MAVE as a high-throughput tool for the characterization of membrane proteins using the example of HpUreI from Helicobacter pylori

Description

Common methods for membrane protein characterization include in vitro assays using purified
protein, in vivo assays, and in silico assays. These methods are complex, time-consuming,
and resource-intensive because each protein mutant must be characterized individually.
Alternatively, membrane proteins can be characterized using Multiplexed Assays for Variant
Effects (MAVE). MAVE allows functional screening of every possible point mutation of a protein
in a single experiment at amino acid resolution in an in vivo system. The key steps of the MAVE
method are: 1) construction of a variant library; 2) transformation of the variant library; 3) yeast
complementation assay to enrich for functional proteins; 4) sequencing of the variant library to
quantify the representation of each variant and 5) functional scoring of each variant. Finally,
amino acid changes are mapped to their functionality in a heat map to visualize gain-of-function
and loss-of-function mutations. The MAVE method was tested and optimized with 20 variants
of UreI, an inner membrane pH-gated urea channel of Helicobacter pylori. The results
demonstrate the potential of MAVE to provide a deeper understanding of channel protein
selectivity, permeability and gating at amino acid resolution in a single experiment for larger
variant libraries, making it a universal high-throughput tool for membrane protein
characterization.

Period	03 Jul 2025
Event title	EBSA 2025 Rom
Event type	Conference
Degree of Recognition	International