Insertions and deletions (indels) enable evolution and cause disease. Due to technical challenges, indels are left out of most mutational scans, limiting our understanding of them in disease, biology, and evolution. We develop a low cost and bias method, DIMPLE, for systematically generating deletions, insertions, and missense mutations in genes, which we test on a range of targets, including Kir2.1. We use DIMPLE to study how indels impact potassium channel structure, disease, and evolution. We find deletions are most disruptive overall, beta sheets are most sensitive to indels, and flexible loops are sensitive to deletions yet tolerate insertions.

This is a protocol for generating and QCing mutagenic libraries using the DIMPLE protocol. This version is updated to include expanded descriptions of QC and to clarify certain steps.

Several evolved properties of adeno-associated virus (AAV), such as broad tropism and immunogenicity in humans, are barriers to AAV-based gene therapy. Most efforts to re-engineer these properties have focused on variable regions near AAV's 3-fold protrusions and capsid protein termini. To comprehensively survey AAV capsids for engineerable hotspots, we determined multiple AAV fitness phenotypes upon insertion of six 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. Insertion permissibility depended strongly on insertion position, domain type, and measured fitness phenotype, which clustered into contiguous structural units that we could link to distinct roles in AAV assembly, stability, and infectivity. We also identified engineerable hotspots of AAV that facilitate the covalent attachment of binding scaffolds, which may represent an alternative approach to re-direct AAV tropism.

Synthetic biology and protein engineering have become major components in developing 21st century medicines for a host of diseases. However, due to a relative paucity of biophysical models of protein domain compatibility, rational design of synthetic multi-domain proteins for biomedical applications remains a significant challenge.

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 cotranslationallyfolded 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.

A long-standing goal in protein science and clinical genetics is to develop quantitative models of sequence, structure, and function relationships to understand how 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 7429 single-residue missense mutations 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 provide 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 surface 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 known 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.

As with many other proteins, ion channels are tremendously complex. In normal physiology for an ion channel to function it must fold, traffic to the surface, respond to stimuli, and conduct ions. In disease, mutations breaks an ion channel by altering one of these stages. However, traditionally, we only study several mutations at a time which limits our ability to mechanistically understand the molecular basis of disease or how a protein normally functions in biology. In the inwardly rectifying potassium channel, Kir2.1, mutations give rise to developmental and cardiac disorder through altering PIP2 dependent activation, folding, and functions.

Inward Rectifier K+ (KIR) channels play key roles in the operation of cells in neuromuscular and other tissue. Pathogenic variants are linked to numerous neurological, cardiovascular, and metabolic disorders. Although some variants cause gating defects in KIR by altering ligand regulation or ion permeation, there is growing evidence that many -perhaps most- variants cause defects in folding and trafficking of KIR. Despite the central role for folding and trafficking in the disease etiology, there have been to date no comprehensive large-scale studies that determine sequence and structural determinants of folding and trafficking robustness in KIR. We recently developed Saturated Programmable Insertion Engineering (SPINE) and differential Domain Insertion Profiling through Sequencing (dDIP-Seq) that allowed us determine the folding and trafficking phenotype of hundreds of thousands variants of Kir2.1, Kir3.1/Kir3.2, and Kir6.2/SUR1 in a single experiment. We will present the SPINE/dDIP-Seq methodology as a generalizable method to study ion channel folding and trafficking, which we have applied (in addition to KIR)to voltage-dependent K+ channels (Kv1.3), acid sensing channels (ASIC1a), and purinoreceptors (P2X3). Focusing on inward rectifiers, we will present our findings that most determinants of folding and trafficking are conserved in KIR and that they correlate with dynamic protein features, such as stiffness. Applying machine learning on the genotype/phenotype datasets plus sequence and structural properties of KIR allowed us to distill the fundamental protein features that determine variant effect. We will also enumerate paralog-specific folding and trafficking determinants and discuss these in the context of adaptive changes in the KIR family that enabled each paralog to respond to different allosteric ligands (e.g., ATP, G proteins, PIP2).

Protein domains are the basic units of protein structure and function. Comparative analysis of genomes and proteomes showed that domain recombination is a main driver of multidomain protein functional diversification and some of the constraining genomic mechanisms are known. Much less is known about biophysical mechanisms that determine whether protein domains can be combined into viable protein folds. Here, we use massively parallel insertional mutagenesis to determine compatibility of over 300,000 domain recombination variants of the Inward Rectifier K+ channel Kir2.1 with channel surface expression. Our data suggest that genomic and biophysical mechanisms acted in concert to favor gain of large, structured domain at protein termini during ion channel evolution. We use machine learning to build a quantitative biophysical model of domain compatibility in Kir2.1 that allows us to derive rudimentary rules for designing domain insertion variants that fold and traffic to the cell surface. Positional Kir2.1 responses to motif insertion clusters into distinct groups that correspond to contiguous structural regions of the channel with distinct biophysical properties tuned towards providing either folding stability or gating transitions. This suggests that insertional profiling is a high-throughput method to annotate function of ion channel structural regions.

Deep mutational scanning (DMS) facilitates data-driven models of protein structure and function. Here, we adapted Saturated Programmable Insertion Engineering (SPINE) as a programmable DMS technique. We validate SPINE with a reference single mutant dataset in the PSD95 PDZ3 domain and then characterize most pairwise double mutants to study 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 that epistatic interactions between these position pairs facilitated the evolutionary expansion and specialization of PDZ domains. We propose that SPINE provides easy experimental access to reveal epistasis signatures in proteins that will improve our understanding of the structural basis for protein function and adaptation.

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. Summary Massively parallel assays reveal interactions between donor domains and recipient proteins govern domain compatibility

Inward rectifier K+ (IRK) channels arose early in cellular life as the combination of a pore and an immunoglobulin (Ig)-like domain. This architecture is conserved throughout the IRK family and enables regulation by different allosteric ligands (lipids, ATP, G proteins). Functional diversity in light of architectural conservation suggests that the required signal transduction functions for different ligands already existed within ancestral IRK channels. The term ‘latent allosteric capacity’ describes how new functions can emerge from conformational flexibility that arise from the combination of specific domains. We determined conformational flexibility across the IRK family using differential Domain Insertion Profiling through Sequencing (dDIPSEQ). The key idea is that ‘permissibility’ to domain insertion is large in conformationally flexible regions (characterized by large values of intrinsic configurational entropy) that are poised to undergo structural changes. Their permissibility is dictated by the specific nature of these structural changes and thus depends crucially on the molecular properties of the inserted domains. Conservation of permissibility profiles closely tracks IRK phylogeny, suggesting that IRK diversification exploited allosteric capacity inherent to this architecture. Using molecular dynamics simulation and machine learning, we show that differential permissibility suggests distinct roles of Kir2.1's tether and interfacial helices in mechanical coupling/uncoupling of distant sites upon PIP2 binding. This data and the established link between protein dynamics and allostery leads us to propose that differential permissibility is a metric of allosteric capacity in IRK. In support of this notion, inserting light-switchable domains at appropriate sites renders Kir2.1 activity sensitive to light, thereby providing a proof of concept for de novo design of ion channels for opto- or chemogenetic applications.

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.

Domain recombination is a key principle in protein evolution and protein engineering, but inserting a donor domain into every position of a target protein is not easily experimentally accessible. Most contemporary domain insertion profiling approaches rely on DNA transposons, which are constrained by sequence bias. Here, we establish Saturated Programmable Insertion Engineering (SPINE), an unbiased, comprehensive, and targeted domain insertion library generation technique using oligo library synthesis and multi-step Golden Gate cloning. Through benchmarking to MuA transposon-mediated library generation on four ion channel genes, we demonstrate that SPINE-generated libraries are enriched for in-frame insertions, have drastically reduced sequence bias as well as near-complete and highly-redundant coverage. Unlike transposon-mediated domain insertion that was severely biased and sparse for some genes, SPINE generated high-quality libraries for all genes tested. Using the Inward Rectifier K+ channel Kir2.1, we validate the practical utility of SPINE by constructing and comparing domain insertion permissibility maps. SPINE is the first technology to enable saturated domain insertion profiling. SPINE could help explore the relationship between domain insertions and protein function, and how this relationship is shaped by evolutionary forces and can be engineered for biomedical applications.

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.

Ion channels are among the most important proteins in neuroscience and serve as drug targets for many brain disorders. During development, learning, disease progression, and other processes, the activity levels of specific ion channels are tuned in a cell-type specific manner. However, it is difficult to assess how cell-specific changes in ion channel activity alter emergent brain functions. We have developed a protein architecture for fully genetically encoded light-activated modulation of endogenous ion channel activity. Fusing a genetically encoded photoswitch and an ion channel-modulating peptide toxin in a computationally designed fashion, this reagent, which we call Lumitoxins, can mediate light-modulation of specific endogenous ion channel activities in targeted cells. The modular lumitoxin architecture may be useful in a diversity of neuroscience tools. Here, we delineate how to construct lumitoxin genes from synthesized components, and provide a general outline for how to test their function in mammalian cell culture.

The natural substrate of hydroxynitrile lyase from rubber tree (HbHNL, Hevea brasiliensis) is acetone cyanohydrin, but synthetic applications usually involve aromatic cyanohydrins such as mandelonitrile. To increase the activity of HbHNL toward this unnatural substrate, we replaced active site residues in HbHNL with the corresponding ones from esterase SABP2 (salicylic acid binding protein 2). Although this enzyme catalyzes a different reaction (hydrolysis of esters), its natural substrate (methyl salicylate) contains an aromatic ring. Three of the eleven single-amino-acid-substitution variants of HbHNL reacted more rapidly with mandelonitrile. The best was HbHNL-L121Y, with a kcat 4.2 times higher and high enantioselectivity. Site-saturation mutagenesis at position 121 identified three other improved variants. We hypothesize that the smaller active site orients the aromatic substrate more productively.

Chemie Ingenieur TechnikVolume 86, Issue 9 p. 1419-1419 PosterFree Access Erhöhung der Reaktionsgeschwindigkeit der Hydroxynitrillyase aus Hevea brasiliensis bezüglich Mandelsäurenitril Dr. J. von Langermann, Corresponding Author Dr. J. von Langermann jan.langermann@uni-rostock.de University of Minnesota, Biotechnology Institute, 1479 Gortner Avenue, 55108 Saint Paul, MN, USA Universität Rostock, Institut für Chemie, Albert-Einstein-Straße 3A, D-18059 Rostock, GermanyUniversity of Minnesota, Biotechnology Institute, 1479 Gortner Avenue, 55108 Saint Paul, MN, USA===Search for more papers by this authorD. M. Nedrud, D. M. Nedrud University of Minnesota, Biotechnology Institute, 1479 Gortner Avenue, 55108 Saint Paul, MN, USASearch for more papers by this authorProf. Dr. R. Kazlauskas, Corresponding Author Prof. Dr. R. Kazlauskas University of Minnesota, Biotechnology Institute, 1479 Gortner Avenue, 55108 Saint Paul, MN, USAUniversity of Minnesota, Biotechnology Institute, 1479 Gortner Avenue, 55108 Saint Paul, MN, USA===Search for more papers by this author Dr. J. von Langermann, Corresponding Author Dr. J. von Langermann jan.langermann@uni-rostock.de University of Minnesota, Biotechnology Institute, 1479 Gortner Avenue, 55108 Saint Paul, MN, USA Universität Rostock, Institut für Chemie, Albert-Einstein-Straße 3A, D-18059 Rostock, GermanyUniversity of Minnesota, Biotechnology Institute, 1479 Gortner Avenue, 55108 Saint Paul, MN, USA===Search for more papers by this authorD. M. Nedrud, D. M. Nedrud University of Minnesota, Biotechnology Institute, 1479 Gortner Avenue, 55108 Saint Paul, MN, USASearch for more papers by this authorProf. Dr. R. Kazlauskas, Corresponding Author Prof. Dr. R. Kazlauskas University of Minnesota, Biotechnology Institute, 1479 Gortner Avenue, 55108 Saint Paul, MN, USAUniversity of Minnesota, Biotechnology Institute, 1479 Gortner Avenue, 55108 Saint Paul, MN, USA===Search for more papers by this author First published: 28 August 2014 https://doi.org/10.1002/cite.201450085AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume86, Issue9Special Issue: ProcessNet-Jahrestagung 2014 und 31. DECHEMA-Jahrestagung der BiotechnologenSeptember, 2014Pages 1419-1419 ReferencesRelatedInformation

Although Glu79 does not contribute to esterase catalysis, it can block esterase catalysis by hydrogen bonding to the active site histidine.