Storage stability of the solution formulation of sCD4 determined by DSC in comparison with two functional assays

Differential scanning calorimetry (DSC) was used, in conjunction with two functional assays that monitor binding, in a storage stability study on a protein of pharmaceutical interest, the soluble form of the T-lymphocyte multidomain surface receptor, sCD4. DSC monitored structural changes in binding and non-binding domains. ELISA, using the monoclonal antibody OKT4a, and frontal elution affinity chromatography, using the HIV surface glycoprotein, gp120, monitored function of the binding domain. The stability of sCD4 in a solution formulation was followed for up to 30 days at five differentpHs ranging from 5.0 to 7.9 and five different temperatures ranging from –70C to 40C. While the overall trends observed with the three techniques were the same, the ELISA data were somewhat less reproducible than those for the other methods. Furthermore, the results suggest that DSC is more sensitive to structural changes that would reduce the protein's bioactivity. The results of this study indicate DSC's utility, in conjunction with quantitative functional analysis, in the formulation of protein-containing pharmaceuticals or foods, especially those containing multiple-domain proteins.This work was supported by NIH Grant AI32687.     

Identification of unknown protein complex members by radiolocalization and analysis of low-abundance complexes resolved using native polyacrylamide gel electrophoresis

Identification of unknown binding partners of a protein of interest can be a difficult process. Current strategies to determine protein binding partners result in a high amount of false-positives, requiring use of several different methods to confirm the accuracy of the apparent association. We have developed and utilized a method that is reliable and easily substantiated. Complexes are isolated from cell extract after exposure to the radiolabeled protein of interest, followed by resolution on a native polyacrylamide gel. Native conformations are preserved, allowing the complex members to maintain associations. By radiolabeling the protein of interest, the complex can be easily identified at detection levels below the threshold of Serva Blue, Coomassie, and silver stains. The visualized radioactive band is analyzed by MS to identify binding partners, which can be subsequently verified by antibody shift and immunoprecipitation of the complex. By using this method we have successfully identified binding partners of two proteins that reside in different locations of a cellular organelle.     

A mass spectrometry screening method for antiaggregatory activity of proteins covalently modified by combinatorial library members: application to sickle hemoglobin

A homogeneous assay, based on electrospray mass spectrometry, is described for identifying compounds in a combinatorial library that covalently modify a protein and thereby enhance its solubility. The technique is based on measuring the distribution of modified proteins in the supernatant versus aggregate. Compounds having the greatest anti-aggregatory activity are those with the highest supernatant/aggregate ratio. Mass is used as a marker to identify which covalent modifier in the library is involved. An exploratory study is presented which demonstrates that the antisickling activity of a family of isothiocyanates, as measured by the standard C(sat) assay, correlates well (r(2) = 0.98) with the mass spectrometry analysis of the supernatant/aggregate distribution. The technique has potential for screening libraries capable of covalently modifying other proteins of clinical interest, e.g., Alzheimer's, Huntington's, and various prion related diseases.     

Methods for the prediction of protein-ligand binding sites for structure-based drug design and virtual ligand screening

Structure Based Drug Design (SBDD) is a computational approach to lead discovery that uses the three-dimensional structure of a protein to fit drug-like molecules into a ligand binding site to modulate function. Identifying the location of the binding site is therefore a vital first step in this process, restricting the search space for SBDD or virtual screening studies. The detection and characterisation of functional sites on proteins has increasingly become an area of interest. Structural genomics projects are increasingly yielding protein structures with unknown functions and binding sites. Binding site prediction was pioneered by pocket detection, since the binding site is often found in the largest pocket. More recent methods involve phylogenetic analysis, identifying structural similarity with proteins of known function and identifying regions on the protein surface with a potential for high binding affinity. Binding site prediction has been used in several SBDD projects and has been incorporated into several docking tools. We discuss different methods of ligand binding site prediction, their strengths and weaknesses, and how they have been used in SBDD.     

Functional assessment of “in vivo” and “in silico” mutations in the guanine binding site of RNase T1: A DFT study

Experimentally, the functional assessment of amino acid side chains in proteins is carried out by comparing parameters such as binding constants for the wild‐type protein and a mutant protein in which the considered side chain is deleted. In the present study, we apply a density functional theory (DFT) methodology to obtain changes in binding energy upon mutations in the enzyme ribonuclease T₁. Mutant structures were either taken directly from crystallographic data (“in vivo”) allowing for conformational changes upon mutation, or derived from the wild‐type (“in silico”). Excluding entropic contributions, the computed interaction energy changes upon mutation in vivo correlate qualitatively well with experimental binding free energy changes. In contrast, the in silico approach does not perform as well, especially for residues that contribute largely to binding. Subsequently, we assessed the applicability of the in vivo approach by analyzing the functional cooperativity between pairs of side chains. © 2004 Wiley Periodicals, Inc. Int J Quantum Chem, 2004     

Targeting norovirus infection-multivalent entry inhibitor design based on NMR experiments

Noroviruses attach to their host cells through histo blood group antigens (HBGAs), and compounds that interfere with this interaction are likely to be of therapeutic or diagnostic interest. It is shown that NMR binding studies can simultaneously identify and differentiate the site for binding HBGA ligands and complementary ligands from a large compound library, thereby facilitating the design of potent heterobifunctional ligands. Saturation transfer difference (STD) NMR experiments, spin-lock filtered NMR experiments, and interligand NOE (ILOE) experiments in the presence of virus-like particles (VLPs), identified compounds that bind to the HBGA binding site of human norovirus. Based on these data two multivalent prototype entry-inhibitors against norovirus infection were synthesized. A surface plasmon resonance based inhibition assay showed avidity gains of 1000 and one million fold over a millimolar univalent ligand. This suggests that further rational design of multivalent inhibitors based on our strategy will identify potent entry-inhibitors against norovirus infections.     

Novel d-seco paclitaxel analogues: synthesis, biological evaluation, and model testing

Four new D-secopaclitaxel analogues were synthesized from paclitaxel. The key step of the synthesis involved the opening of the D-ring by Jones oxidation. Two of the compounds had been predicted to be nearly as active as paclitaxel in a minireceptor model of the binding site on tubulin, but all were biologically inactive in an in vitro cytotoxic assay and a tubulin assembly assay. The biological results identify a weakness in our predictive minireceptor model and suggest a corrective remedy in which additional amino acids are needed to accommodate ligand-protein steric effects around the oxetane ring. These changes to the model lead to correct predictions of the bioactivity. Conformational analysis and dynamics simulations of the compounds showed that the 4-acetyl substituent is as important as the oxetane in determining the A ring conformation.     

Protein-cation interactions: structural and thermodynamic aspects

Cations are specifically recognized by numerous proteins. Cations may play a structural role, as cofactors stabilizing their binding partners, or a functional role, as cofactors activating their binding partners or being themselves involved in enzymatic reactions. Despite their small size, their charge density and their specific interaction with highly charged residues allow them to induce significant conformational changes on their binding proteins. The protein conformational change induced by cation binding may be as large as to account for the complete folding of a protein (as evidenced in Hepatitis C NS3 protease, or human rhinovirus 2A protease), and they may also trigger oligomerization (as in calcium-binding protein 1). Especially intriguing is the ability of cation-binding proteins of discriminating between very similar cations. In particular, calcium and magnesium are recognized by proteins with markedly different binding affinities and cause significantly different conformational changes and stabilization effects in the binding proteins (as in the fifth ligand binding repeat of the LDL receptor binding domain, calcium-binding protein 1, or parvalbumin). This article summarizes recent findings on the structural and energetic impact of cation binding to different proteins. A general framework can be envisaged in which cations can be considered as a special type of allosteric effectors able to modulate the functional properties of proteins, in particular the ability to interact with biological targets, by altering their conformational equilibrium.     

Surface stress changes induced by the conformational change of proteins

A potential binding assay based on conformational-change-induced micromechanical motion is described. Calmodulin was used to modify a microcantilever (MCL) by a self-assembled layer-by-layer approach. The results showed that the modified MCL bent when the proteins changed their conformation upon binding with Ca2+. The cantilever deflection amplitudes were different under different ionic strengths, indicating different degrees of conformational change of the proteins in these conditions. On the contrary, cantilevers modified by proteins, such as hemoglobin and myoglobin, that do not change conformations upon binding with analytes do not cause the cantilever deflection. These results suggest that the conformational changes of proteins may be used to develop cantilever biosensors, and the MCL system has potential for use in label-free, protein-analyte screening applications.     

Limited proteolysis combined with isotope labeling and quantitative LC-MALDI MS for monitoring protein conformational changes: a study on calcium-binding sites of cardiac Troponin C

Studies of protein-protein and protein-ligand interactions are important for understanding biological functions of proteins. A new technique based on the partial proteolysis of proteins combined with quantitative mass spectrometry is developed as a means of tracking structural changes after the formation of a protein-ligand complex. In this technique, a protein of interest with and without the binding of a ligand is digested with an enzyme to generate a set of peptides, followed by separation of the peptides by liquid chromatography. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) is used to identify chromatographically separated peptides, and locate their sequence alignments in the parent protein. Using an isotopically labeled protein as a sample against an unlabeled protein standard, quantitative information can be gathered. This overcomes the inherent lack of quantitative capability of MALDI MS. The utility of the technique to investigate protein-ligand interactions is demonstrated in a model system involving calcium binding to cardiac Troponin C (cTnC). Using this technique, the general location of the three calcium-binding sites of cTnC can be determined by using several different enzymes to generate overlapping peptide maps of cTnC.     

SH3-like fold proteins are structurally conserved and functionally divergent

The folding space for all the protein sequences is limited. Therefore it was observed that many proteins, whose sequences are not related, have similar fold characteristics. The fold databases like SCOP and CATH have classified various protein folds. However, in-depth analysis of the functional features of these folds was not done. We analyzed about twenty unique SH3-like folded proteins in their structural environment and functional characteristics. From our analysis it is apparent that the SH3-like folds could carry out various functions by modulation of loops and the functional region is restricted to one side of a particular sheet helped by two or three loops. The functions vary from oligonucleotide-binding to peptide-binding and other ligand binding. Although certain degree of sequence similarity was observed among the SH3-fold proteins, the similarity was restricted to the beta-strand regions of the proteins.     

Site-specific incorporation of a (19)F-amino acid into proteins as an NMR probe for characterizing protein structure and reactivity

19F NMR is a powerful tool for monitoring protein conformational changes and interactions; however, the inability to site-specifically introduce fluorine labels into proteins of biological interest severely limits its applicability. Using methods for genetically directing incorporation of unnatural amino acids, we have inserted trifluoromethyl-l-phenylalanine (tfm-Phe) into proteins in vivo at TAG nonsense codons with high translational efficiency and fidelity. The binding of substrates, inhibitors, and cofactors, as well as reactions in enzymes, were studied by selective introduction of tfm-Phe and subsequent monitoring of the 19F NMR chemical shifts. Subtle protein conformational changes were detected near the active site and at long distances (25 Angstrom). 19F signal sensitivity and resolution was also sufficient to differentiate protein environments in vivo. Since there has been interest in using 19F-labeled proteins in solid-state membrane protein studies, folding studies, and in vivo studies, this general method for genetically incorporating a 19F-label into proteins of any size in Escherichia coli should have broad application beyond that of monitoring protein conformational changes.     

Drug detection based on the conformational changes of calmodulin and the fluorescence of its enhanced green fluorescent protein fusion partner

A homogeneous phase protein-based assay for the high throughput screening of drugs was developed using enhanced green fluorescent protein (EGFP) as the reporter. For that, a fusion protein between calmodulin (CaM) and EGFP was constructed in order to monitor the conformational changes induced in CaM upon binding to tricyclic anti-depressant drugs. In the presence of Ca²⁺, CaM undergoes a conformational change exposing a hydrophobic pocket that interacts with CaM-binding proteins, peptides, and drugs. Further, the conformational changes induced in CaM upon binding to Ca²⁺ and the target analyte drug, leads to a change in the microenvironment of EGFP concomitant with a change in its fluorescence intensity. The observed change in fluorescence intensity of EGFP can be correlated to the concentration of the analyte present in the sample. Further, the response of CaM–EGFP fusion protein in the presence of Ca²⁺ to increasing concentrations of phenothiazines and structurally related tricyclic anti-depressants was investigated. Dose-response curves for various tricyclic anti-depressants were prepared. Moreover, this assay can serve as a model system for other homogeneous binding assays for pharmaceuticals employing genetically fused binding proteins with reporter proteins and may find applications in the high throughput screening of tricyclic anti-depressants.     

Prediction of small molecule binding property of protein domains with Bayesian classifiers based on Markov chains

Accurate computational methods that can help to predict biological function of a protein from its sequence are of great interest to research biologists and pharmaceutical companies. One approach to assume the function of proteins is to predict the interactions between proteins and other molecules. In this work, we propose a machine learning method that uses a primary sequence of a domain to predict its propensity for interaction with small molecules. By curating the Pfam database with respect to the small molecule binding ability of its component domains, we have constructed a dataset of small molecule binding and non-binding domains. This dataset was then used as training set to learn a Bayesian classifier, which should distinguish members of each class. The domain sequences of both classes are modelled with Markov chains. In a Jack-knife test, our classification procedure achieved the predictive accuracies of 77.2% and 66.7% for binding and non-binding classes respectively. We demonstrate the applicability of our classifier by using it to identify previously unknown small molecule binding domains. Our predictions are available as supplementary material and can provide very useful information to drug discovery specialists. Given the ubiquitous and essential role small molecules play in biological processes, our method is important for identifying pharmaceutically relevant components of complete proteomes. The software is available from the author upon request.     

High-throughput screening assay for the tunable selection of protein ligands

Here, we describe a new protein-ligand binding assay that is amenable to high-throughput screening applications. The assay involves the use of SUPREX (stability of unpurified proteins from rates of H/D exchange), a new H/D exchange and mass spectrometry-based technique we recently developed for the quantitative analysis of protein-ligand binding interactions. As part of this work, we describe a new high-throughput SUPREX protocol, and we demonstrate that this protocol can be used to efficiently screen peptide ligands in a model combinatorial library for binding to a model protein system, the S-protein. The high-throughput SUPREX protocol developed here is generally applicable to a wide variety of protein ligands, including DNA, small molecules, metals, and other proteins. On the basis of the results of the model study in this work, one person with access to one MALDI mass spectrometer should be able to screen approximately 10 000 compounds per 24-h period using the protocol described here. With full automation and the use of a commercially available MALDI mass spectrometer optimized for high-throughput analyses, we estimate that the SUPREX-based assay described here could be used to screen on the order of 100 000 ligands per day.     

Fourier transform ion cyclotron resonance mass spectrometric detection of small Ca2+-induced conformational changes in the regulatory domain of human cardiac troponin C

Troponin C (TnC), a calcium-binding protein of the thin filament of muscle, plays a regulatory role in skeletal and cardiac muscle contraction. NMR reveals a small conformational change in the cardiac regulatory N-terminal domain of TnC (cNTnC) on binding of Ca2+ such that the total exposed hydrophobic surface area increases very slightly from 3090 +/- 86 A2 for apo-cNTnC to 3108 +/- 71 A2 for Ca(2+)-cNTnC. Here, we show that measurement of solvent accessibility for backbone amide protons by means of solution-phase hydrogen/deuterium (H/D) exchange followed by pepsin digestion, high-performance liquid chromatography, and electrospray ionization high-field (9.4 T) Fourier transform Ion cyclotron resonance mass spectrometry is sufficiently sensitive to detect such small ligand binding-induced conformational changes of that protein. The extent of deuterium incorporation increases significantly on binding of Ca2+ for each of four proteolytic segments derived from pepsin digestion of the apo- and Ca(2+)-saturated forms of cNTnC. The present results demonstrate that H/D exchange monitored by mass spectrometry can be sufficiently sensitive to detect and identify even very small conformational changes in proteins, and should therefore be especially informative for proteins too large (or too insoluble or otherwise intractable) for NMR analysis.