Protein Engineering, Design & Selection : PEDS

Antibodies that bind to protein phosphorylation sites are a critical tool for detecting the post-translational modification. The detection of site-specific phosphorylation in the microtubule-associated protein tau is emerging as a means to diagnose and monitor the progression of Alzheimer's Disease (AD) as well as other neurodegenerative diseases. However, the need for reliable phospho-site specific antibodies persists due to a lack of approaches for identifying monoclonal antibodies and characterizing non-specific binding...

No abstract text is available yet for this article.

Biocatalysis has become a powerful alternative for green chemistry. Expanding the range of amino acids used in protein biosynthesis can improve industrially appealing properties such as enantioselectivity, activity and stability. This review will specifically delve into the thermal stability improvements that non-canonical amino acids (ncAAs) can confer to enzymes. Methods to achieve this end, such as the use of halogenated ncAAs, selective immobilization and rational design, will be discussed. Additionally, specific enzyme design considerations using ncAAs are discussed along with the benefits and limitations of the various approaches available to enhance the thermal stability of enzymes...

Lignocellulosic biomass is a renewable source of energy, chemicals, and materials. Many applications of this resource require the depolymerization of one or more of its polymeric constituents. Efficient enzymatic depolymerization of cellulose to glucose by cellulases and accessory enzymes such as lytic polysaccharide monooxygenases (LPMOs) is a prerequisite for economically viable exploitation of this biomass. Microbes produce a remarkably diverse range of cellulases, which consist of glycoside hydrolase (GH) catalytic domains and, although not in all cases, substrate-binding carbohydrate-binding modules (CBMs)...

The Fv region of the antibody (comprising VH and VL domains) is the area responsible for target binding and thus the antibody's specificity. The orientation, or packing, of these two domains relative to each other influences the topography of the Fv region, and therefore can influence the antibody's binding affinity. We present abYpap, an improved method for predicting the packing angle between the VH and VL domains. With the large data set now available, we were able to expand greatly the number of features that could be used compared with our previous work...

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Computational modeling and design of antibodies has become an integral part of today's research and development in antibody therapeutics. Here we describe the Triad Antibody Homology Modeling (TriadAb) package, a functionality of the Triad protein design platform that predicts the structure of any heavy and light chain sequences of an antibody Fv domain using template-based modeling. To gauge the performance of TriadAb, we benchmarked against the results of the Second Antibody Modeling Assessment (AMA-II). On average, TriadAb produced main-chain carbonyl root-mean-square deviations between models and experimentally determined structures at 1...

No abstract text is available yet for this article.

Display technologies are powerful tools for discovering antibodies and other binding proteins against a broad range of biological targets. However, it remains challenging to adapt display technologies for the discovery of proteins that inhibit the enzymatic activities of targets because the phenotypic readout during display screens is binding. The goal of this work is to investigate approaches for discovering inhibitory antibodies in yeast display format using a well-defined series of constructs and the target matrix metalloproteinase-9 (MMP-9)...

Human Transthyretin (TTR) is a homo-tetrameric plasma protein associated with a high percentage of β-sheet forming amyloid fibrils. It accumulates in tissues or extracellular matrices to cause amyloid diseases. Free energy simulations with thermodynamic integration based on all-atom molecular dynamics simulations have been carried out to analyze the effects of the His88 → Ala and Ser mutations on the stability of human TTR. The calculated free energy change differences (ΔΔG) caused by the His88 → Ala and His88 → Ser mutations are -1...

A new format of therapeutic proteins are bispecific antibodies, in which two different heavy chains heterodimerize to obtain two different binding sites. Therefore, it is crucial to understand and optimize the third constant domain (CH3-CH3) interface to favor heterodimerization over homodimerization, and to preserve the physicochemical properties, as thermal stability. Here we use molecular dynamics simulations to investigate the dissociation process of 19 CH3-CH3 crystal structures that differ from each other in few point mutations...

Proline dehydrogenase (PRODH) catalyzes the FAD-dependent oxidation of L-proline to Δ1-pyrroline-5-carboxylate and is a target for inhibitor discovery because of its importance in cancer cell metabolism. Because human PRODH is challenging to purify, the PRODH domains of the bacterial bifunctional enzyme proline utilization A (PutA) have been used for inhibitor development. These systems have limitations due to large polypeptide chain length, conformational flexibility, and the presence of domains unrelated to PRODH activity...

Many glycosylated small molecule natural products and glycoprotein biologics are important in a broad range of therapeutic and industrial applications. The sugar moieties that decorate these compounds often show a profound impact on their biological functions, thus biocatalytic methods for controlling their glycosylation are valuable. Enzymes from nature are useful tools to tailor bioproduct glycosylation but these sometimes have limitations in their catalytic efficiency, substrate specificity, regiospecificity, stereospecificity, or stability...

Enzyme engineering has become a widely adopted practice in research labs and industry. In parallel, the past decades have seen tremendous strides in characterizing the dynamics of proteins, using a growing array of methodologies. Importantly, links have been established between the dynamics of proteins and their function. Characterizing the dynamics of an enzyme prior to, and following, its engineering is beginning to inform on the potential of 'dynamic engineering', i.e., the rational modification of protein dynamics to alter enzyme function...

The field of therapeutic antibodies and especially bi- or multispecific antibodies is growing rapidly. Especially for treating cancers, multispecific antibodies are very promising as there are multiple pathways involved and multispecific antibodies offer the possibility to interfere at two or more sites. Besides being used as therapeutic, multispecific antibodies can be helpful tools in basic research. However, the design and choice of the most appropriate multispecific antibody format is far from trivial. The generation of multispecific antibodies starts with the generation of antibodies directed against the desired targets and then combining the different antigen-binding sites in one molecule...

Enzyme design and engineering strategies are typically constrained by the limited size of nature's genetic alphabet, comprised of only twenty canonical amino acids. In recent years, site-selective incorporation of non-canonical amino acids (ncAAs) via an expanded genetic code has emerged as a powerful means of inserting new functional components into proteins, and hundreds of structurally diverse amino acids are now available. Here, we highlight how the emergence of an expanded repertoire of amino acids has opened new avenues in enzyme design and engineering...

The CRISPR genome editing technology holds great clinical potential for the treatment of monogenetic disorders such as sickle cell disease or muscular dystrophy. The therapeutic in vivo application of the technology relies on targeted delivery methods of the Cas9 and gRNA complex to specific cells and/or tissues. However, such methods are currently limited to direct organ delivery, preventing wide clinical application. Here, we show that monoclonal antibodies can be employed to deliver the Cas9/gRNA complex directly into human cells via cell-surface receptors...

Identifying function-enhancing enzyme variants is a 'holy grail' challenge in protein science because it will allow researchers to expand the biocatalytic toolbox for late-stage functionalization of drug-like molecules, environmental degradation of plastics and other pollutants, and medical treatment of food allergies. Data-driven strategies, including statistical modeling, machine learning, and deep learning, have largely advanced the understanding the sequence-structure-function relationships for enzymes...

Malate dehydrogenase (MDH) catalyzes the reduction of oxaloacetate to L-malate using NADH as a cofactor. Geobacillus stearothermophilus MDH (gs-MDH) is used as a diagnostic reagent; however, gs-MDH is robustly inhibited at high substrate concentrations, which limits its reaction rate. Here, we reduced substrate inhibition of gs-MDH by deleting its C-terminal residues. Computational analysis showed that C-terminal residues regulate the position of the active site loop. C-terminal deletions of gs-MDH successfully increased Ki values by 5- to 8-fold with maintained thermal stability (>90% of the wild-type enzyme), although kcat/Km values were decreased by < 2-fold...

Steroid sulfate esters are important metabolites for anti-doping efforts in sports, pathology, and research. Analysis of these metabolites is facilitated by hydrolysis using either acid or enzymatic catalysis. Although enzymatic hydrolysis is preferred for operating at neutral pH, no known enzyme is capable of hydrolyzing all steroid sulfate metabolites. Pseudomonas aeruginosa arylsulfatase (PaS) is ideal for the hydrolysis of β-configured steroid sulfates but like other known class I sulfatases it is inefficient at hydrolyzing α-configured steroid sulfates...