Structural biology

About: Structural biology is a research topic. Over the lifetime, 2206 publications have been published within this topic receiving 126070 citations.

Industrializing Structural Biology

Abstract: X-ray crystallography is the method of choice for determining molecular structure. The accumulation of molecular structure information has allowed a more complete representation of three-dimensional protein folds, enabling more successful homology modeling of unknown structures. Breakthroughs in genome sequencing projects have underscored the need for concomitant advances in structural biology if we hope to determine the extent of protein folding-space and elucidate how an assembly of proteins constitutes a cellular organism. This essay outlines an industrial approach to high-throughput (HT) determination of molecular structures.

NMR protein structure determination in living E. coli cells using nonlinear sampling

Abstract: The cell is a crowded environment in which proteins interact specifically with other proteins, nucleic acids, cofactors and ligands. Atomic resolution structural explanation of proteins functioning in this environment is a main goal of biochemical research. Recent improvements to nuclear magnetic resonance (NMR) hardware and methodology allow the measurement of high-resolution heteronuclear multidimensional NMR spectra of macromolecules in living cells (in-cell NMR). In this study, we describe a protocol for the stable isotope ((13)C, (15)N and (2)H) labeling and structure determination of proteins overexpressed in Escherichia coli cells exclusively on the basis of information obtained in living cells. The protocol combines the preparation of the protein in E. coli cells, the rapid measurement of the three-dimensional (3D) NMR spectra by nonlinear sampling of the indirectly acquired dimensions, structure calculation and structure refinement. Under favorable circumstances, this in-cell NMR approach can provide high-resolution 3D structures of proteins in living environments. The protocol has been used to solve the first 3D structure of a protein in living cells for the putative heavy metal-binding protein TTHA1718 from Thermus thermophilus HB8 overexpressed in E. coli cells. As no protein purification is necessary, a sample for in-cell NMR measurements can be obtained within 2-3 d. With the nonlinear sampling scheme, the duration of each 3D experiment can be reduced to 2-3 h. Once chemical shift assignments and NOESY peak lists have been prepared, structure calculation with the program CYANA and energy refinement can be completed in less than 1 h on a powerful computer system.

Visualizing conformational dynamics of proteins in solution and at the cell membrane.

Abstract: Conformational dynamics underlie enzyme function, yet are generally inaccessible via traditional structural approaches. FRET has the potential to measure conformational dynamics in vitro and in intact cells, but technical barriers have thus far limited its accuracy, particularly in membrane proteins. Here, we combine amber codon suppression to introduce a donor fluorescent noncanonical amino acid with a new, biocompatible approach for labeling proteins with acceptor transition metals in a method called ACCuRET (Anap Cyclen-Cu2+ resonance energy transfer). We show that ACCuRET measures absolute distances and distance changes with high precision and accuracy using maltose binding protein as a benchmark. Using cell unroofing, we show that ACCuRET can accurately measure rearrangements of proteins in native membranes. Finally, we implement a computational method for correcting the measured distances for the distance distributions observed in proteins. ACCuRET thus provides a flexible, powerful method for measuring conformational dynamics in both soluble proteins and membrane proteins.

Probing the Conformational Diversity of Cancer-Associated Mutations in p53 with Ion-Mobility Mass Spectrometry

Abstract: The tumor suppressor p53 is the most mutated protein in human cancers. It is implicated in lung (70%), colon (60%), and stomach (45%) cancers, respectively. The latest release (R16) of the International Agency for Research on Cancer (IARC) TP53 mutation database contains 29575 somatic mutations (November 2012 ; http://www-p53.iarc.fr/). A distinctive feature of the p53 mutational map is the rate of occurrence of missense mutations. Indeed, these single-point amino acid substitutions in p53 lead to abrogation of protein function, rather than deletions or nonsense mutations, as it is the case with most tumour suppressor proteins. A technique that is able to rapidly distinguish p53 mutants at low concentrations could have marked benefits for cancer screening assays and also for drug discovery. In this study, we used ion-mobility mass spectrometry (IM-MS) for this task. The p53 protein contains 393 amino acids and is divided into several structural and functional domains (Figure 1a): a transactivation domain (TAD, residues 1–61, a proline-rich fragment (PR, residues 62–94) with multiple copies of the PXXP sequence, a DNAbinding domain (DBD, residues 94– 292), a tetramerization domain (TET, residues 325–355), and a strongly basic C-terminal regulatory domain (CT, residues 363–393). Very few mutations have been reported in the Nor C-terminal domains. The central core of the protein, consisting of the DNA-binding domain, is the most highly conserved domain and is required for sequence-specific DNA binding. The majority of tumour-derived mutations (over 95%) are mapped to the DBD, where the cluster of six socalled “hot spots” is located. The structure of the p53DBD was first solved by Cho et al. in 1994. Based on the structure, the “hot spots” were classified as “structural” or “contact” mutations. These residues can affect either the thermodynamic stability and hence the structural integrity of the p53DBD, or the conformation of the protein required for protein–DNA or protein–protein interactions. Herein, we report IM-MS studies on the conformational diversity of wild-type p53 and common cancer-associated p53 mutants. We define “conformational phenotypes” and monitor the variation in these as exhibited by four single-point mutations: R249A, R273H, K292I, and A276Y. Locations of mutated residues used in the studies are depicted schematically on the 3D structure of p53 (Figure 1b). Specifically, we test whether the second-site suppressor mutant from loop L1, H115N, could trigger conformational changes in p53 cancerassociated mutations. In addition we use mass spectrometry as a tool to test the DNA-binding properties of the wild-type (WT) p53 and H115N mutant proteins. IM-MS can provide detailed insights into the structures of macromolecular systems. Measured drift times are recorded as arrival-time distributions (ATDs), which can then be converted into collision cross sections (CCSs). In this study, a Synapt HDMS (Waters Corporation, ManFigure 1. Structure of protein p53. a) Domain structure of full-length p53 with six highlighted mutational “hot spots”. b) Structure of human p53DBD. Locations of residues mutated in the experiments described herein are labeled in red, the zinc atom is represented as a red sphere (PDB: 2FEJ; image generated using the VMD software).

Novel insights into protein misfolding diseases revealed by ion mobility-mass spectrometry

Abstract: Amyloid disorders incorporate a wide range of human diseases arising from the failure of a specific peptide or protein to adopt, or remain in, its native functional conformational state. These pathological conditions, such as Parkinson's disease, Alzheimer's disease and Huntington's disease are highly debilitating, exact enormous costs on both individuals and society, and are predicted to increase in prevalence. Consequently, they form the focus of a topical and rich area of current scientific research. A major goal in attempts to understand and treat protein misfolding diseases is to define the structures and interactions of protein species intermediate between fully folded and aggregated, and extract a description of the aggregation process. This has proven a difficult task due to the inability of traditional structural biology approaches to analyze structurally heterogeneous systems. Continued developments in instrumentation and analytical approaches have seen ion mobility-mass spectrometry (IM-MS) emerge as a complementary approach for protein structure determination, and in some cases, a structural biology tool in its own right. IM-MS is well suited to the study of protein misfolding, and has already yielded significant structural information for selected amyloidogenic systems during the aggregation process. This review describes IM-MS for protein structure investigation, and provides a summary of current research highlighting how this methodology has unequivocally and unprecedentedly provided structural and mechanistic detail pertaining to the oligomerization of a variety of disease related proteins.