Biology subjects

Gall, B.

Publications and source records attributed to Gall, B..

3 recordsLinked to original sources

bioRxiv2025

Examining Selection Dynamics and Limitations in Multi-round Protein Selection of High Diversity Libraries

Proteins and peptides underpin essential biological functions and technological applications, from targeting disease-relevant interactions to providing broad enzymatic activities. However, engineering molecules with desired properties remains difficult, owing to complex sequence-structure-function relationships and the lack of data on specific systems. Experimental selection strategies, including directed evolution, phage display, and mRNA display, address this challenge by leveraging high diversity libraries and iterative enrichment under defined selection pressures. This allows for the identification of candidates without requiring extensive prior knowledge, and can generate extensive datasets for use in machine learning. While many selection systems exist, comparisons across different selection approaches are hindered by the lack of a unifying analytical framework. Here, we present a set of broadly applicable analyses for assessing selection dynamics in multi-round or multi-condition experiments, ranging from position level analysis of sequence properties to full sequence space mappings through protein language model embeddings. Using the toolset to analyze a variety of different datasets in parallel, we explore the potential effects of diversity, coverage, and reproducibility, offering generalizable insights to guide experimental design, interpretation, and troubleshooting across protein and peptide discovery platforms.

molecular biology↗

bioRxiv2024

Exploring large protein sequence space through homology- and representation-based hierarchical clustering

Exploration of protein sequence space can offer insight into protein sequence-function relationships, benefitting both basic science and industrial applications. The use of sequence similarity networks (SSNs) is a standard method for exploring large sequence datasets, but is currently limited when scaling to very large datasets and when viewing more than one level (hierarchy) of homology. Here, we present a sequence analysis pipeline with a number of innovations that address some limitations of traditional SSNs. First, we develop a hierarchical visualization approach that captures the full range of homologies across protein superfamilies. Second, we leverage representations embedded by protein language models as an alternative homology metric to the basic local alignment search tool (BLAST), showing that they produce comparable results when identifying isofunctional protein families. Finally, we demonstrate that unbiased representative sampling of sequences from genetic neighborhoods can be achieved through the use of hidden Markov models (HMMs) or vector representations. The utility of these methods is exemplified by updating the sequence-function analysis of the FMN/F420-binding split barrel superfamily and improving phylogenetic analyses. We provide our sequence exploration pipeline as publicly available code (ProteinClusterTools) and show it to be scalable to large datasets ([~]300k sequences) using desktop computers.

bioinformatics↗

bioRxiv2024

Structure, dynamics and evolution of the Candida albicans multi-drug resistance ABC transporter CDR1

The pleiotropic drug resistance transporter Cdr1 from Candida albicans plays a crucial role in antifungal resistance. Here, we present high-resolution cryo-electron microscopy structures of C. albicans Cdr1 in multiple functional states: nucleotide-bound, substrate-bound, and apo forms. The 3.5 [A] resolution structure of Cdr1 in complex with ATP and ADP reveals the molecular details of its asymmetric nucleotide-binding sites (NBS), with ATP bound to the deviant NBS1 and ADP to the canonical NBS2. Structures of Cdr1 bound to rhodamine 6G (3.4 [A]) and Oregon Green 488 (3.5 [A]) in complex with these nucleotides elucidate the pleiotropic substrate-binding pocket and highlight how nucleotide exchange drives conformational change required for transport. Additionally, we determined a 3.7 [A] resolution structure of Cdr1 in the detergent LMNG without nucleotides, as well as a 3.5 [A] resolution structure with nucleotides but no substrate, representing an apo state. We complemented these structural insights with molecular dynamics simulations to understand substrate binding dynamics, ancestral sequence reconstruction to trace the evolution of key functional motifs, and analysis of clinical isolates from sequence databases to identify potential resistance-associated variations. Comparison of these structures provides new insights into the conformational changes associated with the transport cycle of this asymmetric ABC transporter, revealing how ATP binding at the deviant NBS1 allosterically regulates the canonical NBS2, driving ATPase activity. This work significantly advances our understanding of the molecular mechanisms underlying multidrug resistance in pathogenic fungi and provides a structural and evolutionary framework for the rational design of Cdr1 inhibitors to combat antifungal resistance.

microbiology↗