxiv2026

mCranberry: A rationally engineered monomeric red fluorescent protein with 85% quantum yield

Fluorescent proteins (FPs) are the workhorse of modern-day cellular imaging and biosensing. However, rationally predicting and engineering the photophysical properties of FPs remain challenging, as these are governed by complex three-dimensional microenvironments and post-translational modifications. Here, we present a novel surface-mutation-guided engineering strategy combined with iterative rounds of rational mutagenesis to improve key photophysical properties of red fluorescent proteins (RFPs). This approach generated two bright, monomeric RFP variants from the mFruits lineage, mCranberry and mCranberry2, with quantum yields of 85% and 80%, respectively. Strategic incorporation of charged amino acid residues on the protein surface enhanced fluorescence quantum yield without compromising photostability. Comprehensive photophysical characterization and live-cell microscopy demonstrate enhanced performance of these variants and establish their utility as robust probes for fluorescence imaging. However, the exact mechanism of such improvements is subject to further investigation. Collectively, this work introduces a strategy for optimizing fluorescence properties and provides a framework that may be broadly applicable to other pigment-protein systems such as genetically encoded metal-ion indicators and photosynthetic light-harvesting complexes.

biophysics↗

bioRxiv2026

Dark state-mediated photobleaching in mCherry-basedred fluorescent proteins

Developing bright and photostable red fluorescent proteins (RFPs) is one of the holy grails of the protein engineering community. Despite several attempts, finding such fluorescent proteins (FPs) has remained elusive. One bottleneck to engineering next generation RFPs is our lack of understanding of non-fluorescent or dark state properties in such constructs. Here, we develop a theoretical and experimental framework that describes how photobleaching decays in FPs relates to dark state conversion and ground state recovery. Our systematic photophysical investigation of mCherry and mCherry-d, an RFP with enhanced dark state behavior, showed the presence of photodestructive dark states in such FPs. Molecular dynamics simulations reveal enhanced fluctuation around the imidazolinone-end of the chromophore in mCherry-d, potentially facilitating conversion to non-fluorescent states. Collectively, this work quantifies dark state kinetics and gives insights into engineering dark states in RFPs to develop bright yet photostable molecular probes.

biophysics↗

bioRxiv2023

Energetic driving force for LHCII clustering in plant membranes

Plants protect themselves against photodamage from excess energy using a process known as non-photochemical quenching (NPQ). A significant fraction of NPQ is induced by a {Delta}pH across the membrane, which changes the conformation, composition, and organization of the antenna complexes. In particular, clustering of the major light-harvesting complex (LHCII) has been observed, yet the thermodynamic driving force behind this reorganization has not been determined, largely because measurements of membrane protein interaction energies have not been possible. Here, we introduce a method to quantify membrane protein interaction energies and its application to the thermodynamics of LHCII clusters. By combining single-molecule measurements of LHCII-proteoliposomes at different protein densities and a rigorous analysis of LHCII clusters and photophysics, we quantified the LHCII-LHCII interaction energy to be approximately -5 kBT at neutral pH and at least -7 kBT at acidic pH. From these values, we found the thermodynamic driving force for LHCII clustering was dominated by these enthalpic contributions. Collectively, this work captures the membrane protein-protein interactions responsible for LHCII clustering from the perspective of equilibrium statistical thermodynamics, which has a long and rich tradition in biology.

biophysics↗

bioRxiv2023

Single-molecule acceptor rise time (smART) FRET for nanoscale distance sensitivity

The structure, dynamics, and binding of individual biomolecules have been extensively investigated using single-molecule Forster resonance energy transfer (smFRET) as a spectroscopic ruler. The FRET efficiency between a fluorophore pair is used to measure distances in the several nanometer range. Existing approaches to detect closer distances come at the expense of sensitivity to longer distances. Here, we introduce single-molecule acceptor rise-time (smART) FRET that spans closer and longer distances. The acceptor rise time encodes the FRET rate, which scales polynomially with distance and thus has a steep dependence that expands the working range by 50%. High precision and accuracy is achieved through the spectroscopic separation between the rise time and the photophysical fluctuations that obfuscate other FRET readouts. Using the nanoscale sensitivity, we resolved the architectures of DNA bound to the single-stranded binding protein from E. coli, demonstrating the ability of smART FRET to elucidate the complex behaviors of biomolecules.

biophysics↗

bioRxiv2020

Structure-guided point mutations on FusionRed produce a brighter red fluorescent protein

The development of fluorescent proteins (FPs) has revolutionized biological imaging. FusionRed, a monomeric red FP (RFP), is known for its low cytotoxicity and appropriate localization of target fusion proteins in mammalian cells but is limited in application by low fluorescence brightness. We report a brighter variant of FusionRed, FusionRed-MQV, which exhibits an extended fluorescence lifetime (2.8 ns), enhanced quantum yield (0.53), higher extinction coefficient (~140,000 M-1cm-1), increased radiative rate constant and reduced non-radiative rate constant with respect to its precursor. The properties of FusionRed-MQV derive from three mutations - M42Q, C159V and the previously identified L175M. A structure-guided approach was used to identify and mutate candidate residues around the phenol and the acylimine ends of the chromophore. The C159V mutation was identified via lifetime-based flow cytometry screening of a library in which multiple residues adjacent to the phenol end of the chromophore were mutated. The M42Q mutation is located near the acylimine end of the chromophore and was discovered using site-directed mutagenesis guided by x-ray crystal structures. FusionRed-MQV exhibits 3.4-fold higher molecular brightness and a 5-fold increase in the cellular brightness in HeLa cells (based on FACS) compared to FusionRed. It also retains the low cytotoxicity and high-fidelity localization of FusionRed, as demonstrated through assays in mammalian cells.

biophysics↗

bioRxiv2020

Protein kinase Cϵ activation induces EHD-dependent degradation and downregulation of KATP channels: Implications for glucose stimulated insulin secretion

Pancreatic {beta}-cells have the unique ability to couple glucose metabolism to insulin secretion. This capacity is generally attributed to the ability of ATP to inhibit KATP channels, and the consequent {beta}-cell membrane depolarization and excitation. This notion has recently been challenged by a study which demonstrated that high glucose (HG) downregulates the cell surface KATP channels, and thereby leads to {beta}-cell depolarisation and excitation. The authors attributed the downregulation to HG-induced protein kinase C (PKC) activation and the consequent increase in channel endocytosis. This interpretation, however, is inconsistent with our previous findings that PKC activation does not affect endocytosis. To address this controversy, we revisited the problem: we have used cell biological and electrophysiological approaches combined with the pharmacological activator of PKC, PMA (phorbol 12-myristate 13-acetate). We first confirm that PKC does not play a role in KATP channel endocytosis; instead, it downregulates the channel by promoting lysosomal degradation coupled with reduced recycling. We then show that (i) mutation of the dileucine motif (355LL356) in the C-terminal domain of the Kir6.2 subunit of the KATP channel complex prevents lysosomal degradation; (ii) lysosomal targeting is mediated by the EHD (Eps15 homology domain- containing) proteins; and (iii) the PKC isoform responsible for channel degradation is PKC{varepsilon}. Taken together with the published data, we suggest that HG promotes {beta}-cell excitability via two mechanisms: ATP-dependent channel inhibition and ATP-independent, PKC{varepsilon}-dependent channel degradation. The results likely have implications for glucose induced biphasic insulin secretion.

cell biology↗