Mass spectrometry-from peripheral proteins to membrane motors

That membrane protein complexes could survive in the gas phase had always seemed impossible. The lack of chargeable residues, high hydrophobicity, and poor solubility and the vast excess of detergent contributed to the view that it would not be possible to obtain mass spectra of intact membrane complexes. With the recent success in recording mass spectra of these complexes, first from recombinant sources and later from the cellular environment, many surprising properties of these gas phase membrane complexes have been revealed. The first of these was that the interactions between membrane and soluble subunits could survive in vacuum, without detergent molecules adhering to the complex. The second unexpected feature was that their hydrophobicity and, consequently, lower charge state did not preclude ionization. The final surprising finding was that these gas phase membrane complexes carry with them lipids, bound specifically in subunit interfaces. This provides us with an opportunity to distinguish annular lipids that surround the membrane complexes, from structural lipids that have a role in maintaining structure and subunit interactions. In this perspective, we track these developments and suggest explanations for the various discoveries made during this research.     

Mass spectrometry of macromolecular assemblies: preservation and dissociation

Mass spectrometry not only plays a crucial role in the identification of proteins involved in the intricate interaction networks of the cell, but also is increasingly involved in the characterization of the non-covalent complexes formed by interacting partners. Recent developments have enabled the use of gas phase dissociation to probe oligomeric organization and topology, and increased understanding of the electrospray process is leading to knowledge of the structure of protein assemblies both in solution and in the gas phase.     

Annular lipids determine the ATPase activity of a calcium transport protein complexed with dipalmitoyllecithin.

Pure complexes of dipalmitoyllecithin (DPL, 16:0) which Ca2+, Mg2+ dependent ATPase from sarcoplasmic reticulum are unusual in retaining significant ATPase activity down to about 30 degrees C, well below the transition temperature of the pure lipid at 41 degrees C. A minimum of about 35 lipid molecules per ATPase is required to maintain maximal ATPase activity, but the complexes are progressively and irreversibly inactivated at lower lipid to protein ratios. Complexes containing more than the minimum lipid requirement show very similar temperature profiles of activity about 30 degrees C over a wide range of lipid to protein ratios, up to 1500:1. Spin-label studies indicate that, at lipid to protein ratios of less than about 30 lipids per ATPase, no DPL phase transition can be detected, but at all higher ratios, a phase transition occurs at about 41 degrees C. In all of these complexes there are breaks in the Arrhenius plots of ATPase activity at 27--32 degrees C and at 37.5--38.5 degrees C. Experiments with perturbing agents, such as cholesterol and benzyl alcohol which have well-defined effects on the DPL phase transition, indicate that these breaks in the Arrhenius plots of ATPase activity cannot be attributed to a depressed and broadened phase transition in the lipids near the protein molecules. These results are interpreted as evidence for a phospholipid annulus of at least 30 lipid molecules with interact directly with the ATPase and cannot undergo a phase transition at 41 degrees C. This structural interaction of the ATPase with the annular DPL molecules has a predominant effect in determining the form of the temperature-activity profiles. However, the perturbation of the DPL phase transition does not extend significantly beyond the annulus since a phase transition which starts at 41 degrees C can be detected as soon as extraannular lipid is present in the complexes. We suggest that it may be a general feature of membrane structure that penetrant membrane proteins interact with their immediate lipid environment so as to cause only a minimal perturbation of the lipid bilayer.     

Water structure and interactions with protein surfaces

The structure of liquid water and its interaction with biological molecules is a very active area of experimental and theoretical research. The chemically complex surfaces of protein molecules alter the structure of the surrounding layer of hydrating water molecules. The dynamics of hydration water can be detected by a series of experimental techniques, which show that hydration waters typically have slower correlation times than water in bulk. Specific water-mediated interactions in protein complexes have been studied in detail, and these interactions have been incorporated into potential energy functions for protein folding and design. The subtle changes in the structure of hydration water have been investigated by theoretical studies.     

Probing the energetics of dissociation of carbonic anhydrase-ligand complexes in the gas phase.

This paper describes the use of electrospray ionization-Fourier transform ion cyclotron mass spectrometry (ESI-FTICR-MS) to study the relative stabilities of noncovalent complexes of carbonic anhydrase II (CAII, EC 4.2.1.1) and benzenesulfonamide inhibitors in the gas phase. Sustained off-resonance irradiation collision-induced dissociation (SORI-CID) was used to determine the energetics of dissociation of these CAII-sulfonamide complexes in the gas phase. When two molecules of a benzenesulfonamide (1) were bound simultaneously to one molecule of CAII, one of them was found to exhibit significantly weaker binding (DeltaE50 = 0.4 V, where E50 is defined as the amplitude of sustained off-resonance irradiation when 50% of the protein-ligand complexes are dissociated). In solution, the benzenesulfonamide group coordinates as an anion to a Zn(II) ion bound at the active site of the enzyme. The gas phase stability of the complex with the weakly bound inhibitor was the same as that of the inhibitor complexed with apoCAII (i.e., CAII with the Zn(II) ion removed from the binding site). These results indicate that specific interactions between the sulfonamide group on the inhibitor and the Zn(II) ion on CAII were preserved in the gas phase. Experiments also showed a higher gas phase stability for the complex of para-NO2-benzenesulfonamide-CAII than that for ortho-NO2-benzenesulfonamide-CAII complex. This result further suggests that steric interactions of the inhibitors with the binding pocket of CAII parallel those in solution. Overall, these results are consistent with the hypothesis that CAII retains, at least partially, the structure of its binding pocket in the gas phase on the time scale (seconds to minutes) of the ESI-FTICR measurements.     

Modulating protein-protein interactions with small molecules: the importance of binding hotspots

The modulation of protein-protein interactions (PPIs) by small drug-like molecules is a relatively new area of research and has opened up new opportunities in drug discovery. However, the progress made in this area is limited to a handful of known cases of small molecules that target specific diseases. With the increasing availability of protein structure complexes, it is highly important to devise strategies exploiting homologous structure space on a large scale for discovering putative PPIs that could be attractive drug targets. Here, we propose a scheme that allows performing large-scale screening of all protein complexes and finding putative small-molecule and/or peptide binding sites overlapping with protein-protein binding sites (so-called "multibinding sites"). We find more than 600 nonredundant proteins from 60 protein families with multibinding sites. Moreover, we show that the multibinding sites are mostly observed in transient complexes, largely overlap with the binding hotspots and are more evolutionarily conserved than other interface sites. We investigate possible mechanisms of how small molecules may modulate protein-protein binding and discuss examples of new candidates for drug design.     

Dissection of multi-protein complexes using mass spectrometry: Subunit interactions in transthyretin and retinol-binding protein complexes

Complexes formed between transthyretin and retinol-binding protein prevent loss of retinol from the body through glomerular filtration. The interactions between these proteins have been examined by electrospray ionization combined with time-of-flight mass analysis. Conditions were found whereby complexes of these proteins, containing from four to six protein molecules with up to two ligands, are preserved in the gas phase. Analysis of the mass spectra of these multimeric species gives the overall stoichiometry of the protein subunits and provides estimates for solution dissociation constants of 1.9 ± 1.0 × 10⁻⁷ M for the first and 3.5 ± 1.0 × 10⁻⁵ M for the second retinol-binding protein molecule bound to a transthyretin tetramer. Dissociation of these protein assemblies within the gas phase of the mass spectrometer shows that each retinol-binding protein molecule interacts with three transthyretin molecules. Mass spectral analysis illustrates not only a correlation with solution behavior and crystallographic data of a closely related protein complex but also exemplifies a general method for analysis of multi-protein assemblies. Proteins Suppl. 2:3–11, 1998. © 1998 Wiley-Liss, Inc.     

Elusive affinities

The affinity of two molecules for each other and its temperature dependence are determined by the change in enthalpy, free enthalpy, entropy, and heat capacity upon dissociation. As we know the forces that stabilize-protein–protein or protein–DNA association and the three-dimensional structures of the complex, we can in principle derive values for each one of these parameters. The calculation is done first in gas phase by molecular mechanics, then in solution with the help of hydration parameters calibrated on small molecules. However, estimates of enthalpy and entropy changes in gas phase have excessively large error bars even under the approximation that the components of the complex associate as rigid bodies. No reliable result can be expected at the end. The fit to experimental values derived from binding and calorimetric measurements is poor, except for the dissociation heat capacity. This parameter can be attributed mostly to the hydration step and it correlates with the size of the interface. Many protein–protein complexes have interface areas in the range 1200–2000 Ų and only small conformation changes, so the rigid body approximation applies. It is less generally valid in protein–DNA complexes, which have interfaces covering 2200–3100 Ų, large dissociation heat capacities, and affect both the conformation and the dynamics of their components. © 1995 Wiley-Liss, Inc.     

The particle concept: placing discrete water molecules during protein-ligand docking predictions.

Water is known to play a significant role in the formation of protein-ligand complexes. In this paper, we focus on the influence of water molecules on the structure of protein-ligand complexes. We present an algorithmic approach, called the particle concept, for integrating the placement of single water molecules in the docking algorithm of FLEXX. FLEXX is an incremental construction approach to ligand docking consisting of three phases: the selection of base fragments, the placement of the base fragments, and the incremental reconstruction of the ligand inside the active site of a protein. The goal of the extension is to find water molecules at favorable places in the protein-ligand interface which may guide the placement of the ligand. In a preprocessing phase, favorable positions of water molecules inside the active site are calculated and stored in a list of possible water positions. During the incremental construction phase, water molecules are placed at the precomputed positions if they can form additional hydrogen bonds to the ligand. Steric constraints resulting from the water molecules as well as the geometry of the hydrogen bonds are used to optimize the ligand orientation in the active site during the reconstruction process. We have tested the particle concept on a series of 200 protein-ligand complexes. Although the average improvement of the prediction results is minor, we were able to predict water molecules between the protein and the ligand correctly in several cases. For instance in the case of HIV-1 protease, where a single water molecule between the protein and the ligand is known to be of importance in complex formation, significant improvements can be achieved.     

Examining multiprotein signaling complexes from all angles

Dynamic protein-protein interactions are involved in most physiological processes and, in particular, for the formation of multiprotein signaling complexes at transmembrane receptors, adapter proteins and effector molecules. Because the unregulated induction of signaling complexes has substantial clinical relevance, the investigation of these complexes is an active area of research. These studies strive to answer questions about the composition and function of multiprotein signaling complexes, along with the molecular mechanisms of their formation. In this review, the adapter protein, linker for activation of T cells (LAT), will be employed as a model to exemplify how signaling complexes are characterized using a range of techniques. The intensive investigation of LAT highlights how the systematic use of complementary techniques leads to an integrated understanding of the formation, composition and function of multiprotein signaling complexes that occur at receptors, adapter proteins and effector molecules.     

Mass spectrometry and ion mobility spectrometry of G-quadruplexes. A study of solvent effects on dimer formation and structural transitions in the telomeric DNA sequence d(TAGGGTTAGGGT)

We survey here state of the art mass spectrometry methodologies for investigating G-quadruplexes, and will illustrate them with a new study on a simple model system: the dimeric G-quadruplex of the 12-mer telomeric DNA sequence d(TAGGGTTAGGGT), which can adopt either a parallel or an antiparallel structure. We will discuss the solution conditions compatible with electrospray ionisation, the quantification of complexes using ESI-MS, the interpretation of ammonium ion preservation in the complexes in the gas phase, and the use of ion mobility spectrometry to resolve ambiguities regarding the strand stoichiometry, or separate and characterise different structural isomers. We also describe that adding electrospray-compatible organic co-solvents (methanol, ethanol, isopropanol or acetonitrile) to aqueous ammonium acetate increases the stability and rate of formation of dimeric G-quadruplexes, and causes structural transitions to parallel structures. Structural changes were probed by circular dichroism and ion mobility spectrometry, and the excellent correlation between the two techniques validates the use of ion mobility to investigate G-quadruplex folding. We also demonstrate that parallel G-quadruplex structures are easier to preserve in the gas phase than antiparallel structures.     

Molecular Parameterisation and the Theoretical Calculation of Electrode Potentials

The difference in reduction potentials between ortho and para-benzoquinones has been calculated. The employs gas phase ab initio and semi-empirical computations in combination with free energy perturbation theory applied to gas and solution phase Monte Carlo simulations. The effects on calculated results of altering solute electrostatic parameterisation in solution phase simulations is examined. Atom centred charges derived from the molecular electrostatic potentials, MEPs, from optimised ab initio wavefunctions and charges generated by consideration of hydrogen bonded complexes are considered. Parameterisation of hydroxyl torsions in hydroquinone molecules is treated in a physically realistic manner. The coupled torsional system of the ortho-hydrobenzoquinone molecule is described by a potential energy surface calculated using gas phase AM1 semi-empirical computations rather than the simple torsional energy functions frequently employed in such calculations. Calculated differences in electrode potentials show that the electrostatic interactions of quinone and hydroquinone molecules in aqueous solution are not well described by atom centred charges derived from ab initio calculated MEPs. Moreover, results in good agreement with the experimental reduction potential difference can be obtained by employing high level ab initio calculations and solution phase electrostatic parameters developed by consideration of hydrogen bonded complexes.     

In vivo protein complex topologies: sights through a cross-linking lens

Proteins are a remarkable class of molecules that exhibit wide diversity of shapes or topological features that underpin protein interactions and give rise to biological function. In addition to quantitation of abundance levels of proteins in biological systems under a variety of conditions, the field of proteome research has as a primary mission the assignment of function for proteins and if possible, illumination of factors that enable function. For many years, chemical cross-linking methods have been used to provide structural data on single purified proteins and purified protein complexes. However, these methods also offer the alluring possibility to extend capabilities to complex biological samples such as cell lysates or intact living cells where proteins may exhibit native topological features that do not exist in purified form. Recent efforts are beginning to provide glimpses of protein complexes and topologies in cells that suggest continued development will yield novel capabilities to view functional topological features of many proteins and complexes as they exist in cells, tissues, or other complex samples. This review will describe rationale, challenges, and a few success stories along the path of development of cross-linking technologies for measurement of in vivo protein interaction topologies.     

Solvated protein–DNA docking using HADDOCK

Interfacial water molecules play an important role in many aspects of protein–DNA specificity and recognition. Yet they have been mostly neglected in the computational modeling of these complexes. We present here a solvated docking protocol that allows explicit inclusion of water molecules in the docking of protein–DNA complexes and demonstrate its feasibility on a benchmark of 30 high-resolution protein–DNA complexes containing crystallographically-determined water molecules at their interfaces. Our protocol is capable of reproducing the solvation pattern at the interface and recovers hydrogen-bonded water-mediated contacts in many of the benchmark cases. Solvated docking leads to an overall improvement in the quality of the generated protein–DNA models for cases with limited conformational change of the partners upon complex formation. The applicability of this approach is demonstrated on real cases by docking a representative set of 6 complexes using unbound protein coordinates, model-built DNA and knowledge-based restraints. As HADDOCK supports the inclusion of a variety of NMR restraints, solvated docking is also applicable for NMR-based structure calculations of protein–DNA complexes.     

Protein-nucleic acid complexes and the role of mass spectrometry in their structure determination

Mass spectrometry is now established as a powerful tool for the study of the stoichiometry, interactions, dynamics, and subunit architecture of large protein assemblies and their subcomplexes. Recent evidence has suggested that the 3D structure of protein complexes can be maintained intact in the gas phase, highlighting the potential of ion mobility to contribute to structural biology. A key challenge is to integrate the compositional and structural information from ion mobility mass spectrometry with molecular modelling approaches to produce 3D models of intact protein complexes. In this review, we focus on the mass spectrometry of protein-nucleic acid assemblies with particular attention to the application of ion mobility, an emerging technique in structural studies. We also discuss the challenges that lie ahead for the full integration of ion mobility mass spectrometry with structural biology.     

Protein-nucleic acid complexes and the role of mass spectrometry in their structure determination

Mass spectrometry is now established as a powerful tool for the study of the stoichiometry, interactions, dynamics, and subunit architecture of large protein assemblies and their subcomplexes. Recent evidence has suggested that the 3D structure of protein complexes can be maintained intact in the gas phase, highlighting the potential of ion mobility to contribute to structural biology. A key challenge is to integrate the compositional and structural information from ion mobility mass spectrometry with molecular modelling approaches to produce 3D models of intact protein complexes. In this review, we focus on the mass spectrometry of protein-nucleic acid assemblies with particular attention to the application of ion mobility, an emerging technique in structural studies. We also discuss the challenges that lie ahead for the full integration of ion mobility mass spectrometry with structural biology.     

Diffraction quality crystals of PRD1, a 66-MDa dsDNA virus with an internal membrane

It has proved difficult to obtain well diffracting single crystals of macromolecular complexes rich in lipid. We report here the path that has led to crystals of the bacteriophage PRD1, a particle containing approximately 2,000 protein subunits from 18 different protein species, around 10 of which are integral membrane proteins associated with a host-derived lipid bilayer of some 12,500 lipid molecules. These crystals are capable of diffracting X-rays to Bragg spacings below 4A. It is hoped that some lessons learned from PRD1 will be applicable to other lipidic systems and that these crystals will allow, as a proof of principle, the determination of the structure of the virus in terms of a detailed atomic model.     

多基因表达系统研究进展

细胞中大多数蛋白质以亚基形式与其他蛋白装配成蛋白复合体而发挥功能.大分子蛋白复合物的结构研究和功能分析在后基因组时代成为热点,如何高效地获得多蛋白复合物是研究其功能和结构的前提.利用基因工程技术实现多个蛋白亚基在同一宿主细胞内共表达并装配成复合体是获得多蛋白复合物的有效手段.多基因表达系统在基础和应用研究中正起到越来越...     

几种蓝藻光合NAD（P）H脱氢酶复合体研究的新进展

蓝藻NAD（P）H脱氢酶（NDH-1）是一种重要的光合膜蛋白复合体,参与CO2吸收、围绕光系统I的循环电子传递和细胞呼吸。就几种蓝藻NDH-1复合体的鉴定、结构、生理功能等研究的新进展进行了综述与分析,并对今后NDH-1复合体的研究作了展望。     

Structure of pulmonary surfactant membranes and films: The role of proteins and lipid–protein interactions

The pulmonary surfactant system constitutes an excellent example of how dynamic membrane polymorphism governs some biological functions through specific lipid–lipid, lipid–protein and protein–protein interactions assembled in highly differentiated cells. Lipid–protein surfactant complexes are assembled in alveolar pneumocytes in the form of tightly packed membranes, which are stored in specialized organelles called lamellar bodies (LB). Upon secretion of LBs, surfactant develops a membrane-based network that covers rapidly and efficiently the whole respiratory surface. This membrane-based surface layer is organized in a way that permits efficient gas exchange while optimizing the encounter of many different molecules and cells at the epithelial surface, in a cross-talk essential to keep the whole organism safe from potential pathogenic invaders.The present review summarizes what is known about the structure of the different forms of surfactant, with special emphasis on current models of the molecular organization of surfactant membrane components. The architecture and the behaviour shown by surfactant structures in vivo are interpreted, to some extent, from the interactions and the properties exhibited by different surfactant models as they have been studied in vitro, particularly addressing the possible role played by surfactant proteins. However, the limitations in structural complexity and biophysical performance of surfactant preparations reconstituted in vitro will be highlighted in particular, to allow for a proper evaluation of the significance of the experimental model systems used so far to study structure–function relationships in surfactant, and to define future challenges in the design and production of more efficient clinical surfactants.