Real-time monitoring of protein complexes reveals their quaternary organization and dynamics

The dynamics of protein complexes are crucial for their function yet are challenging to study. Here, we present a nanoelectrospray (nESI) mass spectrometry (MS) approach capable of simultaneously providing structural and dynamical information for protein complexes. We investigate the properties of two small heat shock proteins (sHSPs) and find that these proteins exist as dodecamers composed of dimeric building blocks. Moreover, we show that these proteins exchange dimers on the timescale of minutes, with the rate of exchange being strongly temperature dependent. Because these proteins are expressed in the same cellular compartment, we anticipate that this dynamical behavior is crucial to their function in vivo. Furthermore, we propose that the approach used here is applicable to a range of nonequilibrium systems and is capable of providing both structural and dynamical information necessary for functional genomics.     

Transplant-insert-constrain-relax-assemble (TICRA): protein-ligand complex structure modeling and application to kinases

We introduce TICRA (transplant-insert-constrain-relax-assemble), a method for modeling the structure of unknown protein-ligand complexes using the X-ray crystal structures of homologous proteins and ligands with known activity. We present results from modeling the structures of protein kinase-inhibitor complexes using p38 and Lck as examples. These examples show that the TICRA method may be used prospectively to create and refine models for protein kinase-inhibitor complexes with an overall backbone rmsd of less than 0.75 ? for the kinase domain, when compared to published X-ray crystal structures. Further refinement of the models of the kinase domains of p38 and Lck in complex with their cognate ligands from the published crystal structures was able to improve the rmsd's of the model complexes to below 0.5 ?. Our results show that TICRA is a useful approach to the problem of structure-based drug design in cases where little structural information is available for the target proteins and the binding mode of active compounds is unknown.     

Do Intrinsically Disordered Proteins Possess High Specificity in Protein–Protein Interactions?

Specific protein–protein interactions are critical to cellular function. Structural flexibility and disorder‐to‐order transitions upon binding enable intrinsically disordered proteins (IDPs) to overcome steric restrictions and form complementary binding interfaces, and thus, IDPs are widely considered to have high specificity and low affinity for molecular recognition. However, flexibility may also enable IDPs to form complementary binding interfaces with misbinding partners, resulting in a great number of nonspecific interactions. Consequently, it is questionable whether IDPs really possess high specificity. In this work, we investigated this question from a thermodynamic viewpoint. We collected mutant thermodynamic data for 35 ordered protein complexes and 43 disordered protein complexes. We found that the enthalpy–entropy compensation for disordered protein complexes was more complete than that for ordered protein complexes. We further simulated the binding processes of ordered and disordered protein complexes under mutations. Simulation data confirmed the observation of experimental data analyses and further revealed that disordered protein complexes possessed smaller changes in binding free energy than ordered protein complexes under the same mutation perturbations. Therefore, interactions of IDPs are more malleable than those of ordered proteins due to their structural flexibility in the complex. Our results provide new clues for exploring the relationship between protein flexibility, adaptability, and specificity.     

Application of affinity selection-mass spectrometry assays to purification and affinity-based screening of the chemokine receptor CXCR4

Affinity selection-mass spectrometry (AS-MS) is a sensitive technology for identifying small molecules that bind to target proteins, and assays enabled by AS-MS can be used to delineate relative binding affinities of ligands for proteins. 'Indirect' AS-MS assays employ size-exclusion techniques to separate target-ligand complexes from unbound ligands, and target-associated ligands are then specifically detected by liquid chromatography mass spectrometry. We report how indirect AS-MS binding assays with known reference control compounds were used as guideposts for development of an optimized purification method for CXCR4, a G-protein coupled chemokine receptor, for which we sought novel antagonists. The CXCR4 purification method that was developed was amenable to scale-up and enabled the screening of purified recombinant human CXCR4 against a large combinatorial library of small molecules by high throughput indirect AS-MS. The screen resulted in the discovery of new ligands that competed off binding of reference compounds to CXCR4 in AS-MS binding assays and that antagonized SDF1α-triggered responses and CXCR4-mediated HIV1 viral uptake in cell-based assays. This report provides a methodological paradigm whereby indirect AS-MS-based ligand binding assays may be used to guide optimal integral membrane protein purification methods that enable downstream affinity selection-based applications such as high throughput AS-MS screens.     

Chemical Processes Involving 18-Crown-6-Ether in Activated Noncovalent Complexes with Protonated Peptides

These last decades, it has been widely assumed that 18-crown-6-ether (CE) plays a spectator role during the chemical processes occurring in isolated host-guest complexes between peptides or proteins and CE after activation in mass spectrometers. Our present experimental and theoretical results challenge this hypothesis by showing that CE can abstract a proton or a protonated molecule from protonated peptides after activation by collisions in argon or electron capture/transfer. Furthermore, thanks to comparison between experimental and calculated values of collision cross-sections, we demonstrate that CE can change binding site after electron transfer. We also propose detailed mechanisms for these processes.     

Two-dimensional turbulent flow chromatography coupled on-line to liquid chromatography–mass spectrometry for solution-based ligand screening against multiple proteins

We present herein a novel bioseparation/chemical analysis strategy for protein–ligand screening and affinity ranking in compound mixtures, designed to increase screening rates and improve sensitivity and ruggedness in performance. The strategy is carried out by combining on-line two-dimensional turbulent flow chromatography (2D-TFC) with liquid chromatography–mass spectrometry (LC–MS), and accomplished through the following steps: (1) a reversed-phase TFC stage to separate the protein/ligand complex from the unbound free molecules, (2) an on-line dissociation process to release the bound ligands from the complexes, and (3) a second mixed-mode cation-exchange/reversed-phase TFC stage to trap the bound ligands and to remove the proteins and salts, followed by LC–MS analysis for identification and determination of the binding affinities. The technique can implement an ultra-fast isolation of protein/ligand complex with the retention time of a complex peak in about 5 s, and on-line prepare the “clean” sample to be directly compatible with the LC–MS analysis. The improvement in performance of this 2D-TFC/LC–MS approach over the conventional approach has been demonstrated by determining affinity-selected ligands of the target proteins acetylcholinesterase and butyrylcholinesterase from a small library with known binding affinities and a steroidal alkaloid library composed of structurally similar compounds. Our results show that 2D-TFC/LC–MS is a generic and efficient tool for high-throughput screening of ligands with low-to-high binding affinities, and structure-activity relationship evaluation.     

DNA与生物碱类化合物非共价作用的负离子电喷雾质谱研究(Ⅰ)

目前,临床上使用的许多抗病毒药物均是通过与DNA,RNA发生相互作用破坏其结构,进而影响基因调控与表达的功能,表现出抗病毒活性。因此,核酸与药物分子相互作用的研究对阐述抗病毒药物的作用机理,以及对药物的体外筛选都具有重要的意义．电喷雾电离质谱作为一种软电离手段,可将溶液中生物分子与药物分子的非共价复合物转为气相进行分析,再现其生理状态,使其成为分子水平上进行药物筛选的最佳方法和在分子水平上筛选中药抗病毒活性成分的理想工具,本文选择合成了与SARS病毒相关的DNA片段作为抗病毒药物筛选的靶分子,用电喷雾质谱技术,通过对靶分子与5种生物碱的非共价复合作用,探讨了生物质谱方法用于药物筛选的可行性。     

Flexible protein‐protein docking based on Best‐First search algorithm

We developed a new high resolution protein‐protein docking method based on Best‐First search algorithm that loosely imitates protein‐protein associations. The method operates in two stages: first, we perform a rigid search on the unbound proteins. Second, we search alternately on rigid and flexible degrees of freedom starting from multiple configurations from the rigid search. Both stages use heuristics added to the energy function, which causes the proteins to rapidly approach each other and remain adjacent, while optimizing on the energy. The method deals with backbone flexibility explicitly by searching over ensembles of conformations generated before docking. We ran the rigid docking stage on 66 complexes and grouped the results into four classes according to evaluation criteria used in Critical Assessment of Predicted Interactions (CAPRI; “high,” “medium,” “acceptable,” and “incorrect”). Our method found medium binding conformations for 26% of the complexes and acceptable for additional 44% among the top 10 configurations. Considering all the configurations, we found medium binding conformations for 55% of the complexes and acceptable for additional 39% of the complexes. Introducing side‐chains flexibility in the second stage improves the best found binding conformation but harms the ranking. However, introducing side‐chains and backbone flexibility improve both the best found binding conformation and the best found conformation in the top 10. Our approach is a basis for incorporating multiple flexible motions into protein‐protein docking and is of interest even with the current use of a simple energy function. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2010     

Standard free energy of releasing a localized water molecule from the binding pockets of proteins: double-decoupling method

Localized water molecules in the binding pockets of proteins play an important role in noncovalent association of proteins and small drug compounds. At times, the dominant contribution to the binding free energy comes from the release of localized water molecules in the binding pockets of biomolecules. Therefore, to quantify the energetic importance of these water molecules for drug design purposes, we have used the double-decoupling approach to calculate the standard free energy of tying up a water molecule in the binding pockets of two protein complexes. The double-decoupling approach is based on the underlying principle of statistical thermodynamics. We have calculated the standard free energies of tying up the water molecule in the binding pockets of these complexes to be favorable. These water molecules stabilize the protein-drug complexes by interacting with the ligands and binding pockets. Our results offer ideas that could be used in optimizing protein-drug interactions, by designing ligands that are capable of targeting localized water molecules in protein binding sites. The resulting free energy of ligand binding could benefit from the potential free energy gain accompanying the release of these water molecules. Furthermore, we have examined the theoretical background of the double-decoupling method and its connection to the molecular dynamics thermodynamic integration techniques.     

Peptide‐Guided Assembly of Repeat Protein Fragments

Herein, we present the peptide‐guided assembly of complementary fragments of designed armadillo repeat proteins (dArmRPs) to create proteins that bind peptides not only with high affinity but also with good selectivity. We recently demonstrated that complementary N‐ and C‐terminal fragments of dArmRPs form high‐affinity complexes that resemble the structure of the full‐length protein, and that these complexes bind their target peptides. We now demonstrate that dArmRPs can be split such that the fragments assemble only in the presence of a templating peptide, and that fragment mixtures enrich the combination with the highest affinity for this peptide. The enriched fragment combination discriminates single amino acid variations in the target peptide with high specificity. Our results suggest novel opportunities for the generation of new peptide binders by selection from dArmRP fragment mixtures.     

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.     

Gas-phase binding of non-covalent protein complexes between bovine pancreatic trypsin inhibitor and its target enzymes studied by electrospray ionization tandem mass spectrometry

The potential of electrospray ionization (ESI) mass spectrometry (MS) to detect non-covalent protein complexes has been demonstrated repeatedly. However, questions about correlation of the solution and gas-phase structures of these complexes still produce vigorous scientific discussion. Here, we demonstrate the evaluation of the gas-phase binding of non-covalent protein complexes formed between bovine pancreatic trypsin inhibitor (BPTI) and its target enzymes over a wide range of dissociation constants. Non-covalent protein complexes were detected by ESI-MS. The abundance of the complex ions in the mass spectra is less than expected from the values of the dissociation constants of the complexes in solution. Collisionally activated dissociation (CAD) tandem mass spectrometry (MS/MS) and a collision model for ion activation were used to evaluate the binding of non-covalent complexes in the gas phase. The internal energy required to induce dissociation was calculated for three collision gases (Ne, Ar, Kr) over a wide range of collision gas pressures and energies using an electrospray ionization source. The order of binding energies of the gas-phase ions for non-covalent protein complexes formed by the ESI source and assessed using CAD-MS/MS appears to differ from that of the solution complexes. The implication is that solution structure of these complexes was not preserved in the gas phase.     

Exploring the “intensity fading” phenomenon in the study of noncovalent interactions by MALDI-TOF mass spectrometry

The difficulties to detect intact noncovalent complexes involving proteins and peptides by MALDI-TOF mass spectrometry have hindered a widespread use of this approach. Recently, "intensity fading MS" has been presented as an alternative strategy to detect noncovalent interactions in solution, in which a reduction in the relative signal intensity of low molecular mass binding partners (i.e., protease inhibitors) can be observed when their target protein (i.e., protease) is added to the sample. Here we have performed a systematic study to explore how various experimental conditions affect the intensity fading phenomenon, as well as a comparison with the strategy based on the direct detection of intact complexes by MALDI MS. For this purpose, the study is focused on two different protease-inhibitor complexes naturally occurring in solution, together with a heterogeneous mixture of nonbinding molecules derived from a biological extract, to examine the specificity of the approach, i.e., those of carboxypeptidase A (CPA) bound to potato carboxypeptidase inhibitor (PCI) and of trypsin bound to bovine pancreatic trypsin inhibitor (BPTI). Our results show that the intensity fading phenomenon occurs when the binding assay is carried out in the sub-muM range and the interacting partners are present in complex mixtures of nonbinding compounds. Thus, at these experimental conditions, the specific inhibitor-protease interaction causes a selective reduction in the relative abundance of the inhibitor. Interestingly, we could not detect any gaseous noncovalent inhibitor-protease ions at these conditions, presumably due to the lower high-mass sensitivity of MCP detectors.     

Molecular aptamers for real-time protein-protein interaction study

Protein-protein interactions play critical roles in cellular functions, but current techniques for real-time study of these interactions are limited. We report the real-time monitoring of protein-protein interactions without labeling either of the two interacting proteins; this procedure poses minimum effects on the binding properties of the proteins. Our strategy uses a protein/aptamer complex to probe the interactions in a competitive assay where the binding of an aptamer to its target protein is altered by a second protein that interacts with the target protein. Two signal transduction strategies, fluorescence resonance energy transfer (FRET) and fluorescence anisotropy, have been designed to study the interactions of human alpha-thrombin with different proteins by using two aptamers specific for two binding sites on alpha-thrombin. Our method has been shown to be simple and effective, does not require labeling of proteins, makes use of easily obtainable aptamers, provides detailed protein-protein interaction information and has excellent sensitivity for protein detection and protein-protein interaction studies. The FRET and the fluorescent anisotropy approaches complement each other in providing insight into the kinetics, mechanisms, binding sites and binding dynamics of the interacting proteins.     

Involvement of some large immunophilins and their ligands in the protection and regeneration of neurons: a hypothetical mode of action

The powerful immunosuppressive drugs such as FK506 and its derivatives induce some regeneration and protection of neurons from ischaemic brain injury and some other neurological disorders. The drugs form complexes with diverse FKBPs but apparently the FKBP52/FK506 complex was shown to be involved in the protection and regeneration of neurons. We used several different sequence attributes in searching diverse genomic databases for similar motifs as those present in the FKBPs. A Fortran library of algorithms (Par_Seq) has been designed and used in searching for the similarity of sequence motifs extracted from the multiple sequence alignments of diverse groups of proteins (query motifs) and the target motifs which are encoded in various genomes. The following sequence attributes were used in the establishment of the degree of convergence between: (A) amino acid (AA) sequence similarity (ID) of the query/target motifs and (B) their: (1) AA composition (AAC); (2) hydrophobicity (HI); (3) Jensen-Shannon entropy; and (4) AA propensity to form a particular secondary structure. The sequence hallmark of two different groups of peptidylprolyl cis/trans isomerases (PPIases), namely tetratricopetide repeat (TPR) motifs, which are present in the heat-shock cyclophilins and in the large FK506-binding proteins (FKBPs) were used to search various genomic databases. The Par_Seq algorithm has revealed that the TPR motifs have similar sequence attributes as a number of hydrophobic sequence segments of functionally unrelated membrane proteins, including some of the TMs from diverse G protein-coupled receptors (GPCRs). It is proposed that binding of the FKBP52/FK506 complex to the membranes via the TPR motifs and its interaction with some membrane proteins could be in part responsible for some neuro-regeneration and neuro-protection of the brain during some ischaemia-induced stresses.     

Understanding the recognition mechanism of protein-RNA complexes using energy based approach

Protein-RNA interactions perform diverse functions within the cell. Understanding the recognition mechanism of protein-RNA complexes is a challenging task in molecular and computational biology. In this work, we have developed an energy based approach for identifying the binding sites and important residues for binding in protein-RNA complexes. The new approach considers the repulsive interactions as well as the effect of distance between the atoms in protein and RNA in terms of interaction energy, which are not considered in traditional distance based methods to identify the binding sites. We found that the positively charged, polar and aromatic residues are important for binding. These residues influence to form electrostatic, hydrogen bonding and stacking interactions. Our observation has been verified with the experimental binding specificity of protein-RNA complexes and found good agreement with experiments. Further, the propensities of residues/nucleotides in the binding sites of proteins/RNA and their atomic contributions have been derived. Based on these results we have proposed a novel mechanism for the recognition of protein-RNA complexes: the charged and polar residues in proteins initiate recognition with RNA by making electrostatic and hydrogen bonding interactions between them; the aromatic side chains tend to form aromatic-aromatic interactions and the hydrophobic residues aid to stabilize the complex.     

A carrier protein strategy yields the structure of dalbavancin

Many large natural product antibiotics act by specifically binding and sequestering target molecules found on bacterial cells. We have developed a new strategy to expedite the structural analysis of such antibiotic-target complexes, in which we covalently link the target molecules to carrier proteins, and then crystallize the entire carrier-target-antibiotic complex. Using native chemical ligation, we have linked the Lys-D-Ala-D-Ala binding epitope for glycopeptide antibiotics to three different carrier proteins. We show that recognition of this peptide by multiple antibiotics is not compromised by the presence of the carrier protein partner, and use this approach to determine the first-ever crystal structure for the new therapeutic dalbavancin. We also report the first crystal structure of an asymmetric ristocetin antibiotic dimer, as well as the structure of vancomycin bound to a carrier-target fusion. The dalbavancin structure reveals an antibiotic molecule that has closed around its binding partner; it also suggests mechanisms by which the drug can enhance its half-life by binding to serum proteins, and be targeted to bacterial membranes. Notably, the carrier protein approach is not limited to peptide ligands such as Lys-D-Ala-D-Ala, but is applicable to a diverse range of targets. This strategy is likely to yield structural insights that accelerate new therapeutic development.     

Characterization,stoichiometry, and stability of salivary protein–tannin complexes by ESI-MS and ESI-MS/MS

Numerous protein–polyphenol interactions occur in biological and food domains particularly involving proline-rich proteins, which are representative of the intrinsically unstructured protein group (IUP). Noncovalent protein–ligand complexes are readily detected by electrospray ionization mass spectrometry (ESI-MS), which also gives access to ligand binding stoichiometry. Surprisingly, the study of interactions between polyphenolic molecules and proteins is still an area where ESI-MS has poorly benefited, whereas it has been extensively applied to the detection of noncovalent complexes. Electrospray ionization mass spectrometry has been applied to the detection and the characterization of the complexes formed between tannins and a human salivary proline-rich protein (PRP), namely IB5. The study of the complex stability was achieved by low-energy collision-induced dissociation (CID) measurements, which are commonly implemented using triple quadrupole, hybrid quadrupole time-of-flight, or ion trap instruments. Complexes composed of IB5 bound to a model polyphenol EgCG have been detected by ESI-MS and further analyzed by MS/MS. Mild ESI interface conditions allowed us to observe intact noncovalent PRP–tannin complexes with stoichiometries ranging from 1:1 to 1:5. Thus, ESI-MS shows its efficiency for (1) the study of PRP–tannin interactions, (2) the determination of stoichiometry, and (3) the study of complex stability. We were able to establish unambiguously both their stoichiometries and their overall subunit architecture via tandem mass spectrometry and solution disruption experiments. Our results prove that IB5·EgCG complexes are maintained intact in the gas phase.      

Top-down ESI-ECD-FT-ICR mass spectrometry localizes noncovalent protein-ligand binding sites

Mass spectrometry (MS) with electrospray ionization (ESI) has the capability to measure and detect noncovalent protein-ligand and protein-protein complexes. However, information on the sites of ligand binding is not easily obtained by the ESI-MS methodology. Electron capture dissociation (ECD) favors cleavage of covalent backbone bonds of protein molecules. We show that this characteristic of ECD translates to noncovalent protein-ligand complexes, as covalent backbone bonds of protein complexes are dissociated, but the noncovalent ligand interaction is retained. For the complex formed from 140-residue, 14.5 kDa alpha-synuclein protein, and one molecule of polycationic spermine (202 Da), ECD generates product ions that retain the protein-spermine noncovalent interaction. Spermine binding is localized to residues 106-138; the ECD data are consistent with previous solution NMR studies. Our studies suggest that ECD mass spectrometry can be used to determine directly the sites of ligand binding to protein targets.