Structural Genomics Efforts Research Articles

Establishing a structural genomics platform: The Berlin-based Protein Structure Factory

Structural genomics (structural proteomics) initiatives aiming at the systematic, genome-directed structure analysis of proteins have recently been established in many locations worldwide. As a common international research endeavor, these initiatives thrive at the determination of protein structures representing all sequence families known in the biosphere. To reach this goal, a degree of international coordination of the structural genomics effort, as well as the development of high-throughput structure analysis methods and the provision of suitable research facilities are required. The Protein Structure Factory is a local, Berlin-based structural genomics project aiming at the structure determination of human proteins of presumed structural novelty and medical relevance. This project employs both NMR spectroscopy and X-ray crystallography for protein structure determination and aims at developing automated or semi-automated procedures for protein expression cloning and purification, and NMR spectral assignments. In addition, protein crystallography facilities are being set up at the third-generation synchrotron storage ring BESSY II in Berlin.

Expanding the nitrogen regulatory protein superfamily: Homology detection at below random sequence identity.

Nitrogen regulatory (PII) proteins are signal transduction molecules involved in controlling nitrogen metabolism in prokaryots. PII proteins integrate the signals of intracellular nitrogen and carbon status into the control of enzymes involved in nitrogen assimilation. Using elaborate sequence similarity detection schemes, we show that five clusters of orthologs (COGs) and several small divergent protein groups belong to the PII superfamily and predict their structure to be a (betaalphabeta)(2) ferredoxin-like fold. Proteins from the newly emerged PII superfamily are present in all major phylogenetic lineages. The PII homologs are quite diverse, with below random (as low as 1%) pairwise sequence identities between some members of distant groups. Despite this sequence diversity, evidence suggests that the different subfamilies retain the PII trimeric structure important for ligand-binding site formation and maintain a conservation of conservations at residue positions important for PII function. Because most of the orthologous groups within the PII superfamily are composed entirely of hypothetical proteins, our remote homology-based structure prediction provides the only information about them. Analogous to structural genomics efforts, such prediction gives clues to the biological roles of these proteins and allows us to hypothesize about locations of functional sites on model structures or rationalize about available experimental information. For instance, conserved residues in one of the families map in close proximity to each other on PII structure, allowing for a possible metal-binding site in the proteins coded by the locus known to affect sensitivity to divalent metal ions. Presented analysis pushes the limits of sequence similarity searches and exemplifies one of the extreme cases of reliable sequence-based structure prediction. In conjunction with structural genomics efforts to shed light on protein function, our strategies make it possible to detect homology between highly diverse sequences and are aimed at understanding the most remote evolutionary connections in the protein world.

THEMATICS as a Tool for Functional Genomics

Structural genomics efforts are determining in high throughput the three-dimensional structures of thousands of gene products. Many of these protein structures are of unknown function. The next major step after genome sequencing and protein structure determination is the determination of the function of the thousands of newly-discovered gene products. This presentation focuses on THEMATICS [3, 4], a simple computational predictor of information about the function of a protein. In particular, THEMATICS predicts the location of, and some chemical characteristics of, the active site of an enzyme. Application of THEMATICS requires only the structure of the subject protein. Thus THEMATICS works for proteins that have no similarity in sequence or in structure to any protein of known function. The accuracy of THEMATICS predictions is verified using proteins whose functions and mechanisms have been well studied experimentally.

Residual dipolar couplings: synergy between NMR and structural genomics.

Structural genomics is on a quest for the structure and function of a significant fraction of gene products. Current efforts are focusing on structure determination of single-domain proteins, which can readily be targeted by X-ray crystallography, NMR spectroscopy and computational homology modeling. However, comprehensive association of gene products with functions also requires systematic determination of more complex protein structures and other biomolecules participating in cellular processes such as nucleic acids, and characterization of biomolecular interactions and dynamics relevant to function. Such NMR investigations are becoming more feasible, not only due to recent advances in NMR methodology, but also because structural genomics is providing valuable structural information and new experimental and computational tools. The measurement of residual dipolar couplings in partially oriented systems and other new NMR methods will play an important role in this synergistic relationship between NMR and structural genomics. Both an expansion in the domain of NMR application, and important contributions to future structural genomics efforts can be anticipated.

Browsing gene banks for Fe2S2 ferredoxins and structural modeling of 88 plant-type sequences: an analysis of fold and function.

One-hundred-and-seventy-nine sequences of Fe2S2 ferredoxins and ferredoxin precursors were identified in and retrieved from currently available protein and cDNA databases. On the basis of their cluster-binding patterns, these sequences were divided into three groups: those containing the CX4CX2CXnC pattern (plant-type ferredoxins), those with the CX5CX2CXnC pattern (adrenodoxins), and those with a different pattern. These three groups contain, respectively, 139, 36, and 4 sequences. After excluding ferredoxin precursors in the first group, two subgroups were identified, again based on their cluster-binding patterns: 88 sequences had the CX4CX2CX29C pattern, and 29 had the CX4CX2CXmC (m not equal 29) pattern. The structures of the 88 ferredoxins with the CX4CX2CX29C pattern were modeled based on the available experimental structures of nine proteins within this same group. The modeling procedure was tested by building structural models for the ferredoxins with known structures. The models resulted, on average, in being within 1 A of the backbone root-mean-square deviation from the corresponding experimental structures. In addition, these structural models were shown to be of high quality by using assessment procedures based on energetic and stereochemical parameters. Thus, these models formed a reliable structural database for this group of ferredoxins, which is meaningful within the framework of current structural genomics efforts. From the analysis of the structural database generated it was observed that the secondary structural elements and the overall three-dimensional structures are maintained throughout the superfamily. In particular, the residues in the hydrophobic core of the protein were found to be either absolutely conserved or conservatively substituted. In addition, certain solvent-accessible charged groups, as well as hydrophobic groups, were found to be conserved to the same degree as the core residues. The patterns of conservation of exposed residues identified the regions of the protein that are critical for its function in electron transfer. An extensive analysis of protein-protein interactions is now possible. Some conserved interactions between residues have been identified and related to structural and/or functional features. All this information could not be obtained from the analyses of the primary sequences alone. Finally, the analysis of the sequences of the related subgroup featuring the CX4CX2CXmC (m not equal 29) cluster-binding pattern in the light of the structural and functional insights provided by the inspection of the mentioned structural database affords some hints on the functional features of ferredoxins belonging to this subgroup.

US structural genomics effort needs physicists for success.

US structural genomics effort needs physicists for success.

Object-oriented approach to drug design enabled by NMR SOLVE: first real-time structural tool for characterizing protein-ligand interactions.

As a result of genomics efforts, the number of protein drug targets is expected to increase by an order of magnitude. Functional genomics efforts are identifying these targets, while structural genomics efforts are determining structures for many of them. However, there is a significant gap in going from structural information for a protein target to a high affinity (K(d) < 100 nM) inhibitor, and the problem is multiplied by the sheer number of new targets now available. nature frequently designs proteins in classes that are related by the reuse, through gene duplication events, of cofactor binding domains. This reuse of functional domains is an efficient way to build related proteins in that it is object-oriented. There is a growing realization that the most efficient drug design strategies for attacking the mass of targets coming from genomics efforts will be systems-based approaches that attack groups of related proteins in parallel. We propose that the most effective drug design strategy will be one that parallels the object-oriented manner by which nature designed the gene families themselves. IOPE (Integrated Object-Oriented PharmacoEngineering) is such an approach. It is a three-step technology to build focused combinatorial libraries of potential inhibitors for major families and sub-families of enzymes, using cogent NMR data derived from representatives of these protein families. The NMR SOLVE (Structurally Oriented Library Valency Engineering) data used to design these libraries are gathered in days, and data can be obtained for large proteins (> 170 kDa). Furthermore, the process is fully object-oriented in that once a given bi-ligand is identified for a target, potency is retained if different cofactor mimics are swapped. This gives the drug design process maximum flexibility, allowing for the more facile transition from in vitro potency to in vivo efficacy.

32] Crystallographic analyses of hyperthermophilic proteins

The structure determinations have been undertaken for many of the reasons ‑to understand the structural basis of thermostability; to more fully characterize proteins with unusual cofactors or active centers; for biotechnological purposes; as homologs of eukaryotic proteins, especially those involved in some aspect of macromolecular biosynthesis; and as part of structural genomics efforts, because they either do, or do not, look like some other proteins. As there is an expectation that hyperthermostable proteins should be more robust and potentially better suited for withstanding the solution conditions and time scales of crystallization experiments, these proteins have provided attractive targets for structural studies. This chapter reviews the status and some general implications of crystallographic studies on hyperthermophilic proteins, with emphasis on relevant technical aspects of the structure determination, quality of the crystals, and conclusions about thermal stability and other properties of these fascinating proteins.

A new era

Welcome to the Structural Genomics supplement to Nature Structural Biology . We chose the topic of this special issue for two reasons. First, it is clear that structural biology is entering a new phase — that of high throughput, whole genome analysis — and this change warrants a closer look. Second, although the phrase 'Structural Genomics' has been in use for several years, many are still unsure of what it means exactly. We believe this supplement will go a long way to help this situation — to describe the current state and future plans of Structural Genomics efforts around the world, and to help delineate the possible benefits of this research.

Structural genomics in Europe: slow start, strong finish?

Structural genomics in Europe is slowly coming off the ground. So far, European Commission funding is restricted to methods development projects, whereas structural genomics programs are funded from national sources. The research outlook is more toward techniques for high-throughput structure analysis than toward a systematic coverage of protein fold space. At present, there is little European coordination of the structural genomics effort.

Review of the fourth Johns Hopkins Protein Folding Meeting

Review of the fourth Johns Hopkins Protein Folding Meeting

Book Purchases

Hundreds of genomes have been successfully sequenced to date, and the data are publicly available. At the same time, the advances in large-scale expression and purification of recombinant proteins have paved the way for structural genomics efforts. Frequently, however, little is known about newly expressed proteins calling for large-scale protein characterization to better understand their biochemical roles and to enable structure–function relationship studies. In the Structural Genomics Consortium (SGC), we have established a platform to characterize large numbers of purified proteins. This includes screening for ligands, enzyme assays, peptide arrays and peptide displacement in a 384-well format. In this review, we describe this platform in more detail and report on how our approach significantly increases the success rate for structure determination. Coupled with high-resolution X-ray crystallography and structure-guided methods, this platform can also be used toward the development of chemical probes through screening families of proteins against a variety of chemical series and focused chemical libraries.