Expanding the Genetic Code of Bioelectrocatalysis and Biomaterials.

Genetic code expansion is a promising genetic engineering technology that incorporates noncanonical amino acids into proteins alongside the natural set of 20 amino acids. This enables the precise encoding of non-natural chemical groups in proteins. This review focuses on the applications of genetic code expansion in bioelectrocatalysis and biomaterials. In bioelectrocatalysis, this technique enhances the efficiency and selectivity of bioelectrocatalysts for use in sensors, biofuel cells, and enzymatic electrodes. In biomaterials, incorporating non-natural chemical groups into protein-based polymers facilitates the modification, fine-tuning, or the engineering of new biomaterial properties. The review provides an overview of relevant technologies, discusses applications, and highlights achievements, challenges, and prospects in these fields.

Suppressive cancer nonstop extension mutations increase C-terminal hydrophobicity and disrupt evolutionarily conserved amino acid patterns.

Nonstop extension mutations, a.k.a. stop-lost or stop-loss mutations, convert a stop codon into a sense codon resulting in translation into the 3' untranslated region until the next in-frame stop codon, thereby extending the C-terminus of a protein. In cancer, only nonstop mutations in SMAD4 have been functionally characterized, while the impact of other nonstop mutations remain unknown. Here, we exploit our pan-cancer NonStopDB dataset and test all 2335 C-terminal extensions arising from somatic nonstop mutations in cancer for their impact on protein expression. In a high-throughput screen, 56.1% of the extensions effectively reduce protein abundance. Extensions of multiple tumor suppressor genes like PTEN, APC, B2M, CASP8, CDKN1B and MLH1 are effective and validated for their suppressive impact. Importantly, the effective extensions possess a higher hydrophobicity than the neutral extensions linking C-terminal hydrophobicity with protein destabilization. Analyzing the proteomes of eleven different species reveals conserved patterns of amino acid distribution in the C-terminal regions of all proteins compared to the proteomes like an enrichment of lysine and arginine and a depletion of glycine, leucine, valine and isoleucine across species and kingdoms. These evolutionary selection patterns are disrupted in the cancer-derived effective nonstop extensions.

Identification of a cooperative effect between amino acids 169 and 174 in the rotavirus NSP4 double-layered particle-binding domain.

Segmented RNA viruses are capable of exchanging genome segments via reassortment as a means of immune evasion and to maintain viral fitness. Reassortments of single-genome segments are common among group A rotaviruses. Multiple instances of co-reassortment of two genome segments, GS6(VP6) and GS10(NSP4), have been documented in surveillance. Specifically, a division between NSP4 genotypes has been observed in the NSP4 double-layered particle (DLP)-binding domain. A previously hypothesized mechanism for this co-reassortment has been suggested to be the interaction between VP6 and NSP4 during DLP transport from viroplasms for particle maturation. In this study, we used sequence analysis, RNA secondary structure prediction, molecular dynamics and reverse genetics to form a hypothesis regarding the role of the NSP4 DLP-binding domain. Sequence analysis showed that the polarity of NSP4 DLP-binding domain amino acids 169 and 174 is clearly divided between E1 and E2 NSP4 genotypes. Viruses with E1 NSP4s had 169A/I or 169S/T with 174S. E2 NSP4s had 169R/K and 174A. RNA secondary structure prediction showed that mutation in both 545 (aa169) and 561 (aa174) causes global structure remodelling. Molecular dynamics showed that the NSP4/VP6 interaction stability is increased by mutating both aa positions 169 and 174. Using reverse genetics, we showed that an R169I mutation alone does not prevent rescue. Conversely, 174A to 174S prevented rescue, and rescue could be returned by combining 174S with 169I. When compared to rSA11 NSP4-wt, both rSA11 NSP4-R169I and rSA11 NSP4-R169I/A174S had a negligible but significant reduction in titre at specific time points. This study suggests that amino acid 174 of NSP4 may be essential in maintaining the VP6/NSP4 interaction required for DLP transport. Our results suggest that maintenance of specific polarities of amino acids at positions 169 and 174 may be required for the fitness of rotavirus field strains.

Epidermal Differentiation Genes of the Common Wall Lizard Encode Proteins with Extremely Biased Amino Acid Contents.

The epidermal differentiation complex (EDC) is a cluster of genes that code for protein components of cornified cells on the skin surface of amniotes. Squamates are the most species-rich clade of reptiles with skin adaptations to many different environments. As the genetic regulation of the skin epidermis and its evolution has been characterized for only a few species so far, we aimed to determine the organization of the EDC in a model species of squamates, the common wall lizard (Podarcis muralis). By comparative genomics, we identified EDC genes of the wall lizard and compared them with homologs in other amniotes. We found that the EDC of the wall lizard has undergone a major rearrangement leading to a unique order of three ancestral EDC segments. Several subfamilies of EDC genes, such as those encoding epidermal differentiation proteins containing PCCC motifs (EDPCCC) and loricrins, have expanded by gene duplications. Most of the EDPCCC proteins have cysteine contents higher than 50%, whereas glycine constitutes more than 50% of the amino acid residues of loricrin 1. The extremely biased amino acid compositions indicate unique structural properties of these EDC proteins. This study demonstrates that cornification proteins of the common wall lizard differ from homologous proteins of other reptiles, illustrating the evolutionary dynamics of diversifying evolution in squamates.

An innovative approach to decoding genetic variability in Pseudomonas aeruginosa via amino acid repeats and gene structure profiles.

Pseudomonas aeruginosa, a common pathogen in nosocomial infections, presents significant global health challenges due to its high prevalence and mortality rates. However, the origins and distribution of this bacterium remain unclear, partly due to the lack of effective gene typing methods. This situation necessitates the establishment of trustworthy and high-resolution protocol for differentiating closely related P. aeruginosa strains. In this context, the present study attempted to undertake a comparative genomic analysis of multiple P. aeruginosa strains available in the public database NCBI, with the goal of identifying potential genetic markers for measuring the genetic diversity. The preliminary comparative analysis of 816 P. aeruginosa strains revealed notable variations in two genes-specifically, the CDF family iron/cobalt efflux transporter AitP and the protease modulator HflC-across 44 strains. These variations were associated with single amino acid repeats (SHRs) that responsible for encoding histidine residue. Additionally, comparative gene map analysis revealed differential clustering patterns in the Rsx and TAXI genes among 16 strains. Interestingly, the gene structure pattern observed in TAXI groups displayed a strong correlation with the SHRs pattern in the CDF and HflC groups. In addition, the SHRs pattern of CDF and HflC were strongly correlated with MLST sequence type number. Overall, the study present a novel genetic markers based on SHRs and gene cluster patterns, offering a reliable method for genotyping of P. aeruginosa.

The evolutionary features and roles of single nucleotide variants and charged amino acid mutations in influenza outbreaks during NPI period.

The epidemic and outbreaks of influenza B Victoria lineage (Bv) during 2019-2022 led to an analysis of genetic, epitopes, charged amino acids and Bv outbreaks. Based on the National Influenza Surveillance Network (NISN), the Bv 72 strains isolated during 2019-2022 were selected by spatio-temporal sampling, then were sequenced. Using the Compare Means, Correlate and Cluster, the outbreak data were analyzed, including the single nucleotide variant (SNV), amino acid (AA), epitope, evolutionary rate (ER), Shannon entropy value (SV), charged amino acid and outbreak. With the emergence of COVID-19, the non-pharmaceutical interventions (NPIs) made Less distant transmission and only Bv outbreak. The 2021-2022 strains in the HA genes were located in the same subset, but were distinct from the 2019-2020 strains (P < 0.001). The codon G → A transition in nucleotide was in the highest ratio but the transversion of C → A and T → A made the most significant contribution to the outbreaks, while the increase in amino acid mutations characterized by polar, acidic and basic signatures played a key role in the Bv epidemic in 2021-2022. Both ER and SV were positively correlated in HA genes (R = 0.690) and NA genes (R = 0.711), respectively, however, the number of mutations in the HA genes was 1.59 times higher than that of the NA gene (2.15/1.36) from the beginning of 2020 to 2022. The positively selective sites 174, 199, 214 and 563 in HA genes and the sites 73 and 384 in NA genes were evolutionarily selected in the 2021-2022 influenza outbreaks. Overall, the prevalent factors related to 2021-2022 influenza outbreaks included epidemic timing, Tv, Ts, Tv/Ts, P137 (B → P), P148 (B → P), P199 (P → A), P212 (P → A), P214 (H → P) and P563 (B → P). The preference of amino acid mutations for charge/pH could influence the epidemic/outbreak trends of infectious diseases. Here was a good model of the evolution of infectious disease pathogens. This study, on account of further exploration of virology, genetics, bioinformatics and outbreak information, might facilitate further understanding of their deep interaction mechanisms in the spread of infectious diseases.

Two amino acid residues in the N-terminal region of the polymerase acidic protein determine the virulence of Eurasian avian-like H1N1 swine influenza viruses in mice.

Reassortant Eurasian avian-like H1N1 (rEA H1N1) viruses carrying the internal genes of H1N1/2009 virus have been circulating in pigs for more than 10 years and have caused sporadic human infections. The enhanced virulence phenotype of the rEA H1N1 viruses highlights potential risks to public health. However, the molecular mechanism underlying the viral pathogenicity of the currently circulating rEA H1N1 viruses remains unclear. In this study, we found that two naturally isolated rEA H1N1 swine influenza viruses, A/swine/Liaoning/FX38/2017 (FX38) and A/swine/Liaoning/SY72/2018 (SY72), possessed similar genetic characteristics but exhibited significantly different pathogenicity in a mouse model. Using reverse genetics, we demonstrated that amino acid mutations at positions 100 and 122 in the polymerase acidic (PA) protein had individual and synergistic effects on the polymerase activity and viral replication capacity in vitro, as well as the viral pathogenicity in mice. Furthermore, we revealed that amino acid residue 100 in PA influenced the transcription of viral RNA (vRNA) by altering the endonuclease activity, and amino acid residue 122 affected the synthesis of complementary RNA and messenger RNA by altering the RNA-binding ability and endonuclease activity of the PA protein. Taken together, we identified that two naturally occurring amino acid mutations in PA derived from H1N1/2009 virus are crucial determinants of the virulence of rEA H1N1 viruses and revealed the differential mechanism by which these two mutations affect the transcription and replication of vRNA. These findings will extend our understanding of the roles of PA in the virulence of influenza A viruses.IMPORTANCEMultiple genetic determinants are involved in the virulence of influenza A viruses. In this study, we identified two naturally occurring amino acid mutations, located at residues 100 and 122 in the polymerase acidic (PA) protein, which are associated with viral polymerase activity, replication competence, and pathogenicity in mice. In particular, we clarified the specific mechanism by which the two residues play an important role in viral transcription and replication. These findings will help to improve understanding the functions of amino acid residues in the N-terminal region of the PA protein involved in the pathogenicity of influenza A viruses.

Cracking the Code: Reprogramming the Genetic Script in Prokaryotes and Eukaryotes to Harness the Power of Noncanonical Amino Acids.

Over 500 natural and synthetic amino acids have been genetically encoded in the last two decades. Incorporating these noncanonical amino acids into proteins enables many powerful applications, ranging from basic research to biotechnology, materials science, and medicine. However, major challenges remain to unleash the full potential of genetic code expansion across disciplines. Here, we provide an overview of diverse genetic code expansion methodologies and systems and their final applications in prokaryotes and eukaryotes, represented by Escherichia coli and mammalian cells as the main workhorse model systems. We highlight the power of how new technologies can be first established in simple and then transferred to more complex systems. For example, whole-genome engineering provides an excellent platform in bacteria for enabling transcript-specific genetic code expansion without off-targets in the transcriptome. In contrast, the complexity of a eukaryotic cell poses challenges that require entirely new approaches, such as striving toward establishing novel base pairs or generating orthogonally translating organelles within living cells. We connect the milestones in expanding the genetic code of living cells for encoding novel chemical functionalities to the most recent scientific discoveries, from optimizing the physicochemical properties of noncanonical amino acids to the technological advancements for their in vivo incorporation. This journey offers a glimpse into the promising developments in the years to come.

Triphasic Development of the Genetic Code.

The genetic code contains an alphabet of genetically encoded amino acids. The ten Phase 1 amino acids, including Gly, Ala, Ser, Asp, Glu, Val, Leu, Ile, Pro and Thr, were available from the prebiotic environment, whereas the ten Phase 2 amino acids, including Phe, Tyr, Arg, His, Trp, Asn, Gln, Lys, Cys, and Met, became available only later from amino acid biosyntheses. In the archaeon Methanopyrus kandleri, the oldest organism known, the standard alphabet of 20 amino acids was "frozen" and no additional amino acid was encoded in the subsequent 3 Gyrs. Four decades ago, it was discovered that the code was frozen because all the organisms were so well adapted to the standard amino acids that oligogenic barriers, consisting of genes that are thoroughly dependent on the standard code, would cause loss of viability upon the deletion of any one amino acid from the code. Once the reason for the freezing of the code was ascertained, procedures were devised by scientists worldwide to enable the encoding of novel noncanonical amino acids (ncAAs). These encoded Phase 3 ncAAs now surpass the 20 canonical Phase 2 amino acids in the code.

Amino Acids at Positions 156 and 332 in the E Protein of the West Nile Virus Subtype Kunjin Virus Classical Strain OR393 Are Involved in Plaque Size, Growth, and Pathogenicity in Mice.

The West Nile virus (WNV) subtype Kunjin virus (WNVKUN) is endemic to Australia. Here, we characterized the classical WNVKUN strain, OR393. The original OR393 strain contained two types of viruses: small plaque-forming virus (SP) and large plaque-forming virus (LP). The amino acid residues at positions 156 and 332 in the E protein (E156 and E332) of SP were Ser and Lys (E156S/332K), respectively, whereas those in LP were Phe and Thr (E156F/332T). SP grew slightly faster than LP in vitro. The E protein of SP was N-glycosylated, whereas that of LP was not. Analysis using two recombinant single-mutant LP viruses, rKUNV-LP-EF156S and rKUNV-LP-ET332K, indicated that E156S enlarged plaques formed by LP, but E332K potently reduced them, regardless of the amino acid at E156. rKUNV-LP-EF156S showed significantly higher neuroinvasive ability than LP, SP, and rKUNV-LP-ET332K. Our results indicate that the low-pathogenic classical WNVKUN can easily change its pathogenicity through only a few amino acid substitutions in the E protein. It was also found that Phe at E156 of the rKUNV-LP-ET332K was easily changed to Ser during replication in vitro and in vivo, suggesting that E156S is advantageous for the propagation of WNVKUN in mammalian cells.

Genetic Code Expansion for Mechanistic Studies in Ion Channels: An (Un)natural Union of Chemistry and Biology.

Ion channels play central roles in biology and human health by catalyzing the transmembrane flow of electrical charge. These proteins are ideal targets for genetic code expansion (GCE) methods because it is feasible to measure ion channel activity from miniscule amounts of protein and to analyze the resulting data via rigorous, established biophysical methods. In an ideal scenario, the encoding of synthetic, noncanonical amino acids via GCE allows the experimenter to ask questions inaccessible to traditional methods. For this reason, GCE has been successfully applied to a variety of ligand- and voltage-gated channels wherein extensive structural, functional, and pharmacological data exist. Here, we provide a comprehensive summary of GCE as applied to ion channels. We begin with an overview of the methods used to encode noncanonical amino acids in channels and then describe mechanistic studies wherein GCE was used for photochemistry (cross-linking; caged amino acids) and atomic mutagenesis (isosteric manipulation of charge and aromaticity; backbone mutation). Lastly, we cover recent advances in the encoding of fluorescent amino acids for the real-time study of protein conformational dynamics.

Comparing the complexity of written and molecular symbolic systems.

Symbolic systems (SSs) are uniquely products of living systems, such that symbolism and life may be inextricably intertwined phenomena. Within a given SS, there is a range of symbol complexity over which signaling is functionally optimized. This range exists relative to a complex and potentially infinitely large background of latent, unused symbol space. Understanding how symbol sets sample this latent space is relevant to diverse fields including biochemistry and linguistics. We quantitatively explored the graphic complexity of two biosemiotic systems: genetically encoded amino acids (GEAAs) and written language. Molecular and graphical notions of complexity are highly correlated for GEAAs and written language. Symbol sets are generally neither minimally nor maximally complex relative to their latent spaces, but exist across an objectively definable distribution, with the GEAAs having especially low complexity. The selection pressures guiding these disparate systems are explicable by symbol production and disambiguation efficiency. These selection pressures may be universal, offer a quantifiable metric for comparison, and suggest that all life in the Universe may discover optimal symbol set complexity distributions with respect to their latent spaces. If so, the "complexity" of individual components of SSs may not be as strong a biomarker as symbol set complexity distribution.

The amino acid variation at hemagglutinin sites 145, 153, 164 and 200 modulate antigenicity andreplication of H9N2 avian influenza virus.

H9N2 avian influenza virus (AIV), one of the predominant subtypes circulating in the poultry industry, inflicts substantial economic damage. Mutations in the hemagglutinin (HA) and neuraminidase (NA) proteins of H9N2 frequently alter viral antigenicity and replication. In this paper, we analyzed the HA genetic sequences and antigenic properties of 26 H9N2 isolates obtained from chickens in China between 2012 and 2019. The results showed that these H9N2 viruses all belonged to h9.4.2.5, and were divided into two clades. We assessed the impact of amino acid substitutions at HA sites 145, 149, 153, 164, 167, 168, and 200 on antigenicity, and found that a mutation at site 164 significantly modified antigenic characteristics. Amino acid variations at sites 145, 153, 164 and 200 affected virus's hemagglutination and the growth kinetics in mammalian cells. These results underscore the critical need for ongoing surveillance of the H9N2 virus and provide valuable insights for vaccine development.

Exploring genetic codon expansion for unnatural amino acid incorporation in filamentous fungus Aspergillus nidulans.

Genetic code expansion technology allows the incorporation of unnatural amino acids (UAAs) into proteins, which is useful in protein engineering, synthetic biology, and gene therapy. Despite its potential applications in various species, filamentous fungi remain unexplored. This study aims to address this gap by developing these techniques in Aspergillus nidulans. We introduced an amber stop codon into a specific sequence within the reporter gene expressed in A. nidulans and replaced the anticodon of the fungal tRNATyr with CUA. This resulted in the synthesis of the target protein, confirming the occurrence of amber suppression in the fungus. When exogenous E. coli tRNATyrCUA (Ec. tRNATyrCUA) and E. coli tyrosyl-tRNA (Ec.TyrRS) were introduced into A. nidulans, they successfully synthesized the target protein via amber suppression and were shown to be orthogonal to the fungal translation system. By replacing the wild-type Ec.TyrRS with a mutant with a higher affinity for the UAA O-methyl-L-tyrosine, the fungal system was able to initiate the synthesis of the UAA-labeled protein (UAA-protein). We further increased the expression level of the UAA-protein through several rational modifications. The successful development of a genetic code expansion technique for A. nidulans has introduced a potentially valuable approach to the study of fungal protein structure and function.

Genome-wide SNPs and candidate genes underlying the genetic variations for protein and amino acids in pearl millet (Pennisetum glaucum) germplasm.

MAIN CONCLUSION: A total of 544 significant marker-trait associations and 286 candidate genes associated with total protein and 18 amino acids were identified. Thirty-three candidate genes were found near the strong marker trait associations (- log10P ≥ 5.5). Pearl millet (Pennisetum glaucum) is largely grown as a subsistence crop in South Asia and sub-Saharan Africa. It serves as a major source of daily protein intake in these regions. Despite its importance, no systematic effort has been made to study the genetic variations of protein and amino acid content in pearl millet germplasm. The present study was undertaken to dissect the global genetic variations of total protein and 18 essential and non-essential amino acids in pearl millet, using a set of 435 K Single Nucleotide Polymorphisms (SNPs) and 161 genotypes of the Pearl Millet Inbred Germplasm Association Panel (PMiGAP). A total of 544 significant marker-trait associations (at P < 0.0001; - log10P ≥ 4) were detected and 23 strong marker-trait associations were identified using Bonferroni's correction method. Forty-eight pleiotropic loci were found in the genome for the studied traits. In total, 286 candidate genes associated with total protein and 18 amino acids were identified. Thirty-three candidate genes were found near strongly associated SNPs. The associated markers and the candidate genes provide an insight into the genetic architecture of the traits studied and are going to be useful in breeding improved pearl millet varieties in the future. Availabilities of improved pearl millet varieties possessing higher protein and amino acid compositions will help combat the rising malnutrition problem via diet.

Comparative genome wise analysis of codon usage of Staphylococcus Genus.

The genus Staphylococcus encompasses a diverse array of bacteria with significant implications for human health, including disreputable pathogens such as Staphylococcus aureus and Staphylococcus epidermidis. Understanding the genetic composition and codon usage patterns of Staphylococcus species is crucial for unraveling their evolutionary dynamics, adaptive strategies, and pathogenic potential. In this study, we conducted a comprehensive analysis of codon usage patterns across 48 species within the Staphylococcus genus. Our findings uncovered variations in genomic G-C content across Staphylococcus species, impacting codon usage preferences, with a notable preference for A/T-rich codons observed in pathogenic strains. This preference for A/T-rich codons suggests an energy-saving strategy in pathogenic organisms. Analysis of dinucleotide pair expression patterns unveiled insights into genomic dynamics, with overrepresented codon pairs reflecting trends in dinucleotide expression across genomes. Additionally, a significant correlation between CAI and genomic G-C content underscored the intricate relationship between codon usage patterns and gene expression strategies. Amino acid usage analysis highlighted preferences for energetically cheaper amino acids, suggesting adaptive strategies promoting energy efficiency. This comprehensive analysis sheds light on the evolutionary dynamics and adaptive mechanisms employed by Staphylococcus species, providing valuable insights into their pathogenic potential and clinical implications. Understanding these genomic features is crucial for devising strategies to combat staphylococcal infections and improve public health outcomes.

Genetic Links between Reproductive Traits and Amino Acid Pairwise Distances of Swine Leukocyte Antigen Alleles among Mating Partners in Microminipigs.

Previously, we found that a greater dissimilarity in swine leukocyte antigen (SLA) class I and class II alleles between mating partners resulted in increased farrowing rates in a highly inbred population of Microminipigs (MMPs). In this follow-up study, we have analyzed the effects of dissimilarity in SLA alleles between mating partners for seven different reproductive traits, including litter size and the number of stillborn and live or dead weaned piglets. We determined the relationships among reproductive traits within each mating event and the amino acid distances of SLA alleles as markers of diversity between mating partners. Our results indicate that mating partners with greater amino acid pairwise genetic distances in the SLA-1 class I gene or DQB1 class II gene alleles were associated with significantly larger litter sizes and higher numbers of live piglets at birth and weaning. Also, partners with greater pairwise distances in the SLA-2 class I gene alleles exhibited fewer pre-weaning deaths. These findings suggest that the dissimilarity in SLA class I and class II alleles between mating partners may affect not only farrowing rates but also other key reproductive traits such as litter size and improved piglet survival rates. Consequently, SLA alleles could serve as valuable genetic markers for selecting mating partners in breeding programs and for conducting epistatic studies on various reproductive traits in MMPs.

Noncanonical Amino Acids in Biocatalysis.

In recent years, powerful genetic code reprogramming methods have emerged that allow new functional components to be embedded into proteins as noncanonical amino acid (ncAA) side chains. In this review, we will illustrate how the availability of an expanded set of amino acid building blocks has opened a wealth of new opportunities in enzymology and biocatalysis research. Genetic code reprogramming has provided new insights into enzyme mechanisms by allowing introduction of new spectroscopic probes and the targeted replacement of individual atoms or functional groups. NcAAs have also been used to develop engineered biocatalysts with improved activity, selectivity, and stability, as well as enzymes with artificial regulatory elements that are responsive to external stimuli. Perhaps most ambitiously, the combination of genetic code reprogramming and laboratory evolution has given rise to new classes of enzymes that use ncAAs as key catalytic elements. With the framework for developing ncAA-containing biocatalysts now firmly established, we are optimistic that genetic code reprogramming will become a progressively more powerful tool in the armory of enzyme designers and engineers in the coming years.

High-Performance Workflow for Identifying Site-Specific Crosslinks Originating from a Genetically Incorporated, Photoreactive Amino Acid.

In conventional crosslinking mass spectrometry, proteins are crosslinked using a highly selective, bifunctional chemical reagent, which limits crosslinks to residues that are accessible and reactive to the reagent. Genetically incorporating a photoreactive amino acid offers two key advantages: any site can be targeted, including those that are inaccessible to conventional crosslinking reagents, and photoreactive amino acids can potentially react with a broad range of interaction partners. However, broad reactivity imposes additional challenges for crosslink identification. In this study, we incorporate benzoylphenylalanine (BPA), a photoreactive amino acid, at selected sites in an intrinsically disordered region of the human protein HSPB5. We report and characterize a workflow for identifying and visualizing residue-level interactions originating from BPA. We routinely identify 30 to 300 crosslinked peptide spectral matches with this workflow, which is up to ten times more than existing tools for residue-level BPA crosslink identification. Most identified crosslinks are assigned to a precision of one or two residues, which is supported by a high degree of overlap between replicate analyses. Based on these results, we anticipate that this workflow will support the more general use of genetically incorporated, photoreactive amino acids for characterizing the structures of proteins that have resisted high-resolution characterization.

Fitness landscape of substrate-adaptive mutations in evolved amino acid-polyamine-organocation transporters.

The emergence of new protein functions is crucial for the evolution of organisms. This process has been extensively researched for soluble enzymes, but it is largely unexplored for membrane transporters, even though the ability to acquire new nutrients from a changing environment requires evolvability of transport functions. Here, we demonstrate the importance of environmental pressure in obtaining a new activity or altering a promiscuous activity in members of the amino acid-polyamine-organocation (APC)-type yeast amino acid transporters family. We identify APC members that have broader substrate spectra than previously described. Using in vivo experimental evolution, we evolve two of these transporter genes, AGP1 and PUT4, toward new substrate specificities. Single mutations on these transporters are found to be sufficient for expanding the substrate range of the proteins, while retaining the capacity to transport all original substrates. Nonetheless, each adaptive mutation comes with a distinct effect on the fitness for each of the original substrates, illustrating a trade-off between the ancestral and evolved functions. Collectively, our findings reveal how substrate-adaptive mutations in membrane transporters contribute to fitness and provide insights into how organisms can use transporter evolution to explore new ecological niches.