Structural portrait of filamin interaction mechanisms

We review the most recent findings on human filamin structure, with particular emphasis on the relationships between structure, function, and interaction. Filamin is a cytoskeletal actin-binding protein and it is therefore crucial in providing cells with the necessary mechanical and dynamical properties. Filamentous actin cross-linking by filamin is regulated by a number of other proteins and the molecular mechanisms of this complex interaction network can be understood by highlighting the structural features of isolated filamin moieties and of their complexes with several partners. Here we describe first the structure-function relationships of the isolated filamin, its flexibility, and its dimerization mechanism. Secondly, we illustrate the structural mechanism with which filamin can recognize its partners, both the actin filaments and the regulatory proteins.     

Using Cross-Correlated Spin Relaxation to Characterize Backbone Dihedral Angle Distributions of Flexible Protein Segments

Crucial to the function of proteins is their existence as conformational ensembles sampling numerous and structurally diverse substates. Despite this widely accepted notion there is still a high demand for meaningful and reliable approaches to characterize protein ensembles in solution. As it is usually conducted in solution, NMR spectroscopy offers unique possibilities to address this challenge. Particularly, cross-correlated relaxation (CCR) effects have long been established to encode both protein structure and dynamics in a compelling manner. However, this wealth of information often limits their use in practice as structure and dynamics might prove difficult to disentangle. Using a modern Maximum Entropy (MaxEnt) reweighting approach to interpret CCR rates of Ubiquitin, we demonstrate that these uncertainties do not necessarily impair resolving CCR-encoded structural information. Instead, a suitable balance between complementary CCR experiments and prior information is found to be the most crucial factor in mapping backbone dihedral angle distributions. Experimental and systematic deviations such as oversimplified dynamics appear to be of minor importance. Using Ubiquitin as an example, we demonstrate that CCR rates are capable of characterizing rigid and flexible residues alike, indicating their unharnessed potential in studying disordered proteins.     

原位冷冻电子断层扫描的发展及在生物研究方面的应用

对生物大分子复合物的研究和结构分析对于全面了解其功能和生物学意义至关重要. 冷冻电子显微镜在提供生物大分子结构及大分子分布等方面起到重要的作用. 近年来, 冷冻电子显微镜的硬件和软件的发展进一步提高了冷冻电子显微镜的有效性, 使其对各种生物结构、 蛋白质结构的解析更加准确快捷. 但是, 对于生物系统来说, 蛋白质和大分子复合物等均处于复杂的生理环境中, 因此原位检测生物分子的三维结构对于生物体系和结构生物学具有重要意义. 冷冻电子断层扫描作为一种功能强大的技术, 可以无需标记直接通过冷冻样品的固有衬度识别生物大分子的结构, 并且可在原位生理环境中对生物分子进行纳米级分辨率的三维成像. 本文综述了与冷冻电子断层扫描相关的样品制备和数据处理技术, 并总结了冷冻电子断层扫描技术在分离的大分子复合物和整个细胞或组织中的生物学应用.     

氢氘交换质谱技术在蛋白质和蛋白复合物结构研究中的应用进展

蛋白质是生命功能的执行者,其功能的发挥受自身结构动态变化、与其他生物分子的相互作用及修饰等因素的调节。因此,对蛋白质及蛋白复合物结构的研究有助于揭示重要生命过程中的分子机理与机制。氢氘交换质谱(Hydrogen deuterium exchange mass spectrometry,HDX-MS)是研究蛋白质结构、动态变化和相互作用的强有力工具,也是传统生物物理手段的重要补充。该文综述了HDX-MS的基本原理、机制、实验方法和研究最新进展,并从蛋白质自身动态变化、蛋白质-小分子相互作用、蛋白质-蛋白质相互作用3个方面介绍了近年来HDX-MS在蛋白及蛋白复合物研究中的应用进展。     

Top-down analysis of 30–80 kDa proteins by electron transfer dissociation time-of-flight mass spectrometry

Electron transfer dissociation (ETD)-based top-down mass spectrometry (MS) is the method of choice for in-depth structure characterization of large peptides, small- and medium-sized proteins, and non-covalent protein complexes. Here, we describe the performance of this approach for structural analysis of intact proteins as large as the 80 kDa serotransferrin. Current time-of-flight (TOF) MS technologies ensure adequate resolution and mass accuracy to simultaneously analyze intact 30–80 kDa protein ions and the complex mixture of their ETD product ions. Here, we show that ETD TOF MS is efficient and may provide extensive sequence information for unfolded and highly charged (around 1 charge/kDa) proteins of ～30 kDa and structural motifs embedded in larger proteins. Sequence regions protected by disulfide bonds within intact non-reduced proteins oftentimes remain uncharacterized due to the low efficiency of their fragmentation by ETD. For serotransferrin, reduction of S–S bonds leads to significantly varied ETD fragmentation pattern with higher sequence coverage of N- and C-terminal regions, providing a complementary structural information to top-down analysis of its oxidized form.

Crossing the phase boundary to study protein dynamics and function: combination of amide hydrogen exchange in solution and ion fragmentation in the gas phase

Protein dynamics are the key to understanding their behavior. The static protein structure alone in most cases is insufficient to describe the vast array of complex functions they perform in vivo. Until recently there were relatively few techniques available to investigate the dynamic nature of these proteins. Mass spectrometry has recently emerged as a powerful biophysical method, capable of providing both structural and dynamic information. By utilizing the labile nature of amide hydrogens as a marker of the backbone dynamics in solution, combined with gas-phase dissociation techniques, we now have a high-resolution tool to locate these exchanging hydrogens within the sequence of the protein and to probe the functional importance of its structural elements. In this paper we describe several applications of these methodologies to illustrate the importance of dynamics to the biological functions of proteins.     

Mass spectrometry based tools to investigate protein-ligand interactions for drug discovery

The initial stages of drug discovery are increasingly reliant on development and improvement of analytical methods to investigate protein-protein and protein-ligand interactions. For over 20 years, mass spectrometry (MS) has been recognized as providing a fast, sensitive and high-throughput methodology for analysis of weak non-covalent complexes. Careful control of electrospray ionization conditions has enabled investigation of the structure, stability and interactions of proteins and peptides in a solvent free environment. This critical review covers the use of mass spectrometry for kinetic, dynamic and structural studies of proteins and protein complexes. We discuss how conjunction of mass spectrometry with related techniques and methodologies such as ion mobility, hydrogen-deuterium exchange (HDX), protein footprinting or chemical cross-linking can provide us with structural information useful for drug development. Along with other biophysical techniques, such as NMR or X-ray crystallography, mass spectrometry provides a powerful toolbox for investigation of biological problems of medical relevance (204 references).     

Identifying Protein Conformational Dynamics Using Spin‐label ESR

Spin‐label electron spin resonance (ESR) has emerged as a powerful tool to characterize protein dynamics. One recent advance is the development of ESR for resolving dynamical components that occur or coexist during a biological process. It has been applied to study the complex structural and dynamical aspects of membranes and proteins, such as conformational changes in protein during translocation from cytosol to membrane, conformational exchange between equilibria in response to protein‐protein and protein‐ligand interactions in either soluble or membrane environments, protein oligomerization, and temperature‐ or hydration‐dependent protein dynamics. As these topics are challenging but urgent for understanding the function of a protein on the molecular level, the newly developed ESR methods to capture individual dynamical components, even in low‐populated states, have become a great complement to other existing biophysical tools.     

Diversity in structure and function of tethering complexes: evidence for different mechanisms in vesicular transport regulation

The term 'tethering factor' has been coined for a heterogeneous group of proteins that all are required for protein trafficking prior to vesicle docking and SNARE-mediated membrane fusion. Two groups of tethering factors can be distinguished, long coiled-coil proteins and multi-subunit complexes. To date, eight such protein complexes have been identified in yeast, and they are required for different trafficking steps. Homologous complexes are found in all eukaryotic organisms, but conservation seems to be less strict than for other components of the trafficking machinery. In fact, for most proposed multi-subunit tethers their ability to actually bridge two membranes remains to be shown. Here we discuss recent progress in the structural and functional characterization of tethering complexes and present the emerging view that the different complexes are quite diverse in their structure and the molecular mechanisms underlying their function. TRAPP and the exocyst are the structurally best characterized tethering complexes. Their comparison fails to reveal any similarity on a struc nottural level. Furthermore, the interactions with regulatory Rab GTPases vary, with TRAPP acting as a nucleotide exchange factor and the exocyst being an effector. Considering these differences among the tethering complexes as well as between their yeast and mammalian orthologs which is apparent from recent studies, we suggest that tethering complexes do not mediate a strictly conserved process in vesicular transport but are diverse regulators acting after vesicle budding and prior to membrane fusion.     

How far can we go with structural mass spectrometry of protein complexes?

Physical interactions between proteins and the formation of stable complexes form the basis of most biological functions. Therefore, a critical step toward understanding the integrated workings of the cell is to determine the structure of protein complexes, and reveal how their structural organization dictates function. Studying the three-dimensional organization of protein assemblies, however, represents a major challenge for structural biologists, due to the large size of the complexes, their heterogeneous composition, their flexibility, and their asymmetric structure. In the last decade, mass spectrometry has proven to be a valuable tool for analyzing such noncovalent complexes. Here, I illustrate the breadth of structural information that can be obtained from this approach, and the steps taken to elucidate the stoichiometry, topology, packing, dynamics, and shape of protein complexes. In addition, I illustrate the challenges that lie ahead, and the future directions toward which the field might be heading.     

Proximity‐Triggered Covalent Stabilization of Low‐Affinity Protein Complexes In Vitro and In Vivo

The characterization of low‐affinity protein complexes is challenging due to their dynamic nature. Here, we present a method to stabilize transient protein complexes in vivo by generating a covalent and conformationally flexible bridge between the interaction partners. A highly active pyrrolysyl tRNA synthetase mutant directs the incorporation of unnatural amino acids bearing bromoalkyl moieties (BrCnK) into proteins. We demonstrate for the first time that low‐affinity protein complexes between BrCnK‐containing proteins and their binding partners can be stabilized in vivo in bacterial and mammalian cells. Using this approach, we determined the crystal structure of a transient GDP‐bound complex between a small G‐protein and its nucleotide exchange factor. We envision that this approach will prove valuable as a general tool for validating and characterizing protein–protein interactions in vitro and in vivo.     

Site-specific fluorescent labeling of nascent proteins on the translating ribosome

As newly synthesized proteins emerge from the ribosome, they interact with a variety of cotranslational cellular machineries that facilitate their proper folding, maturation, and localization. These interactions are essential for proper function of the cell, and the ability to study these events is crucial to understanding cellular protein biogenesis. To this end, we have developed a highly efficient method to generate ribosome-nascent chain complexes (RNCs) site-specifically labeled with a fluorescent dye on the nascent polypeptide. The fluorescent RNC provides real-time, quantitative information on its cotranslational interaction with the signal recognition particle and will be a valuable tool in elucidating the role of the translating ribosome in numerous biochemical pathways.     

Structure determination of protein-protein complexes using NMR chemical shifts: case of an endonuclease colicin-immunity protein complex

Nuclear magnetic resonance (NMR) spectroscopy provides a range of powerful techniques for determining the structures and the dynamics of proteins. The high-resolution determination of the structures of protein-protein complexes, however, is still a challenging problem for this approach, since it can normally provide only a limited amount of structural information at protein-protein interfaces. We present here the determination using NMR chemical shifts of the structure (PDB code 2K5X) of the cytotoxic endonuclease domain from bacterial toxin colicin (E9) in complex with its cognate immunity protein (Im9). In order to achieve this result, we introduce the CamDock method, which combines a flexible docking procedure with a refinement that exploits the structural information provided by chemical shifts. The results that we report thus indicate that chemical shifts can be used as structural restraints for the determination of the conformations of protein complexes that are difficult to obtain by more standard NMR approaches.     

Investigation of methods suitable for the matrix-assisted laser desorption/ionization mass spectrometric analysis of proteins from ribonucleoprotein complexes

A variety of protein isolation and purification techniques for ribonucleoprotein (RNP) complexes were investigated for their compatibility with downstream analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Ribosomal proteins from Escherichia coli 70S ribosomes were obtained using methods such as phenol extraction and precipitation by organic solvents or acids. Under optimal conditions, more than 90% of the expected ribosomal proteins were detected in a single MALDI-MS experiment. The most effective approach combined ribosome denaturation by buffer exchange with acid precipitation of the ribosomal ribonucleic acids. An improved acid precipitation approach, involving the sequential additions of acetic and trifluoroacetic acid, yielded more complete protein coverage while minimizing loss of ion signal from lower molecular weight proteins. With phenol extraction, substantial gains in ion abundance of higher molecular weight proteins are noted, although some of the lower molecular weight proteins were not efficiently extracted. These results illustrate several effective approaches for protein isolation from protein complexes such as RNPs that are MALDI-MS compatible, and these approaches should extend the use of MALDI-MS for proteomics-based analyses of other protein-nucleic acid complexes.     

Separation-based approach to study dissociation kinetics of noncovalent DNA-multiple protein complexes

Noncovalent binding of DNA with multiple proteins is pivotal to many regulatory cellular processes. Due to the lack of experimental approaches, the kinetics of assembly and disassembly of DNA-multiple proteins complexes have never been studied. Here, we report on a first method capable of measuring disassembly kinetics of such complexes. The method is based on continuous spatial separation of different complexes. The kinetics of multiple complex dissociation processes are also spatially separated, which in turn facilitates finding their rate constants. Our separation-based approach was compared with a conventional no-separation approach by using computer simulation of dissociation kinetics. It proved to be much more accurate than the no-separation approach and to be a powerful tool for testing hypothetical mechanisms of the disassembly of DNA-multiple proteins complexes. An experimental implementation of the separation-based approach was finally demonstrated by using capillary electrophoresis as a separation method. The interaction between an 80 nucleotide long single-stranded DNA and single-stranded DNA binding protein was studied. DNA-protein complexes with one and two proteins were observed, and rate constants of their dissociation were determined. We foresee that a separation approach will be also developed to study the kinetics of the formation of DNA-multiple protein complexes.     

A GRID-derived water network stabilizes molecular dynamics computer simulations of a protease

Structural water molecules are crucial for the stability and function of proteins. Recently, we presented a molecular dynamics (MD) study on blood coagulation factor Xa (fXa) to investigate the effect of water molecules on the flexibility of the protein structure. We showed that neglecting important water positions at the outset of the simulation leads to severe structural distortions during the MD simulations: A stable trajectory was obtained with a water set that was derived from all 73 X-ray structures of the protein. However, for many proteins of interest, only limited structural data is available, which precludes the merging of information from many X-ray structures. Here, we show that an in silico assembled water network, derived from molecular interaction fields generated with the GRID program, is a viable alternative to X-ray data. MD simulations with the GRID water set show a significantly improved stability over alternative setups without water or the X-ray resolved water molecules in the starting structure. The performance is comparable to a water setup derived from a recently presented clustering approach.     

Rapid 3-dimensional shape determination of globular proteins by mobility capillary electrophoresis and native mass spectrometry

Established high-throughput proteomics methods provide limited information on the stereostructures of proteins. Traditional technologies for protein structure determination typically require laborious steps and cannot be performed in a high-throughput fashion. Here, we report a new medium throughput method by combining mobility capillary electrophoresis (MCE) and native mass spectrometry (MS) for the 3-dimensional (3D) shape determination of globular proteins in the liquid phase, which provides both the geometric structure and molecular mass information of proteins. A theory was established to correlate the ion hydrodynamic radius and charge state distribution in the native mass spectrum with protein geometrical parameters, through which a low-resolution structure (shape) of the protein could be determined. Our test data of 11 different globular proteins showed that this approach allows us to determine the shapes of individual proteins, protein complexes and proteins in a mixture, and to monitor protein conformational changes. Besides providing complementary protein structure information and having mixture analysis capability, this MCE and native MS based method is fast in speed and low in sample consumption, making it potentially applicable in top–down proteomics and structural biology for intact globular protein or protein complex analysis.

Using native mass spectrometry and mobility capillary electrophoresis, the ellipsoid dimensions of globular proteins or protein complexes could be measured efficiently.     

Probing Transient Conformational States of Proteins by Solid‐State R1ρ Relaxation‐Dispersion NMR Spectroscopy

The function of proteins depends on their ability to sample a variety of states differing in structure and free energy. Deciphering how the various thermally accessible conformations are connected, and understanding their structures and relative energies is crucial in rationalizing protein function. Many biomolecular reactions take place within microseconds to milliseconds, and this timescale is therefore of central functional importance. Here we show that R_1ρ relaxation dispersion experiments in magic‐angle‐spinning solid‐state NMR spectroscopy make it possible to investigate the thermodynamics and kinetics of such exchange process, and gain insight into structural features of short‐lived states.     

Painting Proteins with Covalent Labels: What's In the Picture?

Knowledge about the structural and biophysical properties of proteins when they are free in solution and/or in complexes with other molecules is essential for understanding the biological processes that proteins regulate. Such knowledge is also important to drug discovery efforts, particularly those focused on the development of therapeutic agents with protein targets. In the last decade a variety of different covalent labeling techniques have been used in combination with mass spectrometry to probe the solution-phase structures and biophysical properties of proteins and protein—ligand complexes. Highlighted here are five different mass spectrometry—based covalent labeling strategies including: continuous hydrogen/deuterium (H/D) exchange labeling, hydroxyl radical-mediated footprinting, SUPREX (stability of unpurified proteins from rates of H/D exchange), PLIMSTEX (protein-ligand interaction by mass spectrometry, titration, and H/D exchange), and SPROX (stability of proteins from rates of oxidation). The basic experimental protocols used in each of the above-cited methods are summarized along with the kind of biophysical information they generate. Also discussed are the relative strengths and weaknesses of the different methods for probing the wide range of conformational states that proteins and protein-ligand complexes can adopt when they are in solution.