Biology subjects

Rehan, A.

Publications and source records attributed to Rehan, A..

4 recordsLinked to original sources

bioRxiv2025

Design and experimental characterization of specificity-switching mutational paths of WW domains

Specific interactions between proteins and other biomolecules are ubiquitous in cellular processes.How specificity is encoded in the protein sequence and can be modified through a minimal set of concerted mutations is a complex issue. In this work, we focus on the WW protein domain, whose variants specifically bind to different classes of proline-rich peptides. Combining unsupervised learning of homologous WW sequence data with Restricted Boltzmann Machines (RBM) and path-sampling methods, we design mutational paths of putative WW domains interpolating between two natural WW domains with either distinct or similar specificities. Sequences along the designed paths are then experimentally validated with high-throughput in-vitro binding assays against 3 peptides of different classes. The vast majority (93%) of intermediate sequences along the designed paths are responsive to the initial or/and final peptides. On the contrary, domains along scrambled paths, in which the same mutations are introduced in random order are not functional, emphasizing how successful design crucially depends on the ability to model epistatic interactions. Switch in specificity between classes I and IV, whose representative peptides bind to different pockets on the WW domain takes place through intermediates displaying some level of binding cross-reactivity with the tested peptides, contrary to the transition from class I to II, which are associated with the same binding pocket. Lastly, we show that the RBM paths share a high identity with internal nodes obtained from ancestral sequence reconstruction based on the seed WW domains.

bioengineering↗

bioRxiv2025

Parallelization of single-molecule binding kinetic measurements via protein barcode sequencing

Screening protein variants for desired functions has long relied on coupling of genotype (gene sequence) to phenotype (protein function), limiting the use of powerful single-molecule (SM) techniques. Here, we introduce a scalable SM screening method that bypasses this constraint by linking SM functional analysis to protein identity through SM protein sequencing. Protein variants are tagged with unique C-terminal peptide barcodes and loaded onto a semiconductor chip containing millions of nanowells. Protein-ligand interactions are monitored in real time at the SM level, and a dye-cycling strategy extends the measurable dynamic range, enabling quantification of slow dissociation rates typical of high-affinity interactions. After functional analysis, each protein molecule is identified by sequencing its barcode. We apply this method to 20 barcoded nanobodies spanning over 1,000-fold in affinity, yielding results consistent with published values and individual SM measurements. Our approach should accelerate protein engineering by enabling rapid, multiplexed SM screening of protein libraries.

biochemistry↗

bioRxiv2024

Gradients of Recognition Molecules Shape Synaptic Specificity of a Visuomotor Transformation

Converting sensory information into motor commands is fundamental to most of our actions. In Drosophila, visuomotor transformations are mediated by Visual Projection Neurons (VPNs). These neurons convert object location and motion into directional behaviors downstream through a synaptic gradient mechanism. However, the molecular origins of such graded connectivity remain unknown. We addressed this question in a VPN cell type called LPLC2, which integrates looming motion and transforms it into an escape response through two parallel dorsoventral synaptic gradients at its inputs and outputs. We identified two corresponding dorsoventral expression gradients of cell recognition molecules within the LPLC2 population that regulate this synaptic connectivity. Dpr13 determines synaptic outputs of LPLC2 axons by interacting with its binding partner DIP-{varepsilon} expressed in the Giant Fiber, a neuron that mediates escape. Similarly, beat-VI regulates synaptic inputs onto LPLC2 dendrites by interacting with Side-II expressed in upstream motion-detecting neurons. Behavioral, physiological, and molecular experiments demonstrate that these coordinated molecular gradients control differential synaptic connectivity, enabling the accurate transformation of visual features into motor commands. As within-neuronal-type variation in gene expression is also observed in the mammalian brain, graded expression of cell recognition molecules may represent a common mechanism underlying synaptic specificity.

neuroscience↗

bioRxiv2024

Neuronal identity control at the resolution of a single transcription factor isoform

The brain exhibits remarkable neuronal diversity which is critical for its functional integrity. From the sheer number of cell types emerging from extensive transcriptional, morphological, and connectome datasets, the question arises of how the brain is capable of generating so many unique identities. Terminal selectors are transcription factors hypothesized to determine the final identity characteristics in post-mitotic cells. Which transcription factors function as terminal selectors and the level of control they exert over different terminal characteristics are not well defined. Here, we establish a novel role for the transcription factor broad as a terminal selector in Drosophila melanogaster. We capitalize on existing large sequencing and connectomics datasets and employ a comprehensive characterization of terminal characteristics including Perturb-seq and whole-cell electrophysiology. We find a single isoform broad-z4 serves as the switch between the identity of two visual projection neurons LPLC1 and LPLC2. Broad-z4 is natively expressed in LPLC1, and is capable of transforming the transcriptome, morphology, and functional connectivity of LPLC2 cells into LPLC1 cells when perturbed. Our comprehensive work establishes a single isoform as the smallest unit underlying an identity switch, which may serve as a conserved strategy replicated across developmental programs.

neuroscience↗