Structural genomics

Completed genome sequences allow every open reading frame (ORF), the part of a gene that is likely to contain the sequence for the messenger RNA and protein, to be cloned and expressed as protein. These proteins are then purified and crystallized, and then subjected to one of two types of structure determination: X-ray crystallography and nuclear magnetic resonance (NMR). The whole genome sequence allows for the design of every primer required in order to amplify all of the ORFs, clone them into bacteria, and then express them. By using a whole-genome approach to this traditional method of protein structure determination, all of the proteins encoded by the genome can be expressed at once. This approach allows for the structural determination of every protein that is encoded by the genome.

This approach uses protein sequence data and the chemical and physical interactions of the encoded amino acids to predict the 3-D structures of proteins with no homology to solved protein structures. One highly successful method for ab initio modeling is the Rosetta program, which divides the protein into short segments and arranges short polypeptide chain into a low-energy local conformation. Rosetta is available for commercial use and for non-commercial use through its public program, Robetta.

This modeling technique compares the gene sequence of an unknown protein with sequences of proteins with known structures. Depending on the degree of similarity between the sequences, the structure of the known protein can be used as a model for solving the structure of the unknown protein. Highly accurate modeling is considered to require at least 50% amino acid sequence identity between the unknown protein and the solved structure. 30-50% sequence identity gives a model of intermediate-accuracy, and sequence identity below 30% gives low-accuracy models. It has been predicted that at least 16,000 protein structures will need to be determined in order for all structural motifs to be represented at least once and thus allowing the structure of any unknown protein to be solved accurately through modeling.[3] One disadvantage of this method, however, is that structure is more conserved than sequence and thus sequence-based modeling may not be the most accurate way to predict protein structures.

Threading bases structural modeling on fold similarities rather than sequence identity. This method may help identify distantly related proteins and can be used to infer molecular functions.

One current goal of the Joint Center for Structural Genomics (JCSG), a part of the Protein Structure Initiative (PSI) is to solve the structures for all the proteins in Thermotoga maritima, a thermophillic bacterium. T. maritima was selected as a structural genomics target based on its relatively small genome consisting of 1,877 genes and the hypothesis that the proteins expressed by a thermophilic bacterium would be easier to crystallize.

Lesley et al used Escherichia coli to express all the open-reading frames (ORFs) of T. martima. These proteins were then crystallized and structures were determined for successfully crystallized proteins using X-ray crystallography. Among other structures, this structural genomics approach allowed for the determination of the structure of the TM0449 protein, which was found to exhibit a novel fold as it did not share structural homology with any known protein.[4]

The goal of the TB Structural Genomics Consortium is to determine the structures of potential drug targets in Mycobacterium tuberculosis, the bacterium that causes tuberculosis. The development of novel drug therapies against tuberculosis are particularly important given the growing problem of multi-drug-resistant tuberculosis.

The fully sequenced genome of M. tuberculosis has allowed scientists to clone many of these protein targets into expression vectors for purification and structure determination by X-ray crystallography. Studies have identified a number of target proteins for structure determination, including extracellular proteins that may be involved in pathogenesis, iron-regulatory proteins, current drug targets, and proteins predicted to have novel folds. So far, structures have been determined for 708 of the proteins encoded by M. tuberculosis.