A Strategy for Combinatorial Cavity Design in de novo Proteins

Protein sequence space is vast; nature uses only an infinitesimal fraction of possible sequences to sustain life. Are there solutions to biological problems other than those provided by nature? Can we create artificial proteins that sustain life? To investigate this question, the Hecht lab has created combinatorial collections, or libraries, of novel sequences with no homology to those found in living organisms. These libraries were subjected to screens and selections, leading to the identification of sequences with roles in catalysis, modulating gene regulation, and metal homeostasis. However, the resulting functional proteins formed dynamic rather than well-ordered structures. This impeded structural characterization and made it difficult to ascertain a mechanism of action. To address this, my thesis work focuses on developing a new model of libraries based on the de novo protein S-824, a four-helix bundle with a very stable three-dimensional structure. The first part of this research focused on mutagenesis of S-824 and characterization of the resulting proteins, revealing that this scaffold tolerates amino acid substitutions, including buried polar residues and the removal of hydrophobic side chains to create a putative cavity. Distinct from previous libraries, I targeted variability to a specific region of the protein, seeking to create a cavity and potential active site. The second part of this work details the design and creation of a library encoding 1.7 x 10^6 unique proteins, assembled from degenerate oligonucleotides. The third and fourth parts of this work cover the screening effort for a range of activities, both in vitro and in vivo. I found that this collection binds heme readily, leading to abundant peroxidase activity. Hits for lipase and phosphatase activity were also detected. This thesis details the development of a new strategy for creating de novo sequences geared toward function rather than structure. Following my work, these library design principles are being applied to SynF4, a de novo enterobactin esterase, whose structure was recently solved. By diversifying the cavity of SynF4, we hope to create a new family of de novo enzymes. Altogether, this approach represents a step towards creating artificial proteomes capable of carrying out essential biological roles

A library of novel genes with combinatorially diverse cavities, built on a stably folded structural template

Protein sequence space is vast; nature uses only an infinitesimal fraction of possible sequences to sustain life. Are there solutions to biological problems other than those provided by nature? Can we create artificial proteins that sustain life? To investigate this question, the Hecht lab has created combinatorial collections, or libraries, of novel sequences with no homology to those found in living organisms. These libraries were subjected to screens and selections, leading to the identification of sequences with roles in catalysis, modulating gene regulation, and metal homeostasis. However, the resulting functional proteins formed dynamic rather than well-ordered structures. This impeded structural characterization and made it difficult to ascertain a mechanism of action. To address this, Christina Karas's thesis work focuses on developing a new model of libraries based on the de novo protein S-824, a four-helix bundle with a very stable three-dimensional structure. The first part of this research focused on mutagenesis of S-824 and characterization of the resulting proteins, revealing that this scaffold tolerates amino acid substitutions, including buried polar residues and the removal of hydrophobic side chains to create a putative cavity. Distinct from previous libraries, Karas targeted variability to a specific region of the protein, seeking to create a cavity and potential active site. The second part of this work details the design and creation of a library encoding 1.7 x 10^6 unique proteins, assembled from degenerate oligonucleotides. The third and fourth parts of this work cover the screening effort for a range of activities, both in vitro and in vivo. I found that this collection binds heme readily, leading to abundant peroxidase activity. Hits for lipase and phosphatase activity were also detected. This work details the development of a new strategy for creating de novo sequences geared toward function rather than structure.CKavity_Lib_Reference: Reference information explaining the library design at the DNA level and the encoded amino acids at the protein level.Folder 1-Raw_Data_20180425: Contains raw data obtained from sequencing in commonly used FASTQ format. Two separate reads are provided in the contained files.2-Results_Summary: Analyses done on the raw dataANALYSIS: contains text files that can be opened in Excel. Filtered results exclude frameshifts while raw results do not.AA_FREQS: Amino acid frequencies for each of the designed variable codons.NT_FREQS: Nucleotide frequencies for each of the designed variable base positions.PIPELINE: Scripts used in the generation of this data.QC: Quality control information generated during DNA sequencing.REFERENCE: Material to assist in interpretation of the data.amplicon: FASTA DNA file showing the linear PCR product derived from the CKavity library, which was then used for all sequencing.probes: Sequences of the oligonucleotide probes used for Next-Generation Sequencing.3-Random_Invariant: Contains CSV files repeating this analysis for random positions not designed to have combinatorial diversity, for comparison purposes

Selection of a Promiscuous Minimalist cAMP Phosphodiesterase from a Library of De Novo Designed Proteins

The ability of unevolved amino acid sequences to become biological catalysts was key to the emergence of life on earth. However, billions of years of evolution separate complex modern enzymes from their simpler early ancestors. To study how unevolved sequences can develop new functions, we screened for enzymatic activity in a collection of > 1 million novel sequences based on a de novo designed 4-helix bundle library of semi-random sequences. To mirror evolutionary selection for biological function, we screened the collection using ultrahigh-throughput droplet microfluidics to identify features that yield phosphoesterase activity. Characterization of active hits demonstrated that acquiring new function involved a large jump in sequence space: screening enriched for truncations that removed > 40% of the protein chain. This truncated protein dimerized into a dynamic -helical structure, consistent with the idea that gain of function was accompanied by an increase in structural dynamics relative to the parental 4-helix bundle. The purified protein catalyzes the hydrolysis of a range of phosphodiesters, with the greatest activity toward the biological second messenger cyclic AMP (cAMP). The novel cAMPase is a manganese-dependent metalloenzyme and catalyzes cAMP hydrolysis with a rate acceleration of 109 and catalytic proficiency on the order of >1014 M-1, comparable to larger enzymes shaped by billions of years of evolution. These findings suggest that fragmentation and effective shortening of protein length can be an unexpectedly fertile avenue for introducing structural and functional diversity into proteins

Phenotype Driven Analysis of Whole Genome Sequencing Identifies Deep Intronic Variants that Cause Retinal Dystrophies by Aberrant Exonization

International audiencePurpose: To demonstrate the effectiveness of combining retinal phenotyping and focused variant filtering from genome sequencing (GS) in identifying deep intronic disease causing variants in inherited retinal dystrophies.Methods: Affected members from three pedigrees with classical enhanced S-cone syndrome (ESCS; Pedigree 1), congenital stationary night blindness (CSNB; Pedigree 2), and achromatopsia (ACHM; Pedigree 3), respectively, underwent detailed ophthalmologic evaluation, optical coherence tomography, and electroretinography. The probands underwent panel-based genetic testing followed by GS analysis. Minigene constructs (NR2E3, GPR179 and CNGB3) and patient-derived cDNA experiments (NR2E3 and GPR179) were performed to assess the functional effect of the deep intronic variants.Results: The electrophysiological findings confirmed the clinical diagnosis of ESCS, CSNB, and ACHM in the respective pedigrees. Panel-based testing revealed heterozygous pathogenic variants in NR2E3 (NM_014249.3; c.119-2A>C; Pedigree 1) and CNGB3 (NM_019098.4; c.1148delC/p.Thr383Ilefs*13; Pedigree 3). The GS revealed heterozygous deep intronic variants in Pedigrees 1 (NR2E3; c.1100+1124G>A) and 3 (CNGB3; c.852+4751A>T), and a homozygous GPR179 variant in Pedigree 2 (NM_001004334.3; c.903+343G>A). The identified variants segregated with the phenotype in all pedigrees. All deep intronic variants were predicted to generate a splice acceptor gain causing aberrant exonization in NR2E3 [89 base pairs (bp)], GPR179 (197 bp), and CNGB3 (73 bp); splicing defects were validated through patient-derived cDNA experiments and/or minigene constructs and rescued by antisense oligonucleotide treatment.Conclusions: Deep intronic mutations contribute to missing heritability in retinal dystrophies. Combining results from phenotype-directed gene panel testing, GS, and in silico splice prediction tools can help identify these difficult-to-detect pathogenic deep intronic variants

Cosmetic Potential of a Recombinant 50 kDa Protein

Collagen and its derivative proteins have been widely used as a major component for cosmetic formulations as a natural ingredient and moisturizer. Most commercially available collagens are animal-derived collagen type I and other forms of collagen, such as type III collagen, are far less prevalent in animals, making extraction and purification extremely difficult and expensive. Here, we report the production of a 50 kDa protein produced in yeast that is 100% identical to the N-terminus of the human type III collagen. This recombinant protein has a larger molecular weight than most incumbent recombinant collagen proteins available for personal care applications. We report the industrialization of both the fermentation and purification processes to produce a final recombinant protein product. This final protein product was shown to be safe for general applications to human skin and compatible with common formulation protocols, including ethanol-based formulations. This recombinant collagen type III protein was also shown to uniquely stimulate both collagen type I and type III production and secretion by primary human dermal fibroblasts. The unique combination of biostimulation, compatibility with beauty product formulations and demonstrated commercial production, make this novel recombinant type III collagen a good candidate for broad application in the cosmetics industry

Single embryo and oocyte lipid fingerprinting by mass spectrometry[S]

Methods used for lipid analysis in embryos and oocytes usually involve selective lipid extraction from a pool of many samples followed by chemical manipulation, separation and characterization of individual components by chromatographic techniques. Herein we report direct analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of single and intact embryos or oocytes from various species. Biological samples were simply moisturized with the matrix solution and characteristic lipid (represented by phosphatidylcholines, sphingomyelins and triacylglycerols) profiles were obtained via MALDI-MS. As representative examples, human, bovine, sheep and fish oocytes, as well as bovine and insect embryos were analyzed. MALDI-MS is shown to be capable of providing characteristic lipid profiles of gametes and embryos and also to respond to modifications due to developmental stages and in vitro culture conditions of bovine embryos. Investigation in developmental biology of the biological roles of structural and reserve lipids in embryos and oocytes should therefore benefit from these rapid MALDI-MS profiles from single and intact species