Biology subjects

Moya Munoz, G. G.

Publications and source records attributed to Moya Munoz, G. G..

3 recordsLinked to original sources

bioRxiv2024

Determination of Absolute Intramolecular Distances in Proteins by Anomalous X-ray Scattering Interferometry

Biomolecular structures are typically determined using frozen or crystalline samples. Measurement of intramolecular distances in solution can provide additional insights into conformational heterogeneity and dynamics of biological macromolecules and their complexes. The established molecular ruler techniques used for this (NMR, FRET, and EPR) are, however, limited in their dynamic range and require model assumptions to determine absolute distance (distributions). Here, we introduce anomalous X-ray scattering interferometry (AXSI) for intramolecular distance measurements in proteins, which are labeled at two sites with small gold nanoparticles of 0.7 nm radius. We apply AXSI to two different cysteine-variants of maltose binding protein in the presence and absence of its ligand maltose and find distances in quantitative agreement with single-molecule FRET experiments. Our study shows that AXSI enables determination of absolute intramolecular distance distributions under virtually arbitrary solution conditions and we anticipate its broad use to characterize protein conformational ensembles and dynamics.

biophysics↗

bioRxiv2023

Single-molecule detection and super-resolution imaging with a portable and adaptable 3D-printed microscopy platform (Brick-MIC)

Over the past decades, single-molecule and super-resolution microscopy have advanced and represent essential tools for life science research. There is,however, a growing gap between the state-of-the-art and what is accessible to biologists, biochemists, medical researchers or labs with financial constraints. To bridge this gap, we introduce Brick-MIC, a versatile and affordable open-source 3D-printed micro-spectroscopy and imaging platform. Brick-MIC enables the integration of various fluorescence imaging techniques with single-molecule resolution within a single platform and exchange between different modalities within minutes. We here present variants of Brick-MIC that facilitate single-molecule fluorescence detection, fluorescence correlation spectroscopy and super-resolution imaging (STORM and PAINT). Detailed descriptions of the hardware and software components, as well as data analysis routines are provided, to allow non-optics specialist to operate their own Brick-MIC with minimal effort and investments. We foresee that our affordable, flexible, and open-source Brick-MIC platform will be a valuable tool for many laboratories worldwide.

biophysics↗

bioRxiv2023

Labelizer: systematic selection of protein residues for covalent fluorophore labeling

An essential requirement for the use of fluorescent dyes in biomedicine, molecular biology, biochemistry, biophysics and optical imaging is their (covalent) attachment to biomolecules. There is, however, no systematic and automated approach for the selection of suitable labeling sites in macromolecules, which is particular problematic for proteins. Here, we present a general and quantitative strategy to identify optimal residues for protein labeling using a naive Bayes classifier. Based on a literature search and bioinformatics analysis of >100 proteins with ~400 successfully labeled residues, we identified four parameters, which we combined into a labeling score to rank residues for their suitability as a label-site. The utility of our approach for the systematic selection of single residues and of residue pairs for FRET experiments is supported by data from the literature and by new experiments on different proteins. To make the method available to a large community of researchers, we developed a python package called "labelizer", that performs an analysis of a pdb-structure (or structural models), label score calculation, and FRET assay scoring. We further provide a webserver (https://labelizer.bio.lmu.de/) to conveniently apply our approach and to build up a central open-access database of (non-)successfully labeled protein residues to continuously improve and refine the labelizer approach.

biophysics↗