NMR Assessment of Therapeutic Peptides and Proteins: Correlations That Reveal Interactions and Motions

NMR measurements of rotational and translational diffusion are used to characterize the solution behavior of a wide variety of therapeutic proteins and peptides. The timescales of motions sampled in these experiments reveal complicated intrinsic solution behavior such as flexibility, that is central to function, as well as self-interactions, stress-induced conformational changes and other critical attributes that can be discovery and development liabilities. Trends from proton transverse relaxation (R₂) and hydrodynamic radius (R_h) are correlated and used to identify and differentiate intermolecular from intramolecular interactions. In this study, peptide behavior is consistent with complicated multimer self-assembly, while multi-domain protein behavior is dominated by intramolecular interactions. These observations are supplemented by simulations that include effects from slow transient interactions and rapid internal motions. R₂–R_h correlations provide a means to profile protein motions as well as interactions. The approach is completely general and can be applied to therapeutic and target protein characterization.     

Adhesion-Induced Interactions between Micron-Sized Zirconia or Carbon Spheres and Melamine-Cross-Linked Polyester Surfaces

The thermodynamic work of adhesion between micron-sized zirconia or carbon spheres and polyester–melamine surfaces was determined using the Johnson-Kendall-Roberts (JKR) relationship, after first validating the JKR approach for this system. The calculated works of adhesion for both zirconia and carbon were similar for any given substrate composition and were found to be approximately 35 mJ/m² at low melamine concentrations, close to theoretical predictions. The apparent work of adhesion decreased with increasing melamine concentration, most likely due to the presence of a glassy melamine-rich surface layer, which is not representative of the bulk. The value found for low melamine concentration was assumed to be a true value for cocondensed polyester–melamine, and this was used to estimate surface modulus and the amount of excess melamine in the surface as a function of bulk composition.     

Adhesion-Induced Interactions between Micron-Sized Zirconia or Carbon Spheres and Melamine-Cross-Linked Polyester Surfaces

The thermodynamic work of adhesion between micron-sized zirconia or carbon spheres and polyester-melamine surfaces was determined using the Johnson-Kendall-Roberts (JKR) relationship, after first validating the JKR approach for this system. The calculated works of adhesion for both zirconia and carbon were similar for any given substrate composition and were found to be approximately 35 mJ/m² at low melamine concentrations, close to theoretical predictions. The apparent work of adhesion decreased with increasing melamine concentration, most likely due to the presence of a glassy melamine-rich surface layer, which is not representative of the bulk. The value found for low melamine concentration was assumed to be a true value for cocondensed polyester-melamine, and this was used to estimate surface modulus and the amount of excess melamine in the surface as a function of bulk composition.     

Solution NMR Structure of Hypothetical Protein CV_2116 Encoded by a Viral Prophage Element in Chromobacterium violaceum

CV_2116 is a small hypothetical protein of 82 amino acids from the Gram-negative coccobacillus Chromobacterium violaceum. A PSI-BLAST search using the CV_2116 sequence as a query identified only one hit (E = 2e(-07)) corresponding to a hypothetical protein OR16_04617 from Cupriavidus basilensis OR16, which failed to provide insight into the function of CV_2116. The CV_2116 gene was cloned into the p15TvLic expression plasmid, transformed into E. coli, and (13)C- and (15)N-labeled NMR samples of CV_2116 were overexpressed in E. coli and purified for structure determination using NMR spectroscopy. The resulting high-quality solution NMR structure of CV_2116 revealed a novel α + β fold containing two anti-parallel β-sheets in the N-terminal two-thirds of the protein and one α-helix in the C-terminal third of the protein. CV_2116 does not belong to any known protein sequence family and a Dali search indicated that no similar structures exist in the protein data bank. Although no function of CV_2116 could be derived from either sequence or structural similarity searches, the neighboring genes of CV_2116 encode various proteins annotated as similar to bacteriophage tail assembly proteins. Interestingly, C. violaceum exhibits an extensive network of bacteriophage tail-like structures that likely result from lateral gene transfer by incorporation of viral DNA into its genome (prophages) due to bacteriophage infection. Indeed, C. violaceum has been shown to contain four prophage elements and CV_2116 resides in the fourth of these elements. Analysis of the putative operon in which CV_2116 resides indicates that CV_2116 might be a component of the bacteriophage tail-like assembly that occurs in C. violaceum.     

Functionalisation of Inorganic Material Surfaces with Staphylococcus Protein A: A Molecular Dynamics Study

Staphylococcus protein A (SpA) is found in the cell wall of Staphylococcus aureus bacteria. Its ability to bind to the constant Fc regions of antibodies means it is useful for antibody extraction, and further integration with inorganic materials can lead to the development of diagnostics and therapeutics. We have investigated the adsorption of SpA on inorganic surface models such as experimentally relevant negatively charged silica, as well as positively charged and neutral surfaces, by use of fully atomistic molecular dynamics simulations. We have found that SpA, which is itself negatively charged at pH7, is able to adsorb on all our surface models. However, adsorption on charged surfaces is more specific in terms of protein orientation compared to a neutral Au (111) surface, while the protein structure is generally well maintained in all cases. The results indicate that SpA adsorption is optimal on the siloxide-rich silica surface, which is negative at pH7 since this keeps the Fc binding regions free to interact with other species in solution. Due to the dominant role of electrostatics, the results are transferable to other inorganic materials and pave the way for new diagnostic and therapeutic designs where SpA might be used to conjugate antibodies to nanoparticles.     

Solution Structure and Dynamics of the Small Protein HVO_2922 from Haloferax volcanii

Past sequencing campaigns overlooked small proteins as they seemed to be irrelevant due to their small size. However, their occurrence is widespread, and there is growing evidence that these small proteins are in fact functionally very important in organisms found in all kingdoms of life. Within a global proteome analysis for small proteins of the archaeal model organism Haloferax volcanii, the HVO_2922 protein has been identified. It is differentially expressed in response to changes in iron and salt concentrations, thus suggesting that its expression is stress-regulated. The protein is conserved among Haloarchaea and contains an uncharacterized domain of unknown function (DUF1508, UPF0339 family protein). We elucidated the NMR solution structure, which shows that the isolated protein forms a symmetrical dimer. The dimerization is found to be concentration-dependent and essential for protein stability and most likely for its functionality, as mutagenesis at the dimer interface leads to a decrease in stability and protein aggregation.     

Protein interactions in the Escherichia coli cytosol: an impediment to in-cell NMR spectroscopy

Protein science is shifting towards experiments performed under native or native-like conditions. In-cell NMR spectroscopy for instance has the potential to reveal protein structure and dynamics inside cells. However, not all proteins can be studied by this technique. (15)N-labelled cytochrome c (cyt c) over-expressed in Escherichia coli was undetectable by in-cell NMR spectroscopy. When whole-cell lysates were subjected to size-exclusion chromatography (SEC) cyt c was found to elute with an apparent molecular weight of >150 kDa. The presence of high molecular weight species is indicative of complex formation between cyt c and E. coli cytosolic proteins. These interactions were disrupted by charge-inverted mutants in cyt c and by elevated concentrations of NaCl. The physiologically relevant salt, KGlu, was less efficient at disrupting complex formation. Notably, a triple mutant of cyt c could be detected in cell lysates by NMR spectroscopy. The protein, GB1, yields high quality in-cell spectra and SEC analysis of lysates containing GB1 revealed a lack of interaction between GB1 and E. coli proteins. Together these data suggest that protein "stickiness" is a limiting factor in the application of in-cell NMR spectroscopy.     

Transient Interactions of a Cytosolic Protein with Macromolecular and Vesicular Cosolutes: Unspecific and Specific Effects

Cytosolic proteins do not occur as isolated but are exposed to many interactions within a crowded cellular environment. We investigated the associations between a test cytosolic protein, human ileal bile acid binding protein (IBABP), and model cosolutes mimicking macromolecular and lipid membrane intracellular components. Using fluorescence spectroscopy, heteronuclear NMR, and molecular dynamics, we found that IBABP associated weakly with anionic lipid vesicles and experienced transient unspecific contacts with albumin. Localized dynamic perturbations were observed even in the case of apparent unspecific binding. IBABP and ubiquitin did not display mutually attractive forces, whereas IBABP associated specifically with lysozyme. A structural model of the IBABP–lysozyme complex was obtained by data‐driven docking simulation. Presumably, all the interactions shown here contribute to modulating functional communication of a protein in its native environment.     

Using Singular Value Decomposition to Characterize Protein–Protein Interactions by In‐cell NMR Spectroscopy

Distinct differences between how model proteins interact in‐cell and in vitro suggest that the cytosol might have a profound effect in modulating protein–protein and/or protein–ligand interactions that are not observed in vitro. Analyses of in‐cell NMR spectra of target proteins interacting with physiological partners are further complicated by low signal‐to‐noise ratios, and the long overexpression times used in protein–protein interaction studies may lead to changes in the in‐cell spectra over the course of the experiment. To unambiguously resolve the principal binding mode between two interacting species against the dynamic cellular background, we analyzed in‐cell spectral data of a target protein over the time course of overexpression of its interacting partner by using single‐value decomposition (SVD). SVD differentiates between concentration‐dependent and concentration‐independent events and identifies the principal binding mode between the two species. The analysis implicates a set of amino acids involved in the specific interaction that differs from previous NMR analyses but is in good agreement with crystallographic data.     

The Lattice Model of Particles with Orientation-Dependent Interactions at Solid Surfaces: Wetting Scenarios

Wetting phenomena in a lattice model of particles having two chemically different halves (A and B) and being in contact with solid substrates have been studied with Monte Carlo methods. The energy of the interaction between a pair of neighboring particles has been assumed to depend on the degree to which the AA, AB and BB regions face each other. In this work, we have assumed that uAA=−1.0 and considered three series of systems with uAB=uBB, uAB=0 and uBB=0. The phase behavior of bulk systems has been determined. In particular, it has been shown that at sufficiently low temperatures the bulk systems order into the superantiferromagnetic (SAF) phase, or into the antiferromagnetic (AF) phase, depending on the magnitudes of AA, AB and BB interaction energies, uAA, uAB and uBB. The SAF structure occurs whenever ϵ=uAA+uBB−2uAB is lower than zero and the AF structure is stable when ϵ is greater than zero. The wetting behavior has been demonstrated to depend strongly on the structure of the bulk condensed phase, the interactions between fluid particles and the strength of the surface potential. In all series, we have found the dewetting transition, resulting from the limited stability of different ordered structures of surface phases. However, in the systems that exhibit the gas–liquid transition in the bulk, the reentrant wetting transition has been observed at sufficiently high temperatures. The mechanism of dewetting and reentrant wetting transitions has been determined. Moreover, we have also demonstrated, how the dewetting transition in the series with uAB=0 is affected by the wall selectivity, i.e., when the interaction between the parts A and B of fluid particles and the solid is different.     

Interaction between Nanoparticles,Membranes and Proteins: A Surface Plasmon Resonance Study

Regardless of the promising use of nanoparticles (NPs) in biomedical applications, several toxic effects have increased the concerns about the safety of these nanomaterials. Although the pathways for NPs toxicity are diverse and dependent upon many parameters such as the nature of the nanoparticle and the biochemical environment, numerous studies have provided evidence that direct contact between NPs and biomolecules or cell membranes leads to cell inactivation or damage and may be a primary mechanism for cytotoxicity. In such a context, this work focused on developing a fast and accurate method to characterize the interaction between NPs, proteins and lipidic membranes by surface plasmon resonance imaging (SPRi) technique. The interaction of gold NPs with mimetic membranes was evaluated by monitoring the variation of reflectivity after several consecutive gold NPs injections on the lipidic membranes prepared on the SPRi biochip. The interaction on the membranes with varied lipidic composition was compared regarding the total surface concentration density of gold NPs adsorbed on them. Then, the interaction of gold and silver NPs with blood proteins was analyzed regarding their kinetic profile of the association/dissociation and dissociation constants (k_off). The surface concentration density on the membrane composed of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine and cholesterol (POPC/cholesterol) was 2.5 times higher than the value found after the injections of gold NPs on POPC only or with dimethyldioctadecylammonium (POPC/DDAB). Regarding the proteins, gold NPs showed preferential binding to fibrinogen resulting in a value of the variation of reflectivity that was 8 times higher than the value found for the other proteins. Differently, silver NPs showed similar interaction on all the tested proteins but with a variation of reflectivity on immunoglobulin G (IgG) 2 times higher than the value found for the other tested proteins.     

NMR Structure and Biophysical Characterization of Thermophilic Single-Stranded DNA Binding Protein from Sulfolobus Solfataricus

Proteins from Sulfolobus solfataricus (S. solfataricus), an extremophile, are active even at high temperatures. The single-stranded DNA (ssDNA) binding protein of S. solfataricus (SsoSSB) is overexpressed to protect ssDNA during DNA metabolism. Although SsoSSB has the potential to be applied in various areas, its structural and ssDNA binding properties at high temperatures have not been studied. We present the solution structure, backbone dynamics, and ssDNA binding properties of SsoSSB at 50 °C. The overall structure is consistent with the structures previously studied at room temperature. However, the loop between the first two β sheets, which is flexible and is expected to undergo conformational change upon ssDNA binding, shows a difference from the ssDNA bound structure. The ssDNA binding ability was maintained at high temperature, but different interactions were observed depending on the temperature. Backbone dynamics at high temperature showed that the rigidity of the structured region was well maintained. The investigation of an N-terminal deletion mutant revealed that it is important for maintaining thermostability, structure, and ssDNA binding ability. The structural and dynamic properties of SsoSSB observed at high temperature can provide information on the behavior of proteins in thermophiles at the molecular level and guide the development of new experimental techniques.     

Lights,Camera, Interaction: Studying Protein–Protein Interactions of the ER Protein Translocase in Living Cells

Various landmark studies have revealed structures and functions of the Sec61/SecY complex in all domains of live demonstrating the conserved nature of this ancestral protein translocase. While the bacterial homolog of the Sec61 complex resides in the plasma membrane, the eukaryotic counterpart manages the transfer of precursor proteins into or across the membrane of the endoplasmic reticulum (ER). Sec61 complexes are accompanied by a set of dynamically recruited auxiliary proteins assisting the transport of certain precursor polypeptides. TRAP and Sec62/Sec63 are two auxiliary protein complexes in mammalian cells that have been characterized by structural and biochemical methods. Using these ER membrane protein complexes for our proof-of-concept study, we aimed to detect interactions of membrane proteins in living mammalian cells under physiological conditions. Bimolecular luminescence complementation and competition was used to demonstrate multiple protein–protein interactions of different topological layouts. In addition to the interaction of the soluble catalytic and regulatory subunits of the cytosolic protein kinase A, we detected interactions of ER membrane proteins that either belong to the same multimeric protein complex (intra-complex interactions: Sec61α–Sec61β, TRAPα–TRAPβ) or protein complexes in juxtaposition (inter-complex interactions: Sec61α–TRAPα, Sec61α–Sec63, and Sec61β–Sec63). In the process, we established further control elements like synthetic peptide complementation for expression profiling of fusion constructs and protease-mediated reporter degradation demonstrating the cytosolic localization of a reporter complementation. Ease of use and flexibility of the approach presented here will spur further research regarding the dynamics of protein–protein interactions in response to changing cellular conditions in living cells.     

Assessing Carbohydrate–Carbohydrate Interactions by NMR Spectroscopy: The Trisaccharide Epitope from the Marine Sponge Microciona prolifera

Weak recognition processes : Weak calcium‐mediated carbohydrate–carbohydrate interactions have been detected by DOSY and TRNOESY NMR methods by employing a gold glyconanoparticle as a multivalent system. In addition, 3D models of trisaccharide‐Ca^II‐trisaccharide complexes based on results from molecular dynamics simulations are proposed.

     

Huntingtin: A Protein with a Peculiar Solvent Accessible Surface

Taking advantage of the last cryogenic electron microscopy structure of human huntingtin, we explored with computational methods its physicochemical properties, focusing on the solvent accessible surface of the protein and highlighting a quite interesting mix of hydrophobic and hydrophilic patterns, with the prevalence of the latter ones. We then evaluated the probability of exposed residues to be in contact with other proteins, discovering that they tend to cluster in specific regions of the protein. We then found that the remaining portions of the protein surface can contain calcium-binding sites that we propose here as putative mediators for the protein to interact with membranes. Our findings are justified in relation to the present knowledge of huntingtin functional annotation.     

cpxDeepMSA: A Deep Cascade Algorithm for Constructing Multiple Sequence Alignments of Protein–Protein Interactions

Protein–protein interactions (PPIs) are fundamental to many biological processes. The coevolution-based prediction of interacting residues has made great strides in protein complexes that are known to interact. A multiple sequence alignment (MSA) is the basis of coevolution analysis. MSAs have recently made significant progress in the protein monomer sequence analysis. However, no standard or efficient pipelines are available for the sensitive protein complex MSA (cpxMSA) collection. How to generate cpxMSA is one of the most challenging problems of sequence coevolution analysis. Although several methods have been developed to address this problem, no standalone program exists. Furthermore, the number of built-in properties is limited; hence, it is often difficult for users to analyze sequence coevolution according to their desired cpxMSA. In this article, we developed a novel cpxMSA approach (cpxDeepMSA. We used different protein monomer databases and incorporated the three strategies (genomic distance, phylogeny information, and STRING interaction network) used to join the monomer MSA results of protein complexes, which can prevent using a single method fail to the joint two-monomer MSA causing the cpxMSA construction failure. We anticipate that the cpxDeepMSA algorithm will become a useful high-throughput tool in protein complex structure predictions, inter-protein residue-residue contacts, and the biological sequence coevolution analysis.