Biology subjects

Mohona, F. A.

Publications and source records attributed to Mohona, F. A..

3 recordsLinked to original sources

bioRxiv2026

Design of Fluorescent Membrane Scaffold Proteins for Nanodiscs

Nanodiscs are nanoscale lipid bilayer membrane mimetics surrounded by two membrane scaffold proteins (MSP). They are widely used as soluble cassettes for membrane proteins and lipids in diverse applications in structural, functional, and biophysical studies. The original MSP was derived directly from human apolipoprotein A-1, and novel constructs have been adapted from this original design, including fluorescent nanodiscs of varying designs. However, chemical derivatization with fluorophores can be expensive, and prior designs of split fluorescent proteins fused to MSP were limited in the color pallet available. Here, we developed MSPs with a wide range of different fluorescent C-terminal protein tags, including a versatile HaloTag fusion. These fluorescent MSP were purified following typical MSP purification procedures with similar yield. Then, we demonstrate that fluorescent MSPs form nanodiscs with similar structure and stoichiometry to conventional MSP nanodiscs. They are also suitable for assembly of nanodiscs with embedded integral membrane protein. Finally, we apply these constructs to monitor peripheral membrane protein binding to lipids via fluorescence resonance energy transfer. These fluorescent MSP constructs enable a range of different applications and provide a versatile template for future design of nanodiscs with unique functions. For Table of Contents Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/716332v3_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1bf2d2borg.highwire.dtl.DTLVardef@2ea07eorg.highwire.dtl.DTLVardef@12aeccforg.highwire.dtl.DTLVardef@121430d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

Alanine Scanning to Define Membrane Protein-Lipid Interaction Sites Using Native Mass Spectrometry

Lipids surrounding membrane proteins interact with different sites on the protein at varying specificities, ranging from highly specific to weak interactions. These interactions can modulate the structure, function, and stability of membrane proteins. Thus, to better understand membrane protein structure and function, it is important to identify the locations of lipid binding and the relative specificities of lipid binding at these sites. In our previous native mass spectrometry (MS) study, we developed a single and double mutant analysis approach to profile the contribution of specific residues toward lipid binding. Here, we extend this method by screening a broad range of mutants of AqpZ to identify specific lipid binding sites and by measuring binding of different lipid types to measure the selectivity of different lipids at selected binding sites. We complemented these native MS studies with molecular dynamics (MD) simulations to visualize lipid interactions at selected sites. We discovered that AqpZ is selective towards cardiolipins (CL) but only at specific sites. Specifically, CL orients with its headgroup facing the cytoplasmic side, and its acyl chains interact with a hydrophobic pocket located at the monomeric interface within the lipid bilayer. Overall, this integrative approach provides unique insights into lipid binding sites and the selectivity of various lipids towards AqpZ, enabling us to map the AqpZ protein structure based on the lipid affinity.

biophysics↗

bioRxiv2023

Simultaneous Native Mass Spectrometry Analysis of Single and Double Mutants to Probe Lipid Binding to Membrane Proteins

Lipids are critical modulators of membrane protein structure and function. However, it is challenging to investigate the thermodynamics of protein-lipid interactions because lipids can simultaneously bind membrane proteins at different sites with different specificities. Here, we developed a native mass spectrometry (MS) approach using single and double mutants to measure the relative energetic contributions of specific residues on Aquaporin Z (AqpZ) toward cardiolipin (CL) binding. We first mutated potential lipid-binding residues on AqpZ, and mixed mutant and wild-type proteins together with CL. By using native MS to simultaneously resolve lipid binding to the mutant and wild-type proteins in a single spectrum, we directly determined the relative affinities of CL binding, thereby revealing the relative Gibbs free energy change for lipid binding caused by the mutation. Comparing different mutants revealed that the W14 contributes to the tightest CL binding site, with R224 contributing to a lower affinity site. Using double mutant cycling, we investigated the synergy between W14 and R224 sites on CL binding. Overall, this novel native MS approach provides unique insights into lipid binding to specific sites on membrane proteins.

biophysics↗