Protein Engineering, Design & Selection : PEDS

Proline-rich antimicrobial peptides (PrAMPs) are attractive antibiotic candidates that target gram-negative bacteria ribosomes. We elucidated the sequence-function landscape of 43 000 variants of a recently discovered family member, Tur1a, using the validated SAMP-Dep platform that measures intracellular AMP potency in a high-throughput manner via self-depletion of the cellular host. The platform exhibited high replicate reproducibility (ρ = 0.81) and correlation between synonymous genetic variants (R2 = 0...

Many proteins do not fold into a fixed three-dimensional structure, but rather function in a highly disordered state. These intrinsically disordered proteins pose a unique challenge to protein engineering and design: How can proteins be designed de novo if not by tailoring their structure? Here, we will review the nascent field of design of intrinsically disordered proteins with focus on applications in biotechnology and medicine. The design goals should not necessarily be the same as for de novo design of folded proteins as disordered proteins have unique functional strengths and limitations...

SPMweb is the online webserver of the Shortest Path Map (SPM) tool for identifying the key conformationally-relevant positions of a given enzyme structure and dynamics. The server is built on top of the DynaComm.py code and enables the calculation and visualization of the SPM pathways. SPMweb is easy-to-use as it only requires three input files: the three-dimensional structure of the protein of interest, and the two matrices (distance and correlation) previously computed from a Molecular Dynamics simulation...

We developed fluorescent protein sensors for nicotine with improved sensitivity. For iNicSnFR12 at pH 7.4, the proportionality constant for ∆F/F0 vs [nicotine] (δ-slope, 2.7 μM-1) is 6.1-fold higher than the previously reported iNicSnFR3a. The activated state of iNicSnFR12 has a fluorescence quantum yield of at least 0.6. We measured similar dose-response relations for the nicotine-induced absorbance increase and fluorescence increase, suggesting that the absorbance increase leads to the fluorescence increase via the previously described nicotine-induced conformational change, the "candle snuffer" mechanism...

MOTIVATION: Sequence design is a crucial step in the process of designing or engineering proteins. Traditionally, physics-based methods have been used to solve for optimal sequences, with the main disadvantages being that they are computationally intensive for the end user. Deep learning based methods offer an attractive alternative, outperforming physics-based methods at a significantly lower computational cost. RESULTS: In this paper, we explore the application of Convolutional Neural Networks (CNNs) for sequence design...

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Deep learning methods for protein sequence design focus on modeling and sampling the many- dimensional distribution of amino acid sequences conditioned on the backbone structure. To produce physically foldable sequences, inter-residue couplings need to be considered properly. These couplings are treated explicitly in iterative methods or autoregressive methods. Non-autoregressive models treating these couplings implicitly are computationally more efficient, but still await tests by wet experiment. Currently, sequence design methods are evaluated mainly using native sequence recovery rate and native sequence perplexity...

Numerous cellular functions rely on protein-protein interactions. Efforts to comprehensively characterize them remain challenged however by the diversity of molecular recognition mechanisms employed within the proteome. Deep learning has emerged as a promising approach for tackling this problem by exploiting both experimental data and basic biophysical knowledge about protein interactions. Here, we review the growing ecosystem of deep learning methods for modeling protein interactions, highlighting the diversity of these biophysically-informed models and their respective trade-offs...

Enzymatic degradation of plastics is currently limited to the use of engineered natural enzymes. As of yet, all engineering approaches applied to plastic degrading enzymes retain the natural $\alpha $/$\beta $-fold. While mutations can be used to increase thermostability, an inherent maximum likely exists for the $\alpha $/$\beta $-fold. It is thus of interest to introduce catalytic activity toward plastics in a different protein fold to escape the sequence space of plastic degrading enzymes. Here, a method for designing highly thermostable enzymes that can degrade plastics is described...

Organophosphorus (OP) pesticides are still widely applied but pose a severe toxicological threat if misused, including suicidal attempts. For in vivo detoxification, the application of hydrolytic enzymes potentially offers a promising treatment. A well-studied example is the phosphotriesterase of the bacterium Brevundimonas diminuta (BdPTE). Whereas wild-type BdPTE can hydrolyse pesticides like paraoxon, chlorpyrifos-oxon and mevinphos with high catalytic efficiencies, kcat/KM > 2 × 107 M-1 min-1, degradation of malaoxon is unsatisfactory (kcat/KM ≈ 1 × 104 M-1 min-1)...

Most proteins are flexible molecules that coexist in an ensemble of several conformations. Point mutations in the amino acid sequence of a protein can trigger structural changes that drive the protein population to a conformation distinct from the native state. Here, we report a protein engineering approach to better understand protein dynamics and ligand binding of the FK506-binding protein 51 (FKBP51), a prospective target for stress-related diseases, metabolic disorders, some types of cancers and chronic pain...

After approximately 60 years of work, the protein folding problem has recently seen rapid advancement thanks to the inventions of AlphaFold and RoseTTAFold, which are machine-learning algorithms capable of reliably predicting protein structures from their sequences. A key component in their success was the inclusion of pairwise interaction information between residues. As research focus shifts towards developing algorithms to design and engineer binding proteins, it is likely that knowledge of interaction features at protein interfaces can improve predictions...

Self-supervised pretraining on protein sequences has led to state-of-the art performance on protein function and fitness prediction. However, sequence-only methods ignore the rich information contained in experimental and predicted protein structures. Meanwhile, inverse folding methods reconstruct a protein's amino-acid sequence given its structure, but do not take advantage of sequences that do not have known structures. In this study, we train a masked inverse folding protein masked language model parameterized as a structured graph neural network...

Enzyme design is an important application of computational protein design (CPD). It can benefit enormously from the additional chemistries provided by noncanonical amino acids (ncAAs). These can be incorporated into an "expanded" genetic code, and introduced in vivo into target proteins. The key step for genetic code expansion is to engineer an aminoacyl-tRNA synthase (aaRS) and an associated tRNA that handle the ncAA. Experimental directed evolution has been successfully used to engineer aaRSs and incorporate over 200 ncAAs into expanded codes...

Computational protein design promises the ability to build tailor-made proteins de novo. While a range of de novo proteins have been constructed so far, the majority of these designs have idealized topologies that lack larger cavities which are necessary for the incorporation of small molecule binding sites or enzymatic functions. One attractive target for enzyme design is the TIM-barrel fold, due to its ubiquity in nature and capability to host versatile functions. With the successful de novo design of a 4-fold symmetric TIM barrel, sTIM11, an idealized, minimalistic scaffold was created...

Selection by phage display is a popular and widely used technique for the discovery of recombinant protein binders from large protein libraries for therapeutic use. The protein library is displayed on the surface of bacteriophages which are amplified using bacteria, preferably Escherichia coli, to enrich binders in several selection rounds. Traditionally, the so-called panning procedure during which the phages are incubated with the target protein, washed and eluted is done manually, limiting the throughput...

Oxidoreductases catalyze critical essential redox reactions, and many of these enzymes require a diffusible secondary substrate, or cofactor, for electron transport. Nicotinamide adenine dinucleotide NAD(H) and nicotinamide adenine dinucleotide phosphate (NADP(H)) differ by a 2'-phosphate group and their inability to cross-react allows these cofactor pools to be used by a number of cellular processes. Non-canonical cofactor analogs have been explored as a means to create enzymatic reactions that can operate orthogonally to existing metabolic networks...

Monoclonal antibody (mAb) therapies have rapidly become a powerful class of therapeutics with applications covering a diverse range of clinical indications. Though most widely used for the treatment of cancer, mAbs are also playing an increasing role in the defense of viral infections, most recently with palivizumab for prevention and treatment of severe respiratory syncytial virus infections in neonatal and pediatric populations. In addition, during the COVID-19 pandemic, mAbs provided a therapeutic bridge to the rollout of vaccines; however, their continued role as a therapeutic option for those at greatest risk of severe disease has become limited due to the emergence of neutralization resistant Omicron variants...

Numerous technologies are currently in development for use in next-generation protein sequencing platforms. A notable published approach employs fluorescently-tagged binding proteins to identity the N-terminus of immobilized peptides, in-between rounds of digestion. This approach makes use of N-terminal amino acid binder (NAAB) proteins, which would identify amino acids by chemical and shape complementarity. One source of NAABs is the ClpS protein family, which serve to recruit proteins to bacterial proteosomes based on the identity of the N-terminal amino acid...

Gene fusion or co-immobilization are key tools to optimize enzymatic reaction cascades by modulating catalytic features, stability and applicability. Achieving a defined spatial organization between biocatalysts by site-specific applications is complicated by the involvement of oligomeric enzymes. It can lead to activity losses due to disturbances of the quaternary structures and difficulties in stoichiometric control. Thus, a toolkit of active and robust monomeric enzymes is desirable for such applications...