Novel forms of chemical protein diversity -- in nature and in the laboratory

Novel chemical variants of proteins have been found in nature, including potent 'microprotein' natural products and folded protein molecules that contain a cyclic polypeptide chain. Researchers have used chemical synthesis and genetic methods to make these proteins and more: protein catenanes, neoglycoproteins, and artificial protein molecules with novel architectures or made from novel building blocks. De novo design has taken a big step forward with the accurate design and construction of proteins with complex molecular structure. A variety of non-coded amino acids and other building blocks has been used to make increasingly sophisticated protein molecular devices for use as biosensors and for the study of signal transduction inside living cells.     

Deep generative modeling for protein design

Deep learning approaches have produced substantial breakthroughs in fields such as image classification and natural language processing and are making rapid inroads in the area of protein design. Many generative models of proteins have been developed that encompass all known protein sequences, model specific protein families, or extrapolate the dynamics of individual proteins. Those generative models can learn protein representations that are often more informative of protein structure and function than hand-engineered features. Furthermore, they can be used to quickly propose millions of novel proteins that resemble the native counterparts in terms of expression level, stability, or other attributes. The protein design process can further be guided by discriminative oracles to select candidates with the highest probability of having the desired properties. In this review, we discuss five classes of generative models that have been most successful at modeling proteins and provide a framework for model guided protein design.     

Computational design of membrane proteins

Membrane proteins are involved in a wide variety of cellular processes, and are typically part of the first interaction a cell has with extracellular molecules. As a result, these proteins comprise a majority of known drug targets. Membrane proteins are among the most difficult proteins to obtain and characterize, and a structure-based understanding of their properties can be difficult to elucidate. Notwithstanding, the design of membrane proteins can provide stringent tests of our understanding of these crucial biological systems, as well as introduce novel or targeted functionalities. Computational design methods have been particularly helpful in addressing these issues, and this review discusses recent studies that tailor membrane proteins to display specific structures or functions and examines how redesigned membrane proteins are being used to facilitate structural and functional studies.     

Sane in the membrane: designing systems to modulate membrane proteins

Proteins and lipids make sense in rational approaches to the design of systems for the study of membrane proteins. Lipids surround integral membrane proteins in their natural environment. Although lipids have always formed part of investigations into membrane proteins, it has generally been the proteins themselves that have taken the limelight. As knowledge of membrane proteins has increased, so has that of their interactions with lipids. This increased understanding of the interplay of proteins and lipids, together with existing knowledge of lipid properties, is enabling new approaches to be introduced for membrane protein study. The lipids can be used to control protein behaviour and as novel probes of protein motion.     

DARPins and other repeat protein scaffolds: advances in engineering and applications

Antibodies have long been regarded as the only class of binding proteins. With the emergence of protein engineering techniques, new binding proteins based on alternative scaffolds have been designed. Additionally, modern technologies for selection and evolution from libraries are independent of the antibody scaffold and could thus be readily used for obtaining specific binding proteins. One important group of alternative scaffolds is based on repeat proteins. Nature is widely using these proteins to modulate protein-protein interactions, and even in the adaptive immune system of jawless vertebrates; the step to their application as an alternative to antibodies seems therefore logical. In this review, progress on DARPins and other repeat protein scaffolds will be discussed. Advances in their design as well as novel applications will be highlighted.     

Principles of the design and operational considerations of large scale high performance liquid chromatography (HPLC) systems for proteins and peptides purification.

This review introduces concepts of design of large scale HPLC systems for purification of proteins and peptides. It is addressed to users of large scale HPLC systems to aid in system selection and help in customizing the design. Major techniques used for large scale HPLC purification of proteins and peptides are briefly reviewed. Engineering aspects of system design are discussed in detail. The review of selected relevant literature is provided as well as author's experience with the existing designs and his own systems. Commercial publications have been used in preparation of this review but only the most significant are listed as examples. The design process for any new system should be related to the performance of existing systems, if possible of a large scale. Laboratory data are also very useful in aiding the design process since they provide a lead, as to which chromatography techniques may succeed in providing required separation levels. The expertise needed for system design and operation comes from many areas: protein and peptide chemistry, chromatographic theory, mass transfer and hydrodynamics, machine design and material science. All these factors have to be blended together during the system design process. The controls must ensure flexibility in adapting to changing system configuration, depending on the operational requirements for various products. Extensive interfacing with the operator during the process run is essential. This work is focused mostly on system design and operation for reversed-phase chromatography and hydrophobic interaction chromatography, but it also covers aspects associated with other chromatographic techniques. The reviewed design principles would also apply to design other than HPLC large scale chromatography systems for biotechnology and pharmaceutical operations.     

Design and discovery of metamorphic proteins

Metamorphic proteins are single amino acid sequences that reversibly interconvert between multiple, dramatically different native structures, often with distinct functions. Since the discovery of the first metamorphic proteins in the early 2000s, several additional metamorphic proteins have been identified, and it was suggested that up to 4% of proteins in the PDB may switch folds. Metamorphic proteins have been found to share common features such as marginal thermostability and inconsistencies in predicted secondary structures. Outstanding challenges in the field include the search for more metamorphic proteins and the design of new proteins that switch folds. Identification of novel metamorphic proteins in nature will improve therapeutic targeting of fold-switching proteins involved in human pathology and will enhance the design of protein-based therapies. Designed fold switching proteins have applications as biosensors, molecular switches, molecular machines, and self-assembling systems.     

Structural basis of the herpesvirus M3-chemokine interaction

Viruses have been fighting the immune systems of their hosts for millions of years and have evolved evasion strategies to ensure their survival. Viruses can teach us efficient mechanisms to control the immune system, and this information can be used to design new strategies of immune modulation that we might apply to diminish immunopathological responses that cause human diseases. Large DNA viruses, such as poxviruses and herpesviruses, encode proteins that are secreted from infected cells, bind cytokines and neutralize their activity. A subgroup of these viral proteins binds chemokines, a complex family of cytokines that control the recruitment of cells to sites of infection and inflammation. One of the major unresolved questions in the field was to understand how these viral secreted proteins bind chemokines with high affinity, despite having no amino acid sequence similarity to the host chemokine receptors, which are seven-transmembrane-domain proteins that cannot be engineered as soluble proteins.     

Engineering proteins for thermostability: the use of sequence alignments versus rational design and directed evolution

With the advent of directed evolution techniques, protein engineering has received a fresh impetus. Engineering proteins for thermostability is a particularly exciting and challenging field, as it is crucial for broadening the industrial use of recombinant proteins. In addition to directed evolution, a variety of partially successful rational concepts for engineering thermostability have been developed in the past. Recent results suggest that amino acid sequence comparisons of mesophilic proteins alone can be used efficiently to engineer thermostable proteins. The potential benefits of the underlying, semirational 'consensus concept' are compared with those of rational design and directed evolution approaches.     

Towards structure-based protein drug design

Structure-based drug design for chemical molecules has been widely used in drug discovery in the last 30 years. Many successful applications have been reported, especially in the field of virtual screening based on molecular docking. Recently, there has been much progress in fragment-based as well as de novo drug discovery. As many protein-protein interactions can be used as key targets for drug design, one of the solutions is to design protein drugs based directly on the protein complexes or the target structure. Compared with protein-ligand interactions, protein-protein interactions are more complicated and present more challenges for design. Over the last decade, both sampling efficiency and scoring accuracy of protein-protein docking have increased significantly. We have developed several strategies for structure-based protein drug design. A grafting strategy for key interaction residues has been developed and successfully applied in designing erythropoietin receptor-binding proteins. Similarly to small-molecule design, we also tested de novo protein-binder design and a virtual screen of protein binders using protein-protein docking calculations. In comparison with the development of structure-based small-molecule drug design, we believe that structure-based protein drug design has come of age.     

Principles and Methods in Computational Membrane Protein Design

After decades of progress in computational protein design, the design of proteins folding and functioning in lipid membranes appears today as the next frontier. Some notable successes in the de novo design of simplified model membrane protein systems have helped articulate fundamental principles of protein folding, architecture and interaction in the hydrophobic lipid environment. These principles are reviewed here, together with the computational methods and approaches that were used to identify them. We provide an overview of the methodological innovations in the generation of new protein structures and functions and in the development of membrane-specific energy functions. We highlight the opportunities offered by new machine learning approaches applied to protein design, and by new experimental characterization techniques applied to membrane proteins. Although membrane protein design is in its infancy, it appears more reachable than previously thought.     

Modeling backbone flexibility to achieve sequence diversity: the design of novel alpha-helical ligands for Bcl-xL

Computational protein design can be used to select sequences that are compatible with a fixed-backbone template. This strategy has been used in numerous instances to engineer novel proteins. However, the fixed-backbone assumption severely restricts the sequence space that is accessible via design. For challenging problems, such as the design of functional proteins, this may not be acceptable. Here, we present a method for introducing backbone flexibility into protein design calculations and apply it to the design of diverse helical BH3 ligands that bind to the anti-apoptotic protein Bcl-xL, a member of the Bcl-2 protein family. We demonstrate how normal mode analysis can be used to sample different BH3 backbones, and show that this leads to a larger and more diverse set of low-energy solutions than can be achieved using a native high-resolution Bcl-xL complex crystal structure as a template. We tested several of the designed solutions experimentally and found that this approach worked well when normal mode calculations were used to deform a native BH3 helix structure, but less well when they were used to deform an idealized helix. A subsequent round of design and testing identified a likely source of the problem as inadequate sampling of the helix pitch. In all, we tested 17 designed BH3 peptide sequences, including several point mutants. Of these, eight bound well to Bcl-xL and four others showed weak but detectable binding. The successful designs showed a diversity of sequences that would have been difficult or impossible to achieve using only a fixed backbone. Thus, introducing backbone flexibility via normal mode analysis effectively broadened the set of sequences identified by computational design, and provided insight into positions important for binding Bcl-xL.     

Patch engineering: a general approach for creating proteins that have new binding activities

Patch engineering is a technique for creating folded proteins that have new binding activities. Different protein scaffolds are used to present a patch of discontinuous residues on a folded-protein surface. By varying simultaneously the residues in these patches and displaying these mutant proteins on phage, one can select proteins that have new binding activities. Patch engineering is applicable to any protein fold. Novel proteins derived by this approach might replace antibodies in certain applications or provide lead molecules for the design of non-peptide analogues.     

四种结构类型的蛋白质设计方法

给出了以疏水一亲水模型为基础的蛋白质设计方法,该方法以物理学原理为基础,以相对熵作为优化的目标函数。对四种不同结构类型的天然结构的真实蛋白质进行了检测,分析了影响检测成功率的主要因素,结果表明,该方法是普适的,可用于对不同结构类型的蛋白质设计序列。     

Computer-aided design of a PDZ domain to recognize new target sequences

PDZ domains are small globular domains that recognize the last 4-7 amino acids at the C-terminus of target proteins. The specificity of the PDZ-ligand recognition is due to side chain-side chain interactions, as well as the positioning of an alpha-helix involved in ligand binding. We have used computer-aided protein design to produce mutant versions of a Class I PDZ domain that bind to novel Class I and Class II target sequences both in vitro and in vivo, thus providing an alternative to primary antibodies in western blotting, affinity chromatography and pull-down experiments. Our results suggest that by combining different backbone templates with computer-aided protein design, PDZ domains could be engineered to specifically recognize a large number of proteins.     

Fcγ1 fragment of IgG1 as a powerful affinity tag in recombinant Fc-fusion proteins: immunological,biochemical and therapeutic properties

Affinity tags are vital tools for the production of high-throughput recombinant proteins. Several affinity tags, such as the hexahistidine tag, maltose-binding protein, streptavidin-binding peptide tag, calmodulin-binding peptide, c-Myc tag, glutathione S-transferase and FLAG tag, have been introduced for recombinant protein production. The fragment crystallizable (Fc) domain of the IgG1 antibody is one of the useful affinity tags that can facilitate detection, purification and localization of proteins and can improve the immunogenicity, modulatory effects, physicochemical and pharmaceutical properties of proteins. Fcγ recombinant forms a group of recombinant proteins called Fc-fusion proteins (FFPs). FFPs are widely used in drug discovery, drug delivery, vaccine design and experimental research on receptor–ligand interactions. These fusion proteins have become successful alternatives to monoclonal antibodies for drug developments. In this review, the physicochemical, biochemical, immunological, pharmaceutical and therapeutic properties of recombinant FFPs were discussed as a new generation of bioengineering strategies.     

A large scale test of computational protein design: folding and stability of nine completely redesigned globular proteins

A previously developed computer program for protein design, RosettaDesign, was used to predict low free energy sequences for nine naturally occurring protein backbones. RosettaDesign had no knowledge of the naturally occurring sequences and on average 65% of the residues in the designed sequences differ from wild-type. Synthetic genes for ten completely redesigned proteins were generated, and the proteins were expressed, purified, and then characterized using circular dichroism, chemical and temperature denaturation and NMR experiments. Although high-resolution structures have not yet been determined, eight of these proteins appear to be folded and their circular dichroism spectra are similar to those of their wild-type counterparts. Six of the proteins have stabilities equal to or up to 7kcal/mol greater than their wild-type counterparts, and four of the proteins have NMR spectra consistent with a well-packed, rigid structure. These encouraging results indicate that the computational protein design methods can, with significant reliability, identify amino acid sequences compatible with a target protein backbone.     

De novo design of the hydrophobic core of ubiquitin.

We have previously reported the development and evaluation of a computational program to assist in the design of hydrophobic cores of proteins. In an effort to investigate the role of core packing in protein structure, we have used this program, referred to as Repacking of Cores (ROC), to design several variants of the protein ubiquitin. Nine ubiquitin variants containing from three to eight hydrophobic core mutations were constructed, purified, and characterized in terms of their stability and their ability to adopt a uniquely folded native-like conformation. In general, designed ubiquitin variants are more stable than control variants in which the hydrophobic core was chosen randomly. However, in contrast to previous results with 434 cro, all designs are destabilized relative to the wild-type (WT) protein. This raises the possibility that beta-sheet structures have more stringent packing requirements than alpha-helical proteins. A more striking observation is that all variants, including random controls, adopt fairly well-defined conformations, regardless of their stability. This result supports conclusions from the cro studies that non-core residues contribute significantly to the conformational uniqueness of these proteins while core packing largely affects protein stability and has less impact on the nature or uniqueness of the fold. Concurrent with the above work, we used stability data on the nine ubiquitin variants to evaluate and improve the predictive ability of our core packing algorithm. Additional versions of the program were generated that differ in potential function parameters and sampling of side chain conformers. Reasonable correlations between experimental and predicted stabilities suggest the program will be useful in future studies to design variants with stabilities closer to that of the native protein. Taken together, the present study provides further clarification of the role of specific packing interactions in protein structure and stability, and demonstrates the benefit of using systematic computational methods to predict core packing arrangements for the design of proteins.     

Computer assisted simulations and molecular graphics methods in molecular design 2. Proteinases and receptor and transport proteins

A survey is presented of model building techniques and computer-assisted quantum-chemical and molecular-dynamics simulations, as applied to the study of protease and receptor design, to the determination of the properties of related effectors, agonist and antagonist molecules, and to the design of fatty-acid transport proteins and molecular carriers. Studies covering integral membrane protein design have been reviewed: they include porins, ion channels and G protein coupled receptors. In the transport molecule class, hydrophobic ligand transporters, such as serum and cellular retinoid binding proteins, have been reviewed.