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Liu T.,University of California at San Diego | Pantazatos D.,Brown University | Li S.,University of California at San Diego | Hamuro Y.,ExSAR Corporation | And 2 more authors.
Journal of the American Society for Mass Spectrometry | Year: 2012

Peptide amide hydrogen/deuterium exchange mass spectrometry (DXMS) data are often used to qualitatively support models for protein structure. We have developed and validated a method (DXCOREX) by which exchange data can be used to quantitatively assess the accuracy of three-dimensional (3-D) models of protein structure. The method utilizes the COREX algorithm to predict a protein's amide hydrogen exchange rates by reference to a hypothesized structure, and these values are used to generate a virtual data set (deuteron incorporation per peptide) that can be quantitatively compared with the deuteration level of the peptide probes measured by hydrogen exchange experimentation. The accuracy of DXCOREX was established in studies performed with 13 proteins for which both high-resolution structures and experimental data were available. The DXCOREX-calculated and experimental data for each protein was highly correlated. We then employed correlation analysis of DXCOREX-calculated versus DXMS experimental data to assess the accuracy of a recently proposed structural model for the catalytic domain of a Ca2+-independent phospholipase A 2. The model's calculated exchange behavior was highly correlated with the experimental exchange results available for the protein, supporting the accuracy of the proposed model. This method of analysis will substantially increase the precision with which experimental hydrogen exchange data can help decipher challenging questions regarding protein structure and dynamics. © 2011 American Society for Mass Spectrometry. Source


Coales S.J.,ExSAR Corporation
Rapid communications in mass spectrometry : RCM | Year: 2010

Backbone amide hydrogen exchange rates can be used to describe the dynamic properties of a protein. Amide hydrogen exchange rates in a native protein may vary from milliseconds (ms) to several years. Ideally, the rates of all amide hydrogens of the analyte protein can be determined individually. To achieve this goal, monitoring of a wider time window is critical, in addition to high sequence coverage and high sequence resolution. Significant improvements have been made to hydrogen/deuterium exchange mass spectrometry methods in the past decade for better sequence coverage and higher sequence resolution. On the other hand, little effort has been made to expand the experimental time window to accurately determine exchange rates of amide hydrogens. Many fast exchanging amide hydrogens are completely exchanged before completion of a typical short exchange time point (10-30s) and many slow exchanging amide hydrogens do not start exchanging before a typical long exchanging time point (1-3h). Here various experimental conditions, as well as a quenched-flow apparatus, are utilized to monitor cytochrome c amide hydrogen exchange behaviors over more than eight orders of magnitude (0.0044-1000000s), when converted into the standard exchange condition (pH 7 and 23°C). Source


Lewis H.A.,SGX Pharmaceuticals | Wang C.,Columbia University | Zhao X.,SGX Pharmaceuticals | Hamuro Y.,ExSAR Corporation | And 10 more authors.
Journal of Molecular Biology | Year: 2010

The ΔF508 mutation in nucleotide-binding domain 1 (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the predominant cause of cystic fibrosis. Previous biophysical studies on human F508 and ΔF508 domains showed only local structural changes restricted to residues 509-511 and only minor differences in folding rate and stability. These results were remarkable because ΔF508 was widely assumed to perturb domain folding based on the fact that it prevents trafficking of CFTR out of the endoplasmic reticulum. However, the previously reported crystal structures did not come from matched F508 and ΔF508 constructs, and the ΔF508 structure contained additional mutations that were required to obtain sufficient protein solubility. In this article, we present additional biophysical studies of NBD1 designed to address these ambiguities. Mass spectral measurements of backbone amide 1H/2H exchange rates in matched F508 and ΔF508 constructs reveal that ΔF508 increases backbone dynamics at residues 509-511 and the adjacent protein segments but not elsewhere in NBD1. These measurements also confirm a high level of flexibility in the protein segments exhibiting variable conformations in the crystal structures. We additionally present crystal structures of a broader set of human NBD1 constructs, including one harboring the native F508 residue and others harboring the ΔF508 mutation in the presence of fewer and different solubilizing mutations. The only consistent conformational difference is observed at residues 509-511. The side chain of residue V510 in this loop is mostly buried in all non-ΔF508 structures but completely solvent exposed in all ΔF508 structures. These results reinforce the importance of the perturbation ΔF508 causes in the surface topography of NBD1 in a region likely to mediate contact with the transmembrane domains of CFTR. However, they also suggest that increased exposure of the 509-511 loop and increased dynamics in its vicinity could promote aggregation in vitro and aberrant intermolecular interactions that impede trafficking in vivo. © 2009 Elsevier Ltd. Source


Tang L.,ExSAR Corporation | Tang L.,Imclone Systems | Coales S.J.,ExSAR Corporation | Morrow J.A.,Sierra Analytics Inc | And 2 more authors.
ChemBioChem | Year: 2012

An asparagine-to-serine substitution at residue 370 (N370S) in glucocerebrosidase (GCase) is the most prevalent mutation leading to Gaucher's disease, the most common lysosomal storage disorder. Two types of hydrogen/deuterium exchange experiment coupled with proteolysis and liquid chromatography-mass spectrometry (HDX-MS) were used to investigate the dynamic properties and unfolding stability of wt, R495H, and N370S GCases in the presence and absence of ligands. R495H GCase is used for enzyme replacement therapy and is considered to be a wt surrogate, whereas N370S is the most prevalent mutation leading to Gaucher's disease. Time-course HDX experiments of the GCases were performed under near-physiological conditions to detect the protein's local unfolding motions at a submolecular level. In guanidine-titration experiments, HDX reactions were performed with various concentrations of a chemical denaturant to provide the global stability of the proteins. The two types of experiment showed that all three purified GCases, wt, R495H, and N370S, have virtually identical local unfolding motions and global stabilities in solution. Combined with previous X-ray crystallographic studies, which showed indistinguishable backbone conformations for N370S and R495H GCase mutants and very similar melting temperatures for the wt, R495H, and N370S mutants, all three GCases are likely to have virtually identical structural and dynamic properties in solution. The guanidine-titration experiments revealed that the pharmacological chaperone, isofagomine (IFG), interacts more weakly with the N370S mutant than with the R495H GCase; this is consistent with the higher IC50 value of IFG against N370S than against R495H. The time-course experiments showed that IFG restricts the local unfolding motions of N370S in the same way as those of R495H when the ligand saturates the proteins. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source


O'Shannessy D.J.,Morphotek Inc. | Somers E.B.,Morphotek Inc. | Albone E.,Morphotek Inc. | Cheng X.,Morphotek Inc. | And 8 more authors.
Oncotarget | Year: 2011

Folate receptor alpha (FRA) is a cell surface protein whose aberrant expression in malignant cells has resulted in its pursuit as a therapeutic target and marker for diagnosis of cancer. The development of immune-based reagents that can reproducibly detect FRA from patient tissue processed by varying methods has been difficult due to the complex post-translational structure of the protein whereby most reagents developed to date are highly structure-sensitive and have resulted in equivocal expression results across independent studies. The aim of the present study was to generate novel monoclonal antibodies (mAbs) using modified full length FRA protein as immunogen in order to develop a panel of mAbs to various, non-overlapping epitopes that may serve as diagnostic reagents able to robustly detect FRA-positive disease. Here we report the development of a panel of FRA-specific mAbs that are able to specifically detect FRA using an array of diagnostic platforms and methods. In addition, the methods used to develop these mAbs and their diverse binding properties provide additional information on the three dimensional structure of FRA in its native cell surface configuration. © O'Shannessy et al. Source

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