Structural biology

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

NMR spectroscopy: a multifaceted approach to macromolecular structure.

Abstract: 1. Introduction 292. Landmarks in NMR of macromolecules 322.1 Protein structures and methods development 322.1.1 Sequential assignment method 322.1.2 Triple-resonance experiments 342.1.3 Structures of large proteins 362.2 Protein–nucleic acid complexes 372.3 RNA structures 382.4 Membrane-bound systems 393. NMR spectroscopy today 403.1 State-of-the-art structure determination 413.2 New methods 443.2.1 Residual dipolar couplings 443.2.2 Direct detection of hydrogen bonds 443.2.3 Spin labeling 453.2.4 Segmental labeling 463.3 Protein complexes 473.4 Mobility studies 503.5 Determination of time-dependent structures 523.6 Drug discovery 534. The future of NMR 544.1 The ease of structure determination 544.2 The ease of making recombinant protein 554.3 Post-translationally modified proteins 554.4 Approaches to large and/or membrane-bound proteins 564.5 NMR in structural genomics 564.6 Synergy of NMR and crystallography in protein structure determination 565. Conclusion 576. Acknowledgements 577. References 57Since the publication of the first complete solution structure of a protein in 1985 (Williamson et al. 1985), tremendous technological advances have brought nuclear magnetic resonance spectroscopy to the forefront of structural biology. Innovations in magnet design, electronics, pulse sequences, data analysis, and computational methods have combined to make NMR an extremely powerful technique for studying biological macromolecules at atomic resolution (Clore & Gronenborn, 1998). Most recently, new labeling and pulse techniques have been developed that push the fundamental line-width limit for resolution in NMR spectroscopy, making it possible to obtain high-field spectra with better resolution than ever before (Dotsch & Wagner, 1998). These methods are facilitating the study of systems of ever-increasing complexity and molecular weight.

Distance restraints from crosslinking mass spectrometry: Mining a molecular dynamics simulation database to evaluate lysine–lysine distances

Abstract: Integrative structural biology attempts to model the structures of protein complexes that are challenging or intractable by classical structural methods (due to size, dynamics, or heterogeneity) by combining computational structural modeling with data from experimental methods. One such experimental method is chemical crosslinking mass spectrometry (XL-MS), in which protein complexes are crosslinked and characterized using liquid chromatography-mass spectrometry to pinpoint specific amino acid residues in close structural proximity. The commonly used lysine-reactive N-hydroxysuccinimide ester reagents disuccinimidylsuberate (DSS) and bis(sulfosuccinimidyl)suberate (BS3) have a linker arm that is 11.4 A long when fully extended, allowing Cα (alpha carbon of protein backbone) atoms of crosslinked lysine residues to be up to ∼24 A apart. However, XL-MS studies on proteins of known structure frequently report crosslinks that exceed this distance. Typically, a tolerance of ∼3 A is added to the theoretical maximum to account for this observation, with limited justification for the chosen value. We used the Dynameomics database, a repository of high-quality molecular dynamics simulations of 807 proteins representative of diverse protein folds, to investigate the relationship between lysine–lysine distances in experimental starting structures and in simulation ensembles. We conclude that for DSS/BS3, a distance constraint of 26–30 A between Cα atoms is appropriate. This analysis provides a theoretical basis for the widespread practice of adding a tolerance to the crosslinker length when comparing XL-MS results to structures or in modeling. We also discuss the comparison of XL-MS results to MD simulations and known structures as a means to test and validate experimental XL-MS methods.

Solution structure of a protein denatured state and folding intermediate.

Abstract: The most controversial area in protein folding concerns its earliest stages. Questions such as whether there are genuine folding intermediates, and whether the events at the earliest stages are just rearrangements of the denatured state or progress from populated transition states, remain unresolved. The problem is that there is a lack of experimental high-resolution structural information about early folding intermediates and denatured states under conditions that favour folding because competent states spontaneously fold rapidly. Here we have solved directly the solution structure of a true denatured state by nuclear magnetic resonance under conditions that would normally favour folding, and directly studied its equilibrium and kinetic behaviour. We engineered a mutant of Drosophila melanogaster Engrailed homeodomain that folds and unfolds reversibly just by changing ionic strength. At high ionic strength, the mutant L16A is an ultra-fast folding native protein, just like the wild-type protein; however, at physiological ionic strength it is denatured. The denatured state is a well-ordered folding intermediate, poised to fold by docking helices and breaking some non-native interactions. It unfolds relatively progressively with increasingly denaturing conditions, and so superficially resembles a denatured state with properties that vary with conditions. Such ill-defined unfolding is a common feature of early folding intermediate states and accounts for why there are so many controversies about intermediates versus compact denatured states in protein folding.

Structural biology of human H3K9 methyltransferases

Abstract: SET domain methyltransferases deposit methyl marks on specific histone tail lysine residues and play a major role in epigenetic regulation of gene transcription. We solved the structures of the catalytic domains of GLP, G9a, Suv39H2 and PRDM2, four of the eight known human H3K9 methyltransferases in their apo conformation or in complex with the methyl donating cofactor, and peptide substrates. We analyzed the structural determinants for methylation state specificity, and designed a G9a mutant able to tri-methylate H3K9. We show that the I-SET domain acts as a rigid docking platform, while induced-fit of the Post-SET domain is necessary to achieve a catalytically competent conformation. We also propose a model where long-range electrostatics bring enzyme and histone substrate together, while the presence of an arginine upstream of the target lysine is critical for binding and specificity.