Protein Complexes In Solution Research Articles

Structural analysis of clock protein complex in solution by the integrated approach with analytical ultracentrifugation and small-angle scattering

Structural analysis of clock protein complex in solution by the integrated approach with analytical ultracentrifugation and small-angle scattering

Conformational State Estimation of Biological Macromolecules in Solution using BioSAXS

Small-angle X-ray scattering（SAXS）for solution samples of biological macromolecules, recently called BioSAXS, has been used to estimate the conformational state of flexible proteins and unstable protein complexes in solution. With the advent of a new technique called SEC-SAXS, it has become easier to obtain structural information derived only from the target molecule, and it is now possible to estimate the structural state in solution with higher accuracy than 10 years ago. In this article, I will introduce not only some characteristic examples of BioSAXS analysis, but also the recently developing technique of time-resolved measurement.

Characterizing Heterogeneous Mixtures of Assembled States of the Tobacco Mosaic Virus Using Charge Detection Mass Spectrometry.

The tobacco mosaic viral capsid protein (TMV) is a frequent target for derivatization for myriad applications, including drug delivery, biosensing, and light harvesting. However, solutions of the stacked disk assembly state of TMV are difficult to characterize quantitatively due to their large size and multiple assembled states. Charge detection mass spectrometry (CDMS) addresses the need to characterize heterogeneous populations of large protein complexes in solution quickly and accurately. Using CDMS, previously unobserved assembly states of TMV, including 16-monomer disks and odd-numbered disk stacks, have been characterized. We additionally employed a peptide-protein conjugation reaction in conjunction with CDMS to demonstrate that modified TMV proteins do not redistribute between disks. Finally, this technique was used to discriminate between protein complexes of near-identical mass but different configurations. We have gained a greater understanding of the behavior of TMV, a protein used across a broad variety of fields and applications, in the solution state.

Структурные особенности кластеров ДНК-DPS при изменении концентрации 4- гексилрезорцина

The formation of complexes and crystals of bacterial nucleoid DNA inside bacterial cells in response to adverse external influences is of great interest both in biophysics and structural biology, and in various fields of industry. The main role in the condensation and crystallization of DNA in bacteria under stressful conditions (starvation, temperature, oxidative, and other types of stress) is played by DNA-binding proteins DPS. In this work, the dynamic behavior and structure of Escherichia coli DPS protein complexes in solution with short-chain DNA (25 base pairs) are studied using classical molecular dynamics in the all-atom approximation with a change in the concentration of the phenolic lipid 4-hexylresorcinol, which is a chemical analog of bacterial anabiosis inducers. The effect of changing the concentration of 
 4-hexylresorcinol from 0 to 50, 100 and 500 molecules on the DNA-DPS complex is being studied. By searching for the linear interaction energy, data were obtained on the free energies of DNA-protein binding without and in the presence of 4-hexylresorcinol. It has been shown that high concentrations of 
 4-hexylresorcinol promote the formation of DNA complexes with DPS. To identify the features of the dynamic behavior of DNA and protein, the principal component analysis was carried out. The spatial and energy characteristics of the complexes was obtained.

Modeling native state ensembles and macromolecular complexes using hydrogen-deuterium exchange ensemble reweighting

Gaining a high-resolution understanding of the conformational landscape of proteins and protein complexes in solution remains a challenge. Hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) is an insightful approach in probing a protein's structural dynamics in solution. It reports on the structural environment of the backbone amide hydrogens of a protein in a time resolved manner. As such, HDX-MS has now been widely applied to study proteins within various biophysical perspectives including the characterization of native state ensembles of proteins, macromolecular interactions, protein-small molecule interactions or protein folding and unfolding studies.

Single molecule mass photometry reveals the dynamic oligomerization of human and plant peroxiredoxins

SummaryProtein oligomerization is central to biological function and regulation, yet its experimental quantification and measurement of dynamic transitions in solution remain challenging. Here, we show that single molecule mass photometry quantifies affinity and polydispersity of heterogeneous protein complexes in solution. We demonstrate these capabilities by studying the functionally relevant oligomeric equilibria of 2-cysteine peroxiredoxins (2CPs). Comparison of the polydispersity of plant and human 2CPs as a function of concentration and redox state revealed features conserved among all 2CPs. In addition, we also find species-specific differences in oligomeric transitions, the occurrence of intermediates and the formation of high molecular weight complexes, which are associated with chaperone activity or act as a storage pool for more efficient dimers outlining the functional differentiation of human 2CPs. Our results point to a diversified functionality of oligomerization for 2CPs and illustrate the power of mass photometry for characterizing heterogeneous oligomeric protein distributions in near native conditions.

X‐ray Solution Scattering Studies at the Life Sciences X‐ray Scattering (LiX) Beamline

Solution small angle X-ray scattering (SAXS) is a method to characterize the behavior of particles such as proteins and protein complexes in solution. Information about particle size and shape as well as oligomeric state can be determined from the 1D scattering profiles. The life sciences X-ray scattering (LiX) beamline at NSLSII operates in two modes: I) X-ray imaging and II) solution scattering (BioSAXS). Here, we discuss the types of BioSAXS measurements performed, automation, remote collection, data processing and examples of user data. Static SAXS measurements are high throughput and managed with a sample handler, with room for up to 360 samples organized into 18-well holders. Each holder contains two rows that are connected to separate channels of a flow cell that aligns with the X-ray beam. As the sample flows into the channel and is illuminated by the X-ray beam, small angle (SAXS) and wide angle (WAXS) data is collected simultaneously on two detectors with a q-range of 0.006Å-1 to 3.2 Å-1. This type of measurement requires pure, monodisperse sample and for the case when mixed populations exist, such as dimers and monomers, static SAXS will produce poor data quality. Therefore, size exclusion chromatography coupled with SAXS (SEC-SAXS) should be considered to separate each species. At LiX, the center flow channel is connected to an HPLC system, which allows for seamless integration of SEC-SAXS into the workflow without having to make changes to the beamline hardware setup. Once data is collected, it is packaged into HDF5 format and automatically processed. Data is transferred to users via Globus. We now offer remote collection and users can ship samples in holders or 96-well plates. Samples sent in plates are loaded into an Opentrons liquid handling system and transferred into sample holders. Additionally, having the Opentrons allow us to manipulate sample conditions through dilution, mixing and/or screening right before measurement.

Discovery of Potent Charge-Reducing Molecules for Native Ion Mobility Mass Spectrometry Studies.

There is growing interest in the characterization of protein complexes and their interactions with ligands using native ion mobility mass spectrometry. A particular challenge, especially for membrane proteins, is preserving noncovalent interactions and maintaining native-like structures. Different approaches have been developed to minimize activation of protein complexes by manipulating charge on protein complexes in solution and the gas-phase. Here, we report the utility of polyamines that have exceptionally high charge-reducing potencies with some molecules requiring 5-fold less than trimethylamine oxide to elicit the same effect. The charge-reducing molecules do not adduct to membrane protein complexes and are also compatible with ion-mobility mass spectrometry, paving the way for improved methods of charge reduction.

Machine Learning Driven Analysis of Large Scale Simulations Reveals Conformational Characteristics of Ubiquitin Chains.

Understanding the conformational characteristics of protein complexes in solution is crucial for a deeper insight in their biological function. Molecular dynamics simulations performed on high performance computing plants and with modern simulation techniques can be used to obtain large data sets that contain conformational and thermodynamic information about biomolecular systems. While this can in principle give a detailed picture of protein-protein interactions in solution and therefore complement experimental data, it also raises the challenge of processing exceedingly large high-dimensional data sets with several million samples. Here we present a novel method for the characterization of protein-protein interactions, which combines a neural network based dimensionality reduction technique to obtain a two-dimensional representation of the conformational space with a density based clustering algorithm for state detection and a metric which assesses the (dis)similarity between different conformational spaces. This method is highly scalable and therefore makes the analysis of massive data sets computationally tractable. We demonstrate the power of this approach to large scale data analysis by characterizing highly dynamic conformational phase spaces of differently linked ubiquitin (Ub) oligomers from coarse-grained simulations. We are able to extract a protein-protein interaction model for two unlinked Ub proteins which is then used to determine how the Ub-Ub interaction pattern is altered in Ub oligomers by the introduction of a covalent linkage. We find that the Ub chain conformational ensemble depends highly on the linkage type and for some cases also on the Ub chain length. By this, we obtain insight into the conformational characteristics of different Ub chains and how this may contribute to linkage type and chain length specific recognition.

Probing the Broad Time Scale and Heterogeneous Conformational Dynamics in the Catalytic Core of the Arf-GAP ASAP1 via Methyl Adiabatic Relaxation Dispersion.

Methyl-TROSY is one of the most powerful NMR spectroscopic tools for studying structures and conformational dynamics of large protein complexes in solution. In studying conformational dynamics, side chains usually display heterogeneous dynamics, including collective and local motions, that can be difficult to detect and analyze by conventional relaxation dispersion (RD) approaches. The combination of NH-based heteronuclear adiabatic relaxation dispersion (HARD) experiments and a geometric approximation (geoHARD) has been shown to have several advantages over conventional RD in revealing conformational dynamics over a broad time scale. Here, we demonstrate a new technique that has been developed to detect both heterogeneous and wide time scale conformational dynamics in the hydrophobic interior of large macromolecules utilizing methyl-geoHARD. It is shown that methyl-geoHARD will be feasible at ultrahigh magnetic fields (>1 GHz), when this technology becomes available. For the ZA domain of Arf-GAP ASAP1, with a global correlational time of 24 ns at 15 °C, a wide range of conformational dynamics (exhibiting chemical exchange rates (kex) between 102 and 105 s-1) are observed in the methyl groups of isoleucine, leucine, and valine. The dynamics include collective and independent local motions. Furthermore, portions of the collective motions have been confirmed by single-quantum Carr-Purcell-Meiboom-Gill (SQ-CPMG) RD experiments; however, motions outside of the detectable CPMG window (400-8000 s-1) cannot be accurately determined by SQ-CPMG experiments. The methyl-geoHARD experiment allows the dissection of heterogeneous conformational dynamics and pinpoints important motions that, potentially, can be correlated with important biological functions and recognition.

A Cholesterol Analog Induces an Oligomeric Reorganization of VDAC

The oligomeric organization of the voltage-dependent anion-selective channel (VDAC) and its interactions with hexokinase play integral roles in mitochondrially mediated apoptotic signaling. Various small to large assemblies of VDAC are observed in mitochondrial outer membranes, but they do not predominate in detergent-solubilized VDAC samples. In this study, a cholesterol analog, cholesteryl-hemisuccinate (CHS), was shown to induce the formation of detergent-soluble VDAC multimers. The various oligomeric states of VDAC induced by the addition of CHS were deciphered through an integrated biophysics approach using microscale thermophoresis, analytical ultracentrifugation, and size-exclusion chromatography small angle x-ray scattering. Furthermore, CHS stabilizes the interaction between VDAC and hexokinase (Kd of 27 ± 6 μM), confirming the biological relevance of oligomers generated. Thus, sterols such as cholesterol in higher eukaryotes or ergosterol in fungi may regulate the VDAC oligomeric state and may provide a potential target for the modulation of apoptotic signaling by effecting VDAC-VDAC and VDAC-hexokinase interactions. In addition, the integrated biophysical approach described provides a powerful platform for the study of membrane protein complexes in solution.

Localization of Protein Complex Bound Ligands by Surface-Induced Dissociation High-Resolution Mass Spectrometry.

Surface-induced dissociation (SID) is a powerful means of deciphering protein complex quaternary structures due to its capability of yielding dissociation products that reflect the native structures of protein complexes in solution. Here we explore the suitability of SID to locate the ligand binding sites in protein complexes. We studied C-reactive protein (CRP) pentamer, which contains a ligand binding site within each subunit, and cholera toxin B (CTB) pentamer, which contains a ligand binding site between each adjacent subunit. SID dissects ligand-bound CRP into subcomplexes with each subunit carrying predominantly one ligand. In contrast, SID of ligand-bound CTB results in the generation of subcomplexes with a ligand distribution reflective of two subunits contributing to each ligand binding site. SID thus has potential application in localizing sites of small ligand binding for multisubunit protein-ligand complexes.

Merging In-Solution X-ray and Neutron Scattering Data Allows Fine Structural Analysis of Membrane-Protein Detergent Complexes.

In-solution small-angle X-ray and neutron scattering (SAXS/SANS) have become popular methods to characterize the structure of membrane proteins, solubilized by either detergents or nanodiscs. SANS studies of protein-detergent complexes usually require deuterium-labeled proteins or detergents, which in turn often lead to problems in their expression or purification. Here, we report an approach whose novelty is the combined analysis of SAXS and SANS data from an unlabeled membrane protein complex in solution in two complementary ways. First, an explicit atomic analysis, including both protein and detergent molecules, using the program WAXSiS, which has been adapted to predict SANS data. Second, the use of MONSA which allows one to discriminate between detergent head- and tail-groups in an ab initio approach. Our approach is readily applicable to any detergent-solubilized protein and provides more detailed structural information on protein-detergent complexes from unlabeled samples than SAXS or SANS alone.

Capturing dynamic conformational shifts in protein-ligand recognition using integrative structural biology in solution.

In recent years, a dynamic view of the structure and function of biological macromolecules is emerging, highlighting an essential role of dynamic conformational equilibria to understand molecular mechanisms of biological functions. The structure of a biomolecule, i.e. protein or nucleic acid in solution, is often best described as a dynamic ensemble of conformations, rather than a single structural state. Strikingly, the molecular interactions and functions of the biological macromolecule can then involve a shift between conformations that pre-exist in such an ensemble. Upon external cues, such population shifts of pre-existing conformations allow gradually relaying the signal to the downstream biological events. An inherent feature of this principle is conformational dynamics, where intrinsically disordered regions often play important roles to modulate the conformational ensemble. Unequivocally, solution-state NMR spectroscopy is a powerful technique to study the structure and dynamics of such biomolecules in solution. NMR is increasingly combined with complementary techniques, including fluorescence spectroscopy and small angle scattering. The combination of these techniques provides complementary information about the conformation and dynamics in solution and thus affords a comprehensive description of biomolecular functions and regulations. Here, we illustrate how an integrated approach combining complementary techniques can assess the structure and dynamics of proteins and protein complexes in solution.

Synthesis of CID-cleavable protein crosslinking agents containing quaternary amines for structural mass spectrometry.

Two novel cyclic quaternary amine crosslinking probes are synthesized for structural mass spectrometry of protein complexes in solution and for analysis of protein interactions in organellar and whole cell extracts. Each exhibits high aqueous solubility, excellent protein crosslinking efficiencies, low collision induced dissociation (CID) energy fragmentation efficiencies, high stoichiometries of reaction, increased charges of crosslinked peptide ions, and maintenance of overall surface charge balance of crosslinked proteins.

Small-Angle X-ray Scattering Data in Combination with RosettaDock Improves the Docking Energy Landscape.

We have performed a benchmark to evaluate the relative success of using small-angle X-ray scattering (SAXS) data as constraints (hereafter termed SAXSconstrain) in the RosettaDock protocol (hereafter termed RosettaDockSAXS). For this purpose, we have chosen 38 protein complex structures, calculated the theoretical SAXS data for the protein complexes using the program CRYSOL, and then used the SAXS data as constraints. We further considered a few examples where crystal structures and experimental SAXS data are available. SAXSconstrain were added to the protocol in the initial, low-resolution docking step, allowing fast rejection of complexes that violate the shape restraints imposed by the SAXS data. Our results indicate that the implementation of SAXSconstrain in general reduces the sampling space of possible protein-protein complexes significantly and can indeed increase the probability of finding near-native protein complexes. The methodology used is based on rigid-body docking and works for cases where no or minor conformational changes occur upon binding of the docking partner. In a wider perspective, the strength of RosettaDockSAXS lies in the combination of low-resolution structural information on protein complexes in solution from SAXS experiments with protein-protein interaction energies obtained from RosettaDock, which will allow the prediction of unknown three-dimensional atomic structures of protein-protein complexes.

The structural flexibility of the human copper chaperone Atox1: Insights from combined pulsed EPR studies and computations.

Metallochaperones are responsible for shuttling metal ions to target proteins. Thus, a metallochaperone's structure must be sufficiently flexible both to hold onto its ion while traversing the cytoplasm and to transfer the ion to or from a partner protein. Here, we sought to shed light on the structure of Atox1, a metallochaperone involved in the human copper regulation system. Atox1 shuttles copper ions from the main copper transporter, Ctr1, to the ATP7b transporter in the Golgi apparatus. Conventional biophysical tools such as X-ray or NMR cannot always target the various conformational states of metallochaperones, owing to a requirement for crystallography or low sensitivity and resolution. Electron paramagnetic resonance (EPR) spectroscopy has recently emerged as a powerful tool for resolving biological reactions and mechanisms in solution. When coupled with computational methods, EPR with site-directed spin labeling and nanoscale distance measurements can provide structural information on a protein or protein complex in solution. We use these methods to show that Atox1 can accommodate at least four different conformations in the apo state (unbound to copper), and two different conformations in the holo state (bound to copper). We also demonstrate that the structure of Atox1 in the holo form is more compact than in the apo form. Our data provide insight regarding the structural mechanisms through which Atox1 can fulfill its dual role of copper binding and transfer.

Investigating the Interaction between the Neonatal Fc Receptor and Monoclonal Antibody Variants by Hydrogen/Deuterium Exchange Mass Spectrometry

The recycling of immunoglobulins by the neonatal Fc receptor (FcRn) is of crucial importance in the maintenance of antibody levels in plasma and is responsible for the long half-lives of endogenous and recombinant monoclonal antibodies. From a therapeutic point of view there is great interest in understanding and modulating the IgG-FcRn interaction to optimize antibody pharmacokinetics and ultimately improve efficacy and safety. Here we studied the interaction between a full-length human IgG(1) and human FcRn via hydrogen/deuterium exchange mass spectrometry and targeted electron transfer dissociation to map sites perturbed by binding on both partners of the IgG-FcRn complex. Several regions in the antibody Fc region and the FcRn were protected from exchange upon complex formation, in good agreement with previous crystallographic studies of FcRn in complex with the Fc fragment. Interestingly, we found that several regions in the IgG Fab region also showed reduced deuterium uptake. Our findings indicate the presence of hitherto unknown FcRn interaction sites in the Fab region or a possible conformational link between the IgG Fc and Fab regions upon FcRn binding. Further, we investigated the role of IgG glycosylation in the conformational response of the IgG-FcRn interaction. Removal of antibody glycans increased the flexibility of the FcRn binding site in the Fc region. Consequently, FcRn binding did not induce a similar conformational stabilization of deglycosylated IgG as observed for the wild-type glycosylated IgG. Our results provide new molecular insight into the IgG-FcRn interaction and illustrate the capability of hydrogen/deuterium exchange mass spectrometry to advance structural proteomics by providing detailed information on the conformation and dynamics of large protein complexes in solution.

Structural Analysis of Protein-RNA Complexes in Solution Using NMR Paramagnetic Relaxation Enhancements.

Biological activity in the cell is predominantly mediated by large multiprotein and protein-nucleic acid complexes that act together to ensure functional fidelity. Nuclear magnetic resonance (NMR) spectroscopy is the only method that can provide information for high-resolution three-dimensional structures and the conformational dynamics of these complexes in solution. Mapping of binding interfaces and molecular interactions along with the characterization of conformational dynamics is possible for very large protein complexes. In contrast, de novo structure determination by NMR becomes very time consuming and difficult for protein complexes larger than 30 kDa as data are noisy and sparse. Fortunately, high-resolution structures are often available for individual domains or subunits of a protein complex and thus sparse data can be used to define their arrangement and dynamics within the assembled complex. In these cases, NMR can therefore be efficiently combined with complementary solution techniques, such as small-angle X-ray or neutron scattering, to provide a comprehensive description of the structure and dynamics of protein complexes in solution. Particularly useful are NMR-derived paramagnetic relaxation enhancements (PREs), which provide long-range distance restraints (ca. 20Å) for structural analysis of large complexes and also report on conformational dynamics in solution. Here, we describe the use of PREs from sample production to structure calculation, focusing on protein-RNA complexes. On the basis of recent examples from our own research, we demonstrate the utility, present protocols, and discuss potential pitfalls when using PREs for studying the structure and dynamic features of protein-RNA complexes.

The dynamic duo: Combining NMR and small angle scattering in structural biology

Structural biology provides essential information for elucidating molecular mechanisms that underlie biological function. Advances in hardware, sample preparation, experimental methods, and computational approaches now enable structural analysis of protein complexes with increasing complexity that more closely represent biologically entities in the cellular environment. Integrated multidisciplinary approaches are required to overcome limitations of individual methods and take advantage of complementary aspects provided by different structural biology techniques. Although X-ray crystallography remains the method of choice for structural analysis of large complexes, crystallization of flexible systems is often difficult and does typically not provide insights into conformational dynamics present in solution. Nuclear magnetic resonance spectroscopy (NMR) is well-suited to study dynamics at picosecond to second time scales, and to map binding interfaces even of large systems at residue resolution but suffers from poor sensitivity with increasing molecular weight. Small angle scattering (SAS) methods provide low resolution information in solution and can characterize dynamics and conformational equilibria complementary to crystallography and NMR. The combination of NMR, crystallography, and SAS is, thus, very useful for analysis of the structure and conformational dynamics of (large) protein complexes in solution. In high molecular weight systems, where NMR data are often sparse, SAS provides additional structural information and can differentiate between NMR-derived models. Scattering data can also validate the solution conformation of a crystal structure and indicate the presence of conformational equilibria. Here, we review current state-of-the-art approaches for combining NMR, crystallography, and SAS data to characterize protein complexes in solution.