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1.
Protein sequence design with a learned potential.
Anand, N, Eguchi, R, Mathews, II, Perez, CP, Derry, A, Altman, RB, Huang, PS
Nature communications. 2022;(1):746
Abstract
The task of protein sequence design is central to nearly all rational protein engineering problems, and enormous effort has gone into the development of energy functions to guide design. Here, we investigate the capability of a deep neural network model to automate design of sequences onto protein backbones, having learned directly from crystal structure data and without any human-specified priors. The model generalizes to native topologies not seen during training, producing experimentally stable designs. We evaluate the generalizability of our method to a de novo TIM-barrel scaffold. The model produces novel sequences, and high-resolution crystal structures of two designs show excellent agreement with in silico models. Our findings demonstrate the tractability of an entirely learned method for protein sequence design.
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2.
Gene Targeting Facilitated by Engineered Sequence-Specific Nucleases: Potential Applications for Crop Improvement.
Miki, D, Wang, R, Li, J, Kong, D, Zhang, L, Zhu, JK
Plant & cell physiology. 2021;(5):752-765
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Abstract
Humans are currently facing the problem of how to ensure that there is enough food to feed all of the world's population. Ensuring that the food supply is sufficient will likely require the modification of crop genomes to improve their agronomic traits. The development of engineered sequence-specific nucleases (SSNs) paved the way for targeted gene editing in organisms, including plants. SSNs generate a double-strand break (DSB) at the target DNA site in a sequence-specific manner. These DSBs are predominantly repaired via error-prone non-homologous end joining and are only rarely repaired via error-free homology-directed repair if an appropriate donor template is provided. Gene targeting (GT), i.e. the integration or replacement of a particular sequence, can be achieved with combinations of SSNs and repair donor templates. Although its efficiency is extremely low, GT has been achieved in some higher plants. Here, we provide an overview of SSN-facilitated GT in higher plants and discuss the potential of GT as a powerful tool for generating crop plants with desirable features.
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3.
Improving photosynthesis through the enhancement of Rubisco carboxylation capacity.
Iñiguez, C, Aguiló-Nicolau, P, Galmés, J
Biochemical Society transactions. 2021;(5):2007-2019
Abstract
Rising human population, along with the reduction in arable land and the impacts of global change, sets out the need for continuously improving agricultural resource use efficiency and crop yield (CY). Bioengineering approaches for photosynthesis optimization have largely demonstrated the potential for enhancing CY. This review is focused on the improvement of Rubisco functioning, which catalyzes the rate-limiting step of CO2 fixation required for plant growth, but also catalyzes the ribulose-bisphosphate oxygenation initiating the carbon and energy wasteful photorespiration pathway. Rubisco carboxylation capacity can be enhanced by engineering the Rubisco large and/or small subunit genes to improve its catalytic traits, or by engineering the mechanisms that provide enhanced Rubisco expression, activation and/or elevated [CO2] around the active sites to favor carboxylation over oxygenation. Recent advances have been made in the expression, assembly and activation of foreign (either natural or mutant) faster and/or more CO2-specific Rubisco versions. Some components of CO2 concentrating mechanisms (CCMs) from bacteria, algae and C4 plants has been successfully expressed in tobacco and rice. Still, none of the transformed plant lines expressing foreign Rubisco versions and/or simplified CCM components were able to grow faster than wild type plants under present atmospheric [CO2] and optimum conditions. However, the results obtained up to date suggest that it might be achievable in the near future. In addition, photosynthetic and yield improvements have already been observed when manipulating Rubisco quantity and activation degree in crops. Therefore, engineering Rubisco carboxylation capacity continues being a promising target for the improvement in photosynthesis and yield.
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4.
Antisense Peptide Technology for Diagnostic Tests and Bioengineering Research.
Štambuk, N, Konjevoda, P, Pavan, J
International journal of molecular sciences. 2021;(17)
Abstract
Antisense peptide technology (APT) is based on a useful heuristic algorithm for rational peptide design. It was deduced from empirical observations that peptides consisting of complementary (sense and antisense) amino acids interact with higher probability and affinity than the randomly selected ones. This phenomenon is closely related to the structure of the standard genetic code table, and at the same time, is unrelated to the direction of its codon sequence translation. The concept of complementary peptide interaction is discussed, and its possible applications to diagnostic tests and bioengineering research are summarized. Problems and difficulties that may arise using APT are discussed, and possible solutions are proposed. The methodology was tested on the example of SARS-CoV-2. It is shown that the CABS-dock server accurately predicts the binding of antisense peptides to the SARS-CoV-2 receptor binding domain without requiring predefinition of the binding site. It is concluded that the benefits of APT outweigh the costs of random peptide screening and could lead to considerable savings in time and resources, especially if combined with other computational and immunochemical methods.
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5.
De novo sequence redesign of a functional Ras-binding domain globally inverted the surface charge distribution and led to extreme thermostability.
Liu, R, Wang, J, Xiong, P, Chen, Q, Liu, H
Biotechnology and bioengineering. 2021;(5):2031-2042
Abstract
To acquire extremely thermostable proteins of given functions is challenging for conventional protein engineering. Here we applied ABACUS, a statistical energy function we developed for de novo amino acid sequence design, to globally redesign a Ras-binding domain (RBD), and obtained an extremely thermostable RBD that unfolds reversibly at above 110°C, the redesigned RBD experimentally confirmed to have expected structure and Ras-binding interface. Directed evolution of the redesigned RBD improved its Ras-binding affinity to the native protein level without excessive loss of thermostability. The designed amino acid substitutions were mostly at the protein surface. For many substitutions, strong epistasis or significantly differentiated effects on thermostability in the native sequence context relative to the redesigned sequence context were observed, suggesting the globally redesigned sequence to be unreachable through combining beneficial mutations of the native sequence. Further analyses revealed that by replacing 38 of a total of 48 non-interfacial surface residues at once, ABACUS redesign was able to globally "invert" the protein's charge distribution pattern in an optimized way. Our study demonstrates that computational protein design provides powerful new tools to solve challenging protein engineering problems.
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6.
Recapitulation of selective nuclear import and export with a perfectly repeated 12mer GLFG peptide.
Ng, SC, Güttler, T, Görlich, D
Nature communications. 2021;(1):4047
Abstract
The permeability barrier of nuclear pore complexes (NPCs) controls nucleocytoplasmic transport. It retains inert macromolecules while allowing facilitated passage of importins and exportins, which in turn shuttle cargo into or out of cell nuclei. The barrier can be described as a condensed phase assembled from cohesive FG repeat domains. NPCs contain several distinct FG domains, each comprising variable repeats. Nevertheless, we now found that sequence heterogeneity is no fundamental requirement for barrier function. Instead, we succeeded in engineering a perfectly repeated 12mer GLFG peptide that self-assembles into a barrier of exquisite transport selectivity and fast transport kinetics. This barrier recapitulates RanGTPase-controlled importin- and exportin-mediated cargo transport and thus represents an ultimately simplified experimental model system. An alternative proline-free sequence forms an amyloid FG phase. Finally, we discovered that FG phases stain bright with 'DNA-specific' DAPI/ Hoechst probes, and that such dyes allow for a photo-induced block of nuclear transport.
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7.
De Novo-Designed β-Sheet Heme Proteins.
D'Souza, A, Bhattacharjya, S
Biochemistry. 2021;(6):431-439
Abstract
The field of de novo protein design has met with considerable success over the past few decades. Heme, a cofactor, has often been introduced to impart a diverse array of functions to a protein, ranging from electron transport to respiration. In nature, heme is found to occur predominantly in α-helical structures over β-sheets, which has resulted in significant designs of heme proteins utilizing coiled-coil helices. By contrast, there are only a few known β-sheet proteins that bind heme and designs of β-sheets frequently result in amyloid-like aggregates. This review reflects on our success in designing a series of multistranded β-sheet heme binding peptides that are well folded in both aqueous and membrane-like environments. Initially, we designed a β-hairpin peptide that self-assembles to bind heme and performs peroxidase activity in membrane. The β-hairpin was optimized further to accommodate a heme binding pocket within multistranded β-sheets for catalysis and electron transfer in membranes. Furthermore, we de novo designed and characterized β-sheet peptides and miniproteins that are soluble in an aqueous environment capable of binding single and multiple hemes with high affinity and stability. Collectively, these studies highlight the substantial progress made toward the design of functional β-sheets.
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8.
Structural analysis of the β-sheet edge of peptide self-assembly using a model protein.
Shiga, S, Makabe, K
Proteins. 2021;(7):845-852
Abstract
Peptides and proteins self-assemble into β-sheet-rich fibrils, amyloid, which extends its structure by incorporating peptide/protein molecules from solution. At the elongation edge, the peptide/protein molecule binds to the edge of the amyloid β-sheet. Such processes are transient and elusive when observing molecular details by experimental methods. We used a model protein system, peptide self-assembly mimic (PSAM), which mimics an amyloid-like structure within a globular protein by capping both edges of single-layer β sheet (SLB) with certain domains. We constructed a PSAM variant that lacks the capping domain on the C-terminal side to observe the structure of the β-sheet edge of the peptide self-assembly. This variant, which we termed PSAM-edge, proved to be soluble with a monomeric form. Urea-induced unfolding experiments revealed that PSAM-edge displayed two-state cooperative unfolding, indicating the N-terminal capping domain and extended SLB folded as one unit. The crystal structure showed that SLB was almost completely structured except for a few terminal residues. A molecular dynamics simulation results revealed that the SLB structure was retained while the C-terminal four residues fluctuated, which was consistent with the crystal structure. Our findings indicate that SLB is stable even when one side of the β-sheet edge is exposed to a solvent. This stability may prevent the dissociation of the attached peptide from the peptide self-assembly. Because of the scarcity of SLB proteins with exposed β-sheet edges in nature, successful construction of the PSAM-edge expands our understanding of protein folding and design.
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9.
Site-Selective, Chemical Modification of Protein at Aromatic Side Chain and Their Emergent Applications.
Chowdhury, A, Chatterjee, S, Pongen, A, Sarania, D, Tripathi, NM, Bandyopadhyay, A
Protein and peptide letters. 2021;(7):788-808
Abstract
Site-selective chemical modification of protein side chain has probed enormous opportunities in the fundamental understanding of cellular biology and therapeutic applications. Primarily, in the field of biopharmaceuticals, the formulation of bioconjugates has been found to have more potential than an individual constituent. In this regard, Lysine and Cysteine are the most widely used endogenous amino acid for these purposes. Recently, the aromatic side chain residues (Trp, Tyr, and His) that are low abundant in protein have gained more attention in therapeutic applications due to their advantages of chemical reactivity and specificity. This review discusses the site-selective bioconjugation methods for aromatic side chains (Trp, Tyr and His) and highlights the developed strategies in the last three years, along with their applications. Also, the review highlights the prevalent methods published earlier. We have examined that metal-catalyzed and photocatalytic reactions are gaining more attention for bioconjugation, though their practical operation is under development. The review has been summarized with the future perspective of protein and peptide conjugations contemplating therapeutic applications and challenges.
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10.
Structure-based design of a photoswitchable affibody scaffold.
Woloschuk, RM, Reed, PMM, Jaikaran, ASI, Demmans, KZ, Youn, J, Kanelis, V, Uppalapati, M, Woolley, GA
Protein science : a publication of the Protein Society. 2021;(12):2359-2372
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Abstract
Photo-control of affinity reagents offers a general approach for high-resolution spatiotemporal control of diverse molecular processes. In an effort to develop general design principles for a photo-controlled affinity reagent, we took a structure-based approach to the design of a photoswitchable Z-domain, among the simplest of affinity reagent scaffolds. A chimera, designated Z-PYP, of photoactive yellow protein (PYP) and the Z-domain, was designed based on the concept of mutually exclusive folding. NMR analysis indicated that, in the dark, the PYP domain of the chimera was folded, and the Z-domain was unfolded. Blue light caused loss of structure in PYP and a two- to sixfold change in the apparent affinity of Z-PYP for its target as determined using size exclusion chromatography, UV-Vis based assays, and enyzme-linked immunosorbent assay (ELISA). A thermodynamic model indicated that mutations to decrease Z-domain folding energy would alter target affinity without loss of switching. This prediction was confirmed experimentally with a double alanine mutant in helix 3 of the Z-domain of the chimera (Z-PYP-AA) showing >30-fold lower dark-state binding and no loss in switching. The effect of decreased dark-state binding affinity was tested in a two-hybrid transcriptional control format and enabled pronounced light/dark differences in yeast growth in vivo. Finally, the design was transferable to the αZ-Taq affibody enabling tunable light-dependent binding both in vitro and in vivo to the Z-Taq target. This system thus provides a framework for the focused development of light switchable affibodies for a range of targets.