Homology-independent protein engineering

Is structure, rather than sequence, the key to the successful generation of truly novel proteins? While protein evolution by homologous recombination has become an established tool to explore confined regions in sequence space, the generation of functional hybrid proteins by homology-independent methods further expands the scope of protein engineering.     

Active-site assembly in glutaminyl-tRNA synthetase by tRNA-mediated induced fit

Structure-based mutational analysis was employed to probe an unusual intramolecular interaction between partially buried glutamate residues adjacent to the active site of Escherichia coli glutaminyl-tRNA synthetase (GlnRS). The crystal structures of unliganded GlnRS and the GlnRS-tRNA(Gln) complex reveal that the Glu34 and Glu73 side chain carboxylates contact each other only in the tRNA-bound state and that the interaction is formed via mutual induced-fit transitions that occur en route to the ground-state Michaelis complex. Steady-state and transient kinetic analysis of mutant enzymes suggest that the formation of this intermolecular contact is a key event that facilitates the proper formation of the active site. Mutants at both positions destabilize the binding of the substrate glutamine at the opposite side of the active-site cleft, whereas Glu73 appears to play an additional important role by promoting the correct binding of the 3'-acceptor end of tRNA adjacent to both ATP and glutamine. The data suggest the existence of multiple structural pathways by which the binding of tRNA propagates conformational transitions leading to the proper formation of the glutamine binding site. The single-turnover kinetic analysis also establishes that the Glu34 carboxylate does not play a direct enzymatic role as a catalytic base to help deprotonate the tRNA-A76 nucleophilic 2'-hydroxyl group. The elimination of this previously proposed mechanism, together with recent chemical modification experiments in the histidyl-tRNA synthetase system, emphasizes that substrate-assisted catalysis by the phosphate of the aminoacyl adenylate may be a common means by which all tRNA synthetases facilitate the aminoacyl transfer step of the reaction.     

Computer-aided design in protein engineering

The amino acid sequence of a protein can be engineered genetically to yield a molecule with modified or novel properties of clinical or industrial importance, through knowledge of the relationships between sequence, three-dimensional structure and function. Interactive computer graphics can display and model the structural information revealed by protein cyrstallography. In the absence of a suitable crystal structure, computer methods must predict protein conformation from sequence. The most powerful approaches for structure prediction are based on sequence homology or a more general analogy with known crystal structures. Improvements in these methods will require access to data bases containing the basic motifs of protein architecture.     

Non-canonical amino acids in protein engineering

Methods for engineering proteins that contain non-canonical amino acids have advanced rapidly in the past few years. Novel amino acids can be introduced into recombinant proteins in either a residue-specific or site-specific fashion. The methods are complementary: residue-specific incorporation allows engineering of the overall physical and chemical behavior of proteins and protein-like macromolecules, whereas site-specific methods allow mechanistic questions to be probed in atomistic detail. Challenges remain in the engineering of the translational apparatus and in the design of schemes that can be used to encode both canonical and non-canonical amino acids.     

Flexible genetic engineering using RecA protein

With the explosion in genetic information and almost complete sequencing of the human genome, a shift in the experimental goals of molecular biologists is occurring. Instead of focusing on single genes, current attempts seek to divine the interactions of several genes and sequences. This requires increasingly complex genetic constructs and manipulations, often of very large DNA constructs, and these can be made with RecA protein-based techniques. When RecA protein combined with an oligonucleotide acts as a sequence-specific "masking tape" to block DNA from the action of DNA modifying enzymes, and can be used to direct the cleavage of DNA at single predetermined restriction endonuclease sites. This reaction is called RecA-Assisted Restriction Endonuclease (RARE) Cleavage. The reverse reaction, known as RecA-Assisted Ligation, can be used to join any two desired fragments. When one of those fragments is a vector, a desired fragment can be cloned directly without constructing a genomic library. The reagents and equipment needed are relatively inexpensive, and almost any desired genetic construct up to about 300 kb in size can be made in a straightforward manner.     

Cell-free translation systems for protein engineering

Cell-free translation systems have developed significantly over the last two decades and improvements in yield have resulted in their use for protein production in the laboratory. These systems have protein engineering applications, such as the production of proteins containing unnatural amino acids and development of proteins exhibiting novel functions. Recently, it has been suggested that cell-free translation systems might be used as the fundamental basis for cell-like systems. We review recent progress in the field of cell-free translation systems and describe their use as tools for protein production and engineering.     

Putting engineering back into protein engineering: bioinformatic approaches to catalyst design

Complex multivariate engineering problems are commonplace and not unique to protein engineering. Mathematical and data-mining tools developed in other fields of engineering have now been applied to analyze sequence-activity relationships of peptides and proteins and to assist in the design of proteins and peptides with specified properties. Decreasing costs of DNA sequencing in conjunction with methods to quickly synthesize statistically representative sets of proteins allow modern heuristic statistics to be applied to protein engineering. This provides an alternative approach to expensive assays or unreliable high-throughput surrogate screens.     

Rational protein crystallization by mutational surface engineering

Protein crystallization constitutes a limiting step in structure determination by X-ray diffraction. Even if single crystals are available, inadequate physical quality may seriously limit the resolution of the available data and consequently the accuracy of the atomic model. Recent studies show that targeted mutagenesis of surface patches containing residues with large flexible side chains and their replacement with smaller amino acids lead to effective preparation of X-ray quality crystals of proteins otherwise recalcitrant to crystallization. Furthermore, this technique can also be used to obtain crystals of superior quality as compared to those grown for the wild-type protein, sometimes increasing the effective resolution by as much as 1 A or more. Several recent examples of this new methodology suggest that the method has the potential to become a routine tool in protein crystallography.     

Structure-based combinatorial protein engineering (SCOPE)

Presented here is the development a semi-rational protein engineering approach that uses information from protein structure coupled with established DNA manipulation techniques to design and create multiple crossover libraries from non-homologous genes. The utility of structure-based combinatorial protein engineering (SCOPE) was demonstrated by its application to two distantly related members of the X-family of DNA polymerases: rat DNA polymerase beta (Pol beta) and African swine fever virus DNA polymerase X (Pol X). These proteins share similar folds but have low sequence identity, and differ greatly in both size and activity. "Equivalent" subdomain elements of structure were designed on the basis of the tertiary structure of Pol beta and the corresponding regions of Pol X were inferred from homology modeling and sequence alignment analysis. Libraries of chimeric genes with up to five crossovers were synthesized in a series of PCR reactions by employing hybrid oligonucleotides that code for variable connections between structural elements. Genetic complementation in Escherichia coli enabled identification of several novel DNA polymerases with enhanced phenotypes. Both the composition of structural elements and the manner in which they were linked were shown to be essential for this property, indicating the importance of these aspects of design.     

Predicting oligonucleotide-directed mutagenesis failures in protein engineering

Protein engineering uses oligonucleotide-directed mutagenesis to modify DNA sequences through a two-step process of hybridization and enzymatic synthesis. Inefficient reactions confound attempts to introduce mutations, especially for the construction of vast combinatorial protein libraries. This paper applied computational approaches to the problem of inefficient mutagenesis. Several results implicated oligonucleotide annealing to non-target sites, termed ‘cross-hybridization’, as a significant contributor to mutagenesis reaction failures. Test oligonucleotides demonstrated control over reaction outcomes. A novel cross-hybridization score, quickly computable for any plasmid and oligonucleotide mixture, directly correlated with yields of deleterious mutagenesis side products. Cross-hybridization was confirmed conclusively by partial incorporation of an oligonucleotide at a predicted cross-hybridization site, and by modification of putative template secondary structure to control cross-hybridization. Even in low concentrations, cross-hybridizing species in mixtures poisoned reactions. These results provide a basis for improved mutagenesis efficiencies and increased diversities of cognate protein libraries.     

Insertional protein engineering for analytical molecular sensing

The quantitative detection of low analyte concentrations in complex samples is becoming an urgent need in biomedical, food and environmental fields. Biosensors, being hybrid devices composed by a biological receptor and a signal transducer, represent valuable alternatives to non biological analytical instruments because of the high specificity of the biomolecular recognition. The vast range of existing protein ligands enable those macromolecules to be used as efficient receptors to cover a diversity of applications. In addition, appropriate protein engineering approaches enable further improvement of the receptor functioning such as enhancing affinity or specificity in the ligand binding. Recently, several protein-only sensors are being developed, in which either both the receptor and signal transducer are parts of the same protein, or that use the whole cell where the protein is produced as transducer. In both cases, as no further chemical coupling is required, the production process is very convenient. However, protein platforms, being rather rigid, restrict the proper signal transduction that necessarily occurs through ligand-induced conformational changes. In this context, insertional protein engineering offers the possibility to develop new devices, efficiently responding to ligand interaction by dramatic conformational changes, in which the specificity and magnitude of the sensing response can be adjusted up to a convenient level for specific analyte species. In this report we will discuss the major engineering approaches taken for the designing of such instruments as well as the relevant examples of resulting protein-only biosensors.