Biology subjects

Nedrud, D.

Publications and source records attributed to Nedrud, D..

6 recordsLinked to original sources

bioRxiv2023

Multiparametric domain insertional profiling of Adeno-Associated Virus VP1

Evolved properties of Adeno-Associated Virus (AAV), such as broad tropism and immunogenicity in humans, are barriers to AAV-based gene therapy. Previous efforts to re-engineer these properties have focused on variable regions near AAVs 3-fold protrusions and capsid protein termini. To comprehensively survey AAV capsids for engineerable hotspots, we determined multiple AAV fitness phenotypes upon insertion of large, structured protein domains into the entire AAV-DJ capsid protein VP1. This is the largest and most comprehensive AAV domain insertion dataset to date. Our data revealed a surprising robustness of AAV capsids to accommodate large domain insertions. There was strong positional, domain-type, and fitness phenotype dependence of insertion permissibility, which clustered into correlated structural units that we could link to distinct roles in AAV assembly, stability, and infectivity. We also identified new engineerable hotspots of AAV that facilitate the covalent attachment of binding scaffolds, which may represent an alternative approach to re-direct AAV tropism.

biochemistry↗

bioRxiv2022

Deep Insertion, Deletion, and Missense Mutation Libraries for Exploring Protein Variation in Evolution, Disease, and Biology

Insertions and deletions (indels) are a major source of genetic variation in evolution and the cause of nearly 30% of Mendelian disease. Despite their importance, indels are left out of nearly every systematic mutational scan to date due to technical challenges associated with making indel-containing libraries, limiting our understanding of indels in disease, biology, and evolution. Here we present a library generation method, DIMPLE, that generates deletions, insertions, and missense at similar frequencies within any gene. To benchmark DIMPLE, we generated libraries within four genes (Kir2.1, VatD, TRPV1, and OPRM1) of varying length and evolutionary origin. DIMPLE produces libraries that are near complete, low cost, and low bias. We measured how missense mutations and indels of varying length impact the potassium channel Kir2.1 surface expression. Across all Kir2.1s secondary structure, deletions are more disruptive than insertions, beta sheets are extremely sensitive to large deletions, and flexible loops allow insertions far more frequently than deletions. DIMPLEs low bias, ease of use, and low cost will enable high throughput probing of the importance of indels in disease and evolution.

genomics↗

bioRxiv2022

Determinants of trafficking, conduction, and disease within a K+ channel revealed through multiparametric deep mutational scanning

A longstanding goal in protein science and clinical genetics is to develop quantitative models of sequence, structure, and function relationships and delineate the mechanisms by which mutations cause disease. Deep Mutational Scanning (DMS) is a promising strategy to map how amino acids contribute to protein structure and function and to advance clinical variant interpretation. Here, we introduce 7,429 single residue missense mutation into the Inward Rectifier K+ channel Kir2.1 and determine how this affects folding, assembly, and trafficking, as well as regulation by allosteric ligands and ion conduction. Our data provides high-resolution information on a cotranslationally{-}folded biogenic unit, trafficking and quality control signals, and segregated roles of different structural elements in fold-stability and function. We show that Kir2.1 trafficking mutants are underrepresented in variant effect databases, which has implications for clinical practice. By comparing fitness scores with expert-reviewed variant effects, we can predict the pathogenicity of variants of unknown significance and disease mechanisms of know pathogenic mutations. Our study in Kir2.1 provides a blueprint for how multiparametric DMS can help us understand the mechanistic basis of genetic disorders and the structure-function relationships of proteins.

biochemistry↗

bioRxiv2020

The biophysical basis of protein domain compatibility in ion channels

Understanding the biophysical mechanisms that govern the combination of protein domains into viable proteins is essential for advancing synthetic biology and biomedical engineering. Here, we use massively parallel genotype/phenotype assays to determine cell surface expression of over 300,000 variants of the inward rectifier K+ channel Kir2.1 recombined with hundreds of protein motifs. We use machine learning to derive a quantitative biophysical model and practical rules for domain recombination. Insertional fitness depends on nonlinear interactions between the biophysical properties of inserted motifs and the recipient protein, which adds a new dimension to the rational design of fusion proteins. Insertion maps reveal a generalizable hierarchical organization of Kir2.1 and several other ion channels that balances stability needed for folding and dynamics required for function. SummaryMassively parallel assays reveal interactions between donor domains and recipient proteins govern domain compatibility

biophysics↗

bioRxiv2020

A survey of pairwise epistasis supports an outside-in hierarchy of clade-specifying and function-defining residues in PSD95 PDZ3

Deep mutational scanning enables data-driven models of protein structure and function. Here, we adapted Saturated Programmable Insertion Engineering as an economical and programmable deep mutational scanning technique. We validate this approach with an existing single mutant dataset in the PSD95 PDZ3 domain, and further characterize most pairwise double mutants to study how a mutation’s phenotype depends on mutations at other sites, a phenomenon called epistasis. We observe wide-spread proximal negative epistasis, which we attribute to mutations affecting thermodynamic stability, and strong long-range positive epistasis, which is enriched in an evolutionarily conserved and function-defining network of ‘sector’ and clade-specifying residues. Conditional neutrality of mutations in clade-specifying residues compensates for deleterious mutations in sector positions. This suggests an outside-in hierarchy of interactions through which positive epistasis between clade-specifying residues and the PDZ sector facilitated the evolutionary expansion and specialization of PDZ domains.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗

bioRxiv2019

Efficient mesoscale phenotypic screening in cultured primary neuron culture

A longstanding question in neuroscience is how the activity of ion channels shapes neuronal activity and, as a result, computation in circuits and networks. Optogenetic reagents are tools to answer this question by enabling precise and dynamic perturbation of cellular states. However, development of these reagents can be hampered by low-throughput assays in non-physiological contexts. Here, we develop an all optical phenotypic screen in cultured primary hippocampal neurons that enables the functional assessment of large libraries of genetically encoded optogenetic actuators. Combining real-time analysis and data reduction methods allows for continuous observation of several thousand neurons for several days without onerous data storage overhead. This screening system may be useful in a diversity of research questions that can be coupled to optical perturbation and sensing.

neuroscience↗