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At least 235 records · Page 13

A chloroplast protein atlas reveals punctate structures and spatial organization of biosynthetic pathways

Chloroplasts are eukaryotic photosynthetic organelles that drive the global carbon cycle. Despite their importance, our understanding of their protein composition, function, and spatial organization remains limited. Here, we determined the localizations of 1,034 candidate chloroplast proteins using fluorescent protein tagging in the model alga Chlamydomonas reinhardtii. The localizations provide insights into the functions of poorly characterized proteins; identify novel components of nucleoids, plastoglobules, and the pyrenoid; and reveal widespread protein targeting to multiple compartments. We discovered and further characterized cellular organizational features, including eleven chloroplast punctate structures, cytosolic crescent structures, and unexpected spatial distributions of enzymes within the chloroplast. We also used machine learning to predict the localizations of other nuclear-encoded Chlamydomonas proteins. The strains and localization atlas developed here will serve as a resource to accelerate studies of chloroplast architecture and functions.

59 BASIC BIOLOGICAL SCIENCES↗

A functional role of Rv1738 in Mycobacterium tuberculosis persistence suggested by racemic protein crystallography

Racemic protein crystallography was used to determine the X-ray structure of the predicted Mycobacterium tuberculosis protein Rv1738, which had been completely recalcitrant to crystallization in its natural L-form. Native chemical ligation was used to synthesize both L-protein and D-protein enantiomers of Rv1738. Crystallization of the racemic {D-protein + L-protein} mixture was immediately successful. The resulting crystals diffracted to high resolution and also enabled facile structure determination because of the quantized phases of the data from centrosymmetric crystals. The X-ray structure of Rv1738 revealed striking similarity with bacterial hibernation factors, despite minimal sequence similarity. As a result, we predict that Rv1738, which is highly up-regulated in conditions that mimic the onset of persistence, helps trigger dormancy by association with the bacterial ribosome.

59 BASIC BIOLOGICAL SCIENCES↗

Functional genomics of the rapidly replicating bacterium Vibrio natriegens by CRISPRi

We present that the fast-growing Gram-negative bacterium Vibrio natriegens is an attractive microbial system for molecular biology and biotechnology due to its remarkably short generation time and metabolic prowess. However, efforts to uncover and utilize the mechanisms underlying its rapid growth are hampered by the scarcity of functional genomic data. Here, we develop a pooled genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) screen to identify a minimal set of genes required for rapid wild-type growth. Targeting 4,565 (99.7%) of predicted protein-coding genes, our screen uncovered core genes comprising putative essential and growth-supporting genes that are enriched for respiratory pathways. We found that 96% of core genes were located on the larger chromosome 1, with growth-neutral duplicates of core genes located primarily on chromosome 2. Lastly, our screen also refines metabolic pathway annotations by distinguishing functional biosynthetic enzymes from those predicted on the basis of comparative genomics. Taken together, this work provides a broadly applicable platform for high-throughput functional genomics to accelerate biological studies and engineering of V. natriegens.

59 BASIC BIOLOGICAL SCIENCES↗

Comparative Virulence and Genomic Analysis of Streptococcus suis Isolates

Streptococcus suis is a zoonotic bacterial swine pathogen causing substantial economic and health burdens to the pork industry. Mechanisms used by S. suis to colonize and cause disease remain unknown and vaccines and/or intervention strategies currently do not exist. Studies addressing virulence mechanisms used by S. suis have been complicated because different isolates can cause a spectrum of disease outcomes ranging from lethal systemic disease to asymptomatic carriage. The objectives of this study were to evaluate the virulence capacity of nine United States S. suis isolates following intranasal challenge in swine and then perform comparative genomic analyses to identify genomic attributes associated with swine-virulent phenotypes. No correlation was found between the capacity to cause disease in swine and the functional characteristics of genome size, serotype, sequence type (ST), or in vitro virulence-associated phenotypes. A search for orthologs found in highly virulent isolates and not found in non-virulent isolates revealed numerous predicted protein coding sequences specific to each category. While none of these predicted protein coding sequences have been previously characterized as potential virulence factors, this analysis does provide a reliable one-to-one assignment of specific genes of interest that could prove useful in future allelic replacement and/or functional genomic studies. Collectively, this report provides a framework for future allelic replacement and/or functional genomic studies investigating genetic characteristics underlying the spectrum of disease outcomes caused by S. suis isolates.

59 BASIC BIOLOGICAL SCIENCES↗

Electronic coupling through natural amino acids

Myriad scientific domains concern themselves with biological electron transfer (ET) events that span across vast scales of rate and efficiency through a remarkably fine-tuned integration of amino acid (AA) sequences, electronic structure, dynamics, and environment interactions. Within this intricate scheme, many questions persist as to how proteins modulate electron-tunneling properties. To help elucidate these principles, we develop a model set of peptides representing the common α-helix and β-strand motifs including all natural AAs within implicit protein-environment solvation. Using an effective Hamiltonian strategy with density functional theory, we characterize the electronic coupling through these peptides, furthermore considering side-chain dynamics. For both motifs, predictions consistently show that backbone-mediated electronic coupling is distinctly sensitive to AA type (aliphatic, polar, aromatic, negatively charged and positively charged), and to side-chain orientation. The unique properties of these residues may be employed to design activated, deactivated, or switch-like superexchange pathways. Electronic structure calculations and Green’s function analyses indicate that localized shifts in the electron density along the peptide play a role in modulating these pathways, and further substantiate the experimentally observed behavior of proline residues as superbridges. The distinct sensitivities of tunneling pathways to sequence and conformation revealed in this electronic coupling database help improve our fundamental understanding of the broad diversity of ET reactivity and provide guiding principles for peptide design.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Synthase-Selective Exploration of a Tunicate Microbiome by Activity-Guided Single-Cell Genomics

While thousands of environmental metagenomes have been mined for the presence of novel biosynthetic gene clusters, such computational predictions do not provide evidence of their in vivo biosynthetic functionality. Using fluorescent in situ enzyme assay targeting carrier proteins common to polyketide (PKS) and nonribosomal peptide synthetases (NRPS), we applied fluorescence-activated cell sorting to tunicate microbiome to enrich for microbes with active secondary metabolic capabilities. Single-cell genomics uncovered the genetic basis for a wide biosynthetic diversity in the enzyme-active cells and revealed a member of marine Oceanospirillales harboring a novel NRPS gene cluster with high similarity to phylogenetically distant marine and terrestrial bacteria. Interestingly, this synthase belongs to a larger class of siderophore biosynthetic gene clusters commonly associated with pestilence and disease. Furthermore, this demonstrates activity-guided single-cell genomics as a tool to guide novel biosynthetic discovery.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Strategies to identify and edit improvements in synthetic genome segments episomally

Genome engineering projects often utilize bacterial artificial chromosomes (BACs) to carry multi-kilobase DNA segments at low copy number. However, all stages of whole-genome engineering have the potential to impose mutations on the synthetic genome that can reduce or eliminate the fitness of the final strain. Here, we describe improvements to a multiplex automated genome engineering (MAGE) protocol to improve recombineering frequency and multiplexability. This protocol was applied to recoding an Escherichia coli strain to replace seven codons with synonymous alternatives genome wide. Ten 44 402–47 179 bp de novo synthesized DNA segments contained in a BAC from the recoded strain were unable to complement deletion of the corresponding 33–61 wild-type genes using a single antibiotic resistance marker. Next-generation sequencing (NGS) was used to identify 1–7 non-recoding mutations in essential genes per segment, and MAGE in turn proved a useful strategy to repair these mutations on the recoded segment contained in the BAC when both the recoded and wild-type copies of the mutated genes had to exist by necessity during the repair process. Finally, two web-based tools were used to predict the impact of a subset of non-recoding missense mutations on strain fitness using protein structure and function calls.

59 BASIC BIOLOGICAL SCIENCES↗

Evolutionary Variation in MADS Box Dimerization Affects Floral Development and Protein Abundance in Maize

Interactions between MADS-box transcription factors are critical in the regulation of floral development, and shifting MADS-box protein-protein interactions are predicted to have influenced floral evolution. However, precisely how evolutionary variation in protein-protein interactions affects MADS-box protein function remains unknown. To assess the impact of changing MADS-box protein-protein interactions on transcription factor function, we turned to the grasses, where interactions between B-class MADS-box proteins vary. We tested the functional consequences of this evolutionary variability using maize as an experimental system. We found that differential B-class dimerization was associated with subtle, quantitative differences in stamen shape. In contrast, differential dimerization resulted in large-scale changes to downstream gene expression. Differential dimerization also affected B-class complex composition and abundance, independent of transcript levels. This indicates that differential B-class dimerization affects protein degradation, revealing an important consequence for evolutionary variability in MADS-box interactions. Our results highlight complexity in the evolution of developmental gene networks - changing protein-protein interactions could affect not only the composition of transcription factor complexes, but also their degradation and persistence in developing flowers. Here, our results also show how coding change in a pleiotropic master regulator could have small, quantitative effects on development.

59 BASIC BIOLOGICAL SCIENCES↗

The histone demethylase PHF8 is an oncogenic protein in human non-small cell lung cancer

Highlights: • PHF8 overexpresses in human NSCLC and predicts poor survival. • PHF8 regulates lung cancer cell growth and transformation. • PHF8 regulates apoptosis in human lung cancer cells. • PHF8 promotes miR-21 expression in human lung cancer. • MiR-21 is critically essential for PHF8 function in human lung cancer cells. - Abstract: PHF8 is a JmjC domain-containing protein and erases repressive histone marks including H4K20me1 and H3K9me1/2. It binds to H3K4me3, an active histone mark usually located at transcription start sites (TSSs), through its plant homeo-domain, and is thus recruited and enriched in gene promoters. PHF8 is involved in the development of several types of cancer, including leukemia, prostate cancer, and esophageal squamous cell carcinoma. Herein we report that PHF8 is an oncogenic protein in human non-small cell lung cancer (NSCLC). PHF8 is up-regulated in human NSCLC tissues, and high PHF8 expression predicts poor survival. Our in vitro and in vivo evidence demonstrate that PHF8 regulates lung cancer cell proliferation and cellular transformation. We found that PHF8 knockdown induces DNA damage and apoptosis in lung cancer cells. PHF8 promotes miR-21 expression in human lung cancer, and miR-21 knockdown blocks the effects of PHF8 on proliferation and apoptosis of lung cancer cells. In summary, PHF8 promotes lung cancer cell growth and survival by regulating miR-21.

60 APPLIED LIFE SCIENCES↗

The standard operating procedure of the DOE-JGI Metagenome Annotation Pipeline (MAP v.4)

Abstract The DOE-JGI Metagenome Annotation Pipeline (MAP v.4) performs structural and functional annotation for metagenomic sequences that are submitted to the Integrated Microbial Genomes with Microbiomes (IMG/M) system for comparative analysis. The pipeline runs on nucleotide sequences provided via the IMG submission site. Users must first define their analysis projects in GOLD and then submit the associated sequence datasets consisting of scaffolds/contigs with optional coverage information and/or unassembled reads in fasta and fastq file formats. The MAP processing consists of feature prediction including identification of protein-coding genes, non-coding RNAs and regulatory RNAs, as well as CRISPR elements. Structural annotation is followed by functional annotation including assignment of protein product names and connection to various protein family databases.

59 BASIC BIOLOGICAL SCIENCES↗

Modeling the Activity of Single Genes

The central dogma of molecular biology states that information is stored in DNA, transcribed to messenger RNA (mRNA) and then translated into proteins. This picture is significantly augmentated when we consider the action of certain proteins in regulating transcription. These transcription factors provide a feedback pathway by which genes can regulate one another's expression as mRNA and then as protein. To review: DNA, RNA and proteins have different functions. DNA is the molecular storehouse of genetic information. When cells divide, the DNA is replicated, so that each daughter cell maintains the same genetic information as the mother cell. RNA acts as a go-between from DNA to proteins. Only a single copy of DNA is present, but multiple copies of the same piece of RNA may be present, allowing cells to make huge amounts of protein. In eukaryotes (organisms with a nucleus), DNA is found in the nucleus only. RNA is copied in the nucleus then translocates(moves) outside the nucleus, where it is transcribed into proteins. Along the way, the RNA may be spliced, i.e., may have pieces cut out. RNA then attaches to ribosomes and is translated to proteins. Proteins are the machinery of the cell other than DNA and RNA, all the complex molecules of the cell are proteins. Proteins are specialized machines, each of which fulfills its own task, which may be transporting oxygen, catalyzing reactions, or responding to extracellular signals, just to name a few. One of the more interesting functions a protein may have is binding directly or indirectly to DNA to perform transcriptional regulation, thus forming a closed feedback loop of gene regulation. The structure of DNA and the central dogma were understood in the 50s; in the early 80s it became possible to make arbitrary modifications to DNA and use cellular machinery to transcribe and translate the resulting genes; more recently, genomes (i.e., the complete DNA sequence) of many organisms have been sequenced. This large-scale sequencing began with simple organisms, viruses and bacteria, progressed to eukaryotes such as yeast, and more recently (1998) progressed to a multi-cellular animal, the nematode Caenorhabditis elegans. Sequencers have now moved on to the fruit fly Drosophila melanogaster, whose sequence is slated for completion by the end of 1999. The human genome project is expected to determine the complete sequence of all 3 billion bases of human DNA within the next five years. In the wake of genome-scale sequencing, further instrumentation is being developed to assay gene expression and function on a comparably large scale. Much of the work in computational biology focuses on computational tools used in sequencing, finding genes that are related to a particular gene, finding which parts of the DNA code for proteins and which do not, understanding what proteins will be formed from a given length of DNA, predicting how the proteins will fold from a one-dimensional structure into a three dimensional structure, and so on. Much less computational work has been done regarding the function of proteins. One reason for this is that different proteins function very differently, and so work on protein function is very specific to certain classes of proteins. There are, for example, proteins such enzymes that catalyze various intracellular reactions, receptors that respond to extracellular signals and ion channels that regulate the flow of charged particles into and out of the cell. In this chapter, we will consider a particular class of proteins called transcription factors(TFs), which are responsible for regulating when a certain gene is expressed in a certain cell, which cells it is express in, and how much is expressed. Understanding these processes will involve developing a deeper understanding of transcription, translation, and the cellular processes that control those processes. All of these elements fall under the aegis of gene regulation or more narrowly transcriptional regulation. Some of the key questions in gene regulation are: What genes are expressed in a certain cell at a certain time? How does gene expression differ from cell to cell in a multicellular organism? Which proteins act as transcription factors, i.e., are important in regulating gene expression? From questions like these, we hope to understand which genes are important for various macroscopic processes. Nearly all of the cells of a multicellular organism contain the same DNA. Yet this same genetic information yields a large number of different cell types. The fundamental difference between a neuron and a liver cell, for example, is which genes are expressed. Thus understanding gene regulation is an important step in understanding development. Furthermore, understanding the usual genes that are expressed in cells may give important clues about various diseases. Some diseases, such as sickle cell anemia and cystic fibrosis, are caused by defects in single, non-regulatory genes; others, such as certain cancers, are caused when the cellular control circuitry malfunctions - an understanding of these diseases will involve pathways of multiple interacting gene products. There are numerous challenges in the area of understanding and modeling gene regulation. First and foremost, biologists would like to develop a deeper understanding of the processes involved, including which genes and families of genes are important, how they interact, etc. From a computation point of view, there has been embarrassingly little work done. In this chapter there are many areas in which we can phrase meaningful, non-trivial computational questions, but questions that have not been addressed. Some of these are purely computational (what is a good algorithm for dealing with a model of type X) and others are more mathematical (given a system with certain characteristics, what sort of model can one use? How does one find biochemical parameters from system-level behavior using as few experiments as possible?). In addition to biological and algorithmic problems, there is also the ever-present issue of theoretical biology - what general principles can be derived from these systems, what can one do with models other than just simulate time-courses, what can be deduced about a class of systems without knowing all the details? The fundamental challenge to computationalists and theorists is to add value to the biology - to use models, modeling techniques and algorithms to understand the biology in new ways.

Mjolsness, Eric↗

Critical lysine residues of Klf4 required for protein stabilization and degradation

Highlights: • Klf4 undergoes the 26S proteasomal degradation by ubiquitination on its multiple lysine residues. • Essential Klf4 ubiquitination sites are accumulated between 190–263 amino acids. • A mutation of lysine at 232 on Klf4 elongates protein turnover. • Klf4 mutants dramatically suppress p53 expression both under normal and UV irradiated conditions. - Abstract: The transcription factor, Krüppel-like factor 4 (Klf4) plays a crucial role in generating induced pluripotent stem cells (iPSCs). As the ubiquitination and degradation of the Klf4 protein have been suggested to play an important role in its function, the identification of specific lysine sites that are responsible for protein degradation is of prime interest to improve protein stability and function. However, the molecular mechanism regulating proteasomal degradation of the Klf4 is poorly understood. In this study, both the analysis of Klf4 ubiquitination sites using several Klf4 deletion fragments and bioinformatics predictions showed that the lysine sites which are signaling for Klf4 protein degradation lie in its N-terminal domain (aa 1–296). The results also showed that Lys32, 52, 232, and 252 of Klf4 are responsible for the proteolysis of the Klf4 protein. These results suggest that Klf4 undergoes proteasomal degradation and that these lysine residues are critical for Klf4 ubiquitination.

60 APPLIED LIFE SCIENCES↗

PET-FBA: A lightweight enzyme allocation and thermodynamics-constrained flux analysis approach to explore Escherichia coli metabolic adaptation to intracellular acidification

Escherichia coli employs diverse strategies to adapt to acidic environments that disrupt enzyme activity and the thermodynamic feasibility of essential reactions. To understand the impact of pH stress on cell metabolism, we present the PET-FBA (pH-, Enzyme protein allocation-, and Thermodynamics-constrained Flux Balance Analysis) framework. PET-FBA extends genome-scale modeling by integrating enzyme protein costs and reaction Gibbs free energy changes. Additionally, by incorporating pH-dependent enzyme kinetics in response to intracellular acidification, this framework enables the simulation of E. coli's metabolic adjustments across varying external pH levels. The model's accuracy is validated by comparing in silico growth simulations with experimental measurements under both anaerobic and aerobic conditions, as well as in silico gene knockouts of essential genes. By explicitly incorporating pH effects, our model accurately replicates the metabolic shift towards lactate production as the primary fermentation product at low pH in anaerobic conditions. This shift is only predicted when enzyme kinetics are dynamically adjusted as a function of pH. Further analysis revealed that this shift can be attributed to the reduced protein efficiency of the acetyl-CoA branch compared to lactate dehydrogenase under acidic stress, which then becomes crucial for maintaining NAD regeneration and cell growth at low pH. Furthermore, we identified strategies for enhancing cell growth under acidic anaerobic conditions by improving the enzyme activity of lactate dehydrogenase and pyruvate formate lyase, which increases NAD production efficiency and reduces enzyme protein allocation costs. Designed as a lightweight yet versatile framework, PET-FBA enables efficient genome-scale metabolic analysis. Using E. coli as a model system, our framework provides a systematic approach to understanding metabolic responses to environmental stress, pinpointing key metabolic bottlenecks, and identifying potential targets for strain optimization.

42 ENGINEERING↗

Nitrogen remobilization and conservation, and underlying senescence-associated gene expression in the perennial switchgrass Panicum virgatum

Improving nitrogen (N) remobilization from aboveground to underground organs during yearly shoot senescence is an important goal for sustainable production of switchgrass (Panicum virgatum) as a biofuel crop. Little is known about the genetic control of senescence and N use efficiency in perennial grasses such as switchgrass, which limits our ability to improve the process. Switchgrass aboveground organs (leaves, stems and inflorescences) and underground organs (crowns and roots) were harvested every month over a 3-yr period. Transcriptome analysis was performed to identify genes differentially expressed in various organs during development. Total N content in aboveground organs increased from spring until the end of summer, then decreased concomitant with senescence, while N content in underground organs exhibited an increase roughly matching the decrease in shoot N during fall. Hundreds of senescence-associated genes were identified in leaves and stems. Functional grouping indicated that regulation of transcription and protein degradation play important roles in shoot senescence. Co-expression networks predict important roles for five switchgrass NAC (NAM, ATAF1,2, CUC2) transcription factors (TFs) and other TF family members in orchestrating metabolism of carbohydrates, N and lipids, protein modification/degradation, and transport processes during senescence. This study establishes a molecular basis for understanding and enhancing N remobilization and conservation in switchgrass.

09 BIOMASS FUELS↗

Draft genome sequence of Pseudomonas extremaustralis strain USBA-GBX 515 isolated from Superparamo soil samples in Colombian Andes

Abstract Here we present the physiological features of Pseudomonas extremaustralis strain USBA-GBX-515 ( CMPUJU 515 ), isolated from soils in Superparamo ecosystems, > 4000 m.a.s.l, in the northern Andes of South America, as well as the thorough analysis of the draft genome. Strain USBA-GBX-515 is a Gram-negative rod shaped bacterium of 1.0–3.0 μm × 0.5–1 μm, motile and unable to form spores, it grows aerobically and cells show one single flagellum. Several genetic indices, the phylogenetic analysis of the 16S rRNA gene sequence and the phenotypic characterization confirmed that USBA-GBX-515 is a member of Pseudomonas genus and, the similarity of the 16S rDNA sequence was 100% with P. extremaustralis strain CT14–3 T . The draft genome of P. extremaustralis strain USBA-GBX-515 consisted of 6,143,638 Mb with a G + C content of 60.9 mol%. A total of 5665 genes were predicted and of those, 5544 were protein coding genes and 121 were RNA genes. The distribution of genes into COG functional categories showed that most genes were classified in the category of amino acid transport and metabolism (10.5%) followed by transcription (8.4%) and signal transduction mechanisms (7.3%). We performed experimental analyses of the lipolytic activity and results showed activity mainly on short chain fatty acids. The genome analysis demonstrated the existence of two genes, lip515A and est515A, related to a triacylglycerol lipase and carboxylesterase, respectively. Ammonification genes were also observed, mainly nitrate reductase genes. Genes related with synthesis of poly-hydroxyalkanoates (PHAs), especially poly-hydroxybutyrates (PHBs), were detected. The phaABC and phbABC operons also appeared complete in the genome. P. extremaustralis strain USBA-GBX-515 conserves the same gene organization of the type strain CT14–3 T . We also thoroughly analyzed the potential for production of secondary metabolites finding close to 400 genes in 32 biosynthetic gene clusters involved in their production.

16S rRNA↗

Rhesus macaque and mouse models for down-selecting circumsporozoite protein based malaria vaccines differ significantly in immunogenicity and functional outcomes

Background: Non-human primates, such as the rhesus macaques, are the preferred model for down-selecting human malaria vaccine formulations, but the rhesus model is expensive and does not allow for direct efficacy testing of human malaria vaccines. Transgenic rodent parasites expressing genes of human Plasmodium are now routinely used for efficacy studies of human malaria vaccines. Mice have however rarely predicted success in human malaria trials and there is scepticism whether mouse studies alone are sufficient to move a vaccine candidate into the clinic. Methods: A comparison of immunogenicity, fine-specificity and functional activity of two Alum-adjuvanted Plasmodium falciparum circumsporozoite protein (CSP)-based vaccines was conducted in mouse and rhesus models. One vaccine was a soluble recombinant protein (CSP) and the other was the same CSP covalently conjugated to the Qβ phage particle (Qβ-CSP). Results: Mice showed different kinetics of antibody responses and different sensitivity to the NANP-repeat and N-terminal epitopes as compared to rhesus. While mice failed to discern differences between the protective efficacy of CSP versus Qβ-CSP vaccine following direct challenge with transgenic Plasmodium berghei parasites, rhesus serum from the Qβ-CSP-vaccinated animals induced higher in vivo sporozoite neutralization activity. Conclusions: Despite some immunologic parallels between models, these data demonstrate that differences between the immune responses induced in the two models risk conflicting decisions regarding potential vaccine utility in humans. In combination with historical observations, the data presented here suggest that although murine models may be useful for some purposes, non-human primate models may be more likely to predict the human response to investigational vaccines.

60 APPLIED LIFE SCIENCES↗

Computation-guided optimization of split protein systems

Splitting bioactive proteins into conditionally reconstituting fragments is a powerful strategy for building tools to study and control biological systems. However, split proteins often exhibit a high propensity to reconstitute, even without the conditional trigger, limiting their utility. Current approaches for tuning reconstitution propensity are laborious, context-specific or often ineffective. Here, we report a computational design strategy grounded in fundamental protein biophysics to guide experimental evaluation of a sparse set of mutants to identify an optimal functional window. We hypothesized that testing a limited set of mutants would direct subsequent mutagenesis efforts by predicting desirable mutant combinations from a vast mutational landscape. This strategy varies the degree of interfacial destabilization while preserving stability and catalytic activity. We validate our method by solving two distinct split protein design challenges, generating both design and mechanistic insights. Furthermore, this new technology will streamline the generation and use of split protein systems for diverse applications.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Energetic Basis and Design of Enzyme Function Demonstrated Using GFP, an Excited-State Enzyme

We report the past decades have witnessed an explosion of de novo protein designs with a remarkable range of scaffolds. It remains challenging, however, to design catalytic functions that are competitive with naturally occurring counterparts as well as biomimetic or nonbiological catalysts. Although directed evolution often offers efficient solutions, the fitness landscape remains opaque. Green fluorescent protein (GFP), which has revolutionized biological imaging and assays, is one of the most redesigned proteins. While not an enzyme in the conventional sense, GFPs feature competing excited-state decay pathways with the same steric and electrostatic origins as conventional ground-state catalysts, and they exert exquisite control over multiple reaction outcomes through the same principles. Thus, GFP is an “excited-state enzyme”. Herein we show that rationally designed mutants and hybrids that contain environmental mutations and substituted chromophores provide the basis for a quantitative model and prediction that describes the influence of sterics and electrostatics on excited-state catalysis of GFPs. As both perturbations can selectively bias photoisomerization pathways, GFPs with fluorescence quantum yields (FQYs) and photoswitching characteristics tailored for specific applications could be predicted and then demonstrated. The underlying energetic landscape, readily accessible via spectroscopy for GFPs, offers an important missing link in the design of protein function that is generalizable to catalyst design.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗