Protein design

Protein design is the rational design of new protein molecules to design novel activity, behavior, or purpose, and to advance basic understanding of protein function. Proteins can be designed from scratch (de novo design) or by making calculated variants of a known protein structure and its sequence (termed protein redesign). Rational protein design approaches make protein-sequence predictions that will fold to specific structures. These predicted sequences can then be validated experimentally through methods such as peptide synthesis, site-directed mutagenesis, or artificial gene synthesis. Rational protein design dates back to the mid-1970s. Recently, however, there were numerous examples of successful rational design of water-soluble and even transmembrane peptides and proteins, in part due to a better understanding of different factors contributing to protein structure stability and development of better computational methods. Overview and history The goal in rational pr

Integrating Gene Synthesis and MITOMI for Rapid Protein Engineering

Matthew Christopher Blackburn

The research described within this thesis is primarily motivated by two fields of science with reversed, yet complementary, approaches to addressing the same essential objective: manipulating and understanding the complex program of life. Whereas synthetic biology has to do with the bottom-up construction of living systems from a library of âpartsâ, systems biology takes a top-down, reverse-engineering analysis of the same processes within functioning cells to elucidate the crucial elements and interactions. Both perspectives have led to important contributions in learning how biological systems operate. Discoveries from these studies can have far-reaching implications, notably in medicine, manufacturing, energy, and the environment. One exciting outcome of the research efforts within synthetic biology is the ability to design genetic constructs encoding proteins with novel, optimized functions. Although advances have been made in predicting how a proteinâs amino acid sequence relates to its higher order structural form and behavior, experimental methods for protein engineering remain invaluable for evaluating computationally derived designs. Unfortunately, the design-build-test cycle for rational protein development is commonly a time, labor and resource intensive endeavor. This is primarily due to limitations in the ability to generate libraries of gene variants and bottlenecks in cell-based expression and quantitative screening of protein products. This thesis describes the development of a solid-phase gene assembly technique and its integration with microfluidic protein analysis to accelerate the prototyping of novel protein designs. The gene assembly method utilizes commodity-scale, chemically manufactured DNA, and operates without the need for ligase or restriction enzymes. As a bench top process amenable to scale up, the modularity of the assembly process allows the efficient and economical generation of expression-ready gene variants. We directly coupled this gene assembly pipeline to a microfluidic device to enable high-throughput, on-chip, cell-free protein expression, purification, and characterization. By circumventing molecular cloning and cell-based steps, the lag time between protein design and quantitative analysis was dramatically reduced. As a proof of concept, this protein engineering platform was applied towards the construction of over 400 artificial, engineered variants of C2H2 zinc finger (ZF) proteins. ZF protein domains are the most prevalent form of transcription factor (TF) in humans, and are found throughout the tree of life. They provide a convenient structure for refactoring due to their relatively small size and composability, and are an ideal model for exploring the biophysics of TF-DNA specificity. The ability to engineer ZF proteins makes them useful as programmable, DNA-targeting units for applications in biotechnology and synthetic biology. We demonstrate that although ZFPs can be readily engineered to recognize a particular DNA target, engineering the precise binding energy landscape remains a challenge. Additionally, we show that ZF-DNA binding affinity can be tuned independently of sequence specificity. Together, these results demonstrate the versatility of the coupled gene assembly and microfluidic analysis platform as a new tool for rational protein engineering.

EPFL2016

Explicit solvent effects on protein physics - modeling lattice proteins in aqueous environments

Protein folding stands as one of the major interdisciplinary challenges of the last ten years, involving biology, chemistry, medicine and physics. In this PhD thesis, we investigate the solvent effects and the resulting hydrophobic effect on proteins. In particular, we focus on a simple lattice model of proteins (the HPW model), where the solvent is (semi)explicitly taken into account (by means of the model of Muller, Lee and Graziano (MLG)), investigating thermodynamical quantities of the modeled proteins and comparing the results with those of real proteins. We approach the protein design problem using the HPW model and we investigate some statistical and thermodynamic properties of the designed sequences. In particular we show that the HPW model is able to capture, within a single framework, both warm and cold denaturation of proteins. We further adapt the MLG model model to include chaotrope cosolvents in the solution. We show that the alteration of the network of hydrogen bonds between water molecules due to the presence of chaotropic agents induces destabilization of proteins. Moreover, we find that the presence of chaotropes originates an effective interaction between the chaotropes and the protein. In addition, we use a common method to extract effective amino-acid interactions on real proteins: a perceptron algorithm. The method applied to proteins designed with the HPW model reveals that the solvent effects can not be reproduced by effective residue interactions. Indeed, we show that it is not possible to determine a set of global interaction values able to stabilize all proteins at once.

EPFL2004

Integrating Gene Synthesis and MITOMI for Rapid Protein Engineering

Matthew Christopher Blackburn

EPFL2016

Protein design: progress toward the synthesis of a 16-residue peptide designed to contain an N-terminal cystein and to form an amphiphilic alpha-helix.

Integrating Gene Synthesis and MITOMI for Rapid Protein Engineering

Explicit solvent effects on protein physics - modeling lattice proteins in aqueous environments

Protein design: progress toward the synthesis of a 16-residue peptide designed to contain an N-terminal cystein and to form an amphiphilic alpha-helix.

Explicit solvent effects on protein physics - modeling lattice proteins in aqueous environments

Integrating Gene Synthesis and MITOMI for Rapid Protein Engineering