Annual review of biophysics and biomolecular structure最新文献

Accurate methods of computing the affinity of a small molecule with a protein are needed to speed the discovery of new medications and biological probes. This paper reviews physics-based models of binding, beginning with a summary of the changes in potential energy, solvation energy, and configurational entropy that influence affinity, and a theoretical overview to frame the discussion of specific computational approaches. Important advances are reported in modeling protein-ligand energetics, such as the incorporation of electronic polarization and the use of quantum mechanical methods. Recent calculations suggest that changes in configurational entropy strongly oppose binding and must be included if accurate affinities are to be obtained. The linear interaction energy (LIE) and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) methods are analyzed, as are free energy pathway methods, which show promise and may be ready for more extensive testing. Ultimately, major improvements in modeling accuracy will likely require advances on multiple fronts, as well as continued validation against experiment.

Single-molecule biophysics has been serving biology for more than two decades. Fluorescence microscopy is one of the most commonly used tools to identify molecules of interest and to visualize biological events. Here we describe some of the most commonly used fluorescence imaging tools to measure nanoscale movements and the rotational dynamics of biomolecules.

Computational studies of large macromolecular assemblages have come a long way during the past 10 years. With the explosion of computer power and parallel computing, timescales of molecular dynamics simulations have been extended far beyond the hundreds of picoseconds timescale. However, limitations remain for studies of large-scale conformational changes occurring on timescales beyond nanoseconds, especially for large macromolecules. In this review, we describe recent methods based on normal mode analysis that have enabled us to study dynamics on the microsecond timescale for large macromolecules using different levels of coarse graining, from atomically detailed models to those employing only low-resolution structural information. Emerging from such studies is a control principle for robustness in Nature's machines. We discuss this idea in the context of large-scale functional reorganization of the ribosome, virus particles, and the muscle protein myosin.

Over the past 10 years there has been tremendous success in the area of computational protein design. Protein design software has been used to stabilize proteins, solubilize membrane proteins, design intermolecular interactions, and design new protein structures. A key motivation for these studies is that they test our understanding of protein energetics and structure. De novo design of novel structures is a particularly rigorous test because the protein backbone must be designed in addition to the amino acid side chains. A priori it is not guaranteed that the target backbone is even designable. To address this issue, researchers have developed a variety of methods for generating protein-like scaffolds and for optimizing the protein backbone in conjunction with the amino acid sequence. These protocols have been used to design proteins from scratch and to explore sequence space for naturally occurring protein folds.

Three-dimensional structure determination of small proteins and oligonucleotides by solution NMR is established. With the development of novel NMR and labeling techniques, structure determination is now feasible for proteins with a molecular mass of up to approximately 100 kDa and RNAs of up to 35 kDa. Beyond these molecular masses special techniques and approaches are required for applying NMR as a multiprobe method for structural investigations of proteins and RNAs. It is the aim of this review to summarize the NMR techniques and approaches available to advance the molecular mass limit of NMR both for proteins (up to 1 MDa) and RNAs (up to 100 kDa). Physical pictures of the novel techniques, their experimental applications, as well as labeling and assignment strategies are discussed and accompanied by future perspectives.

Structural proteomics approaches using mass spectrometry are increasingly used in biology to examine the composition and structure of macromolecules. Hydroxyl radical-mediated protein footprinting using mass spectrometry has recently been developed to define structure, assembly, and conformational changes of macromolecules in solution based on measurements of reactivity of amino acid side chain groups with covalent modification reagents. Accurate measurements of side chain reactivity are achieved using quantitative liquid-chromatography-coupled mass spectrometry, whereas the side chain modification sites are identified using tandem mass spectrometry. In addition, the use of footprinting data in conjunction with computational modeling approaches is a powerful new method for testing and refining structural models of macromolecules and their complexes. In this review, we discuss the basic chemistry of hydroxyl radical reactions with peptides and proteins, highlight various approaches to map protein structure using radical oxidation methods, and describe state-of-the-art approaches to combine computational and footprinting data.

The plasma membrane of most animal cells conforms to the cytoskeleton and only occasionally separates to form blebs. Previous studies indicated that many weak interactions between cytoskeleton and the lipid bilayer kept the surfaces together to counteract the normal outward pressure of cytoplasm. Either the loss of adhesion strength or the formation of gaps in the cytoskeleton enables the pressure to form blebs. Membrane-associated cytoskeleton proteins, such as spectrin and filamin, can control the movement and aggregation of membrane proteins and lipids, e.g., phosphoinositol phospholipids (PIPs), as well as blebbing. At the same time, lipids (particularly PIPs) and membrane proteins affect cytoskeleton and signaling dynamics. We consider here the roles of the major phosphatidylinositol-4,5-diphosphate (PIP2) binding protein, MARCKS, and PIP2 levels in controlling cytoskeleton dynamics. Further understanding of dynamics will provide important clues about how membrane-cytoskeleton adhesion rapidly adjusts to cytoskeleton and membrane dynamics.

Splicing is an essential step of gene expression in which introns are removed from pre-mRNA to generate mature mRNA that can be translated by the ribosome. This reaction is catalyzed by a large and dynamic macromolecular RNP complex called the spliceosome. The spliceosome is formed by the stepwise integration of five snRNPs composed of U1, U2, U4, U5, and U6 snRNAs and more than 150 proteins binding sequentially to pre-mRNA. To study the structure of this particularly dynamic RNP machine that undergoes many changes in composition and conformation, single-particle cryo-electron microscopy (cryo-EM) is currently the method of choice. In this review, we present the results of these cryo-EM studies along with some new perspectives on structural and functional aspects of splicing, and we outline the perspectives and limitations of the cryo-EM technique in obtaining structural information about macromolecular complexes, such as the spliceosome, involved in splicing.

Structural, compositional, and material (elastic) properties of lipid bilayers exert strong influences on the interactions of water-soluble proteins and peptides with membranes, the distribution of transmembrane proteins in the plane of the membrane, and the function of specific membrane channels. Theoretical and experimental studies show that the binding of either cytoplasmic proteins or extracellular peptides to membranes is regulated by the presence of charged lipids and that the sorting of transmembrane proteins into or out of membrane microdomains (rafts) depends on several factors, including bilayer material properties governed by the presence of cholesterol. Recent studies have also shown that bilayer material properties modify the permeability of membrane pores, formed either by protein channels or by cell-lytic peptides.

Complex cellular events commonly depend on the activity of molecular "machines" that efficiently couple enzymatic and regulatory functions within a multiprotein assembly. An essential and expanding subset of these assemblies comprises proteins of the ATPases associated with diverse cellular activities (AAA+) family. The defining feature of AAA+ proteins is a structurally conserved ATP-binding module that oligomerizes into active arrays. ATP binding and hydrolysis events at the interface of neighboring subunits drive conformational changes within the AAA+ assembly that direct translocation or remodeling of target substrates. In this review, we describe the critical features of the AAA+ domain, summarize our current knowledge of how this versatile element is incorporated into larger assemblies, and discuss specific adaptations of the AAA+ fold that allow complex molecular manipulations to be carried out for a highly diverse set of macromolecular targets.