Quantitative Assessment of Protein Structural Mode

Authors: Tong Liu Dennis Pantazatos Sheng Li Yoshitomo Hamuro Vincent J Hilser Virgil L Woods

Publish Date: 2011/10/20

Volume: 23, Issue: 1, Pages: 43-56

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Abstract

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 threedimensional 3D 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 highresolution structures and experimental data were available The DXCOREXcalculated and experimental data for each protein was highly correlated We then employed correlation analysis of DXCOREXcalculated versus DXMS experimental data to assess the accuracy of a recently proposed structural model for the catalytic domain of a Ca2+independent phospholipase A2 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 dynamicsPeptide amide hydrogen/deuterium exchange mass spectrometry DXMS is an increasingly important tool for the study of protein dynamics and structure 1 2 3 4 Amide hydrogens in proteins exchange with hydrogen atoms from solvent water molecules surrounding the protein with exchange rates that depend on the details of the protein’s structure and thermodynamic stability In general amide hydrogens that only exchange as a result of largescale unfolding of the protein exchange the slowest while amide hydrogens in loop regions or within thermodynamically less stable regions of the protein tend to exchange faster Beyond such generalizations interpretation of exchange data in terms of protein structure remains a great challenge for the field DXMS is an exceptionally powerful approach for examining the exchange behavior of proteins and data can be obtained on otherwise problematic proteins that are too large limited in quantity or of limited solubility to be purified or crystallized in structurally competent form Thus hydrogen exchange data is frequently used to support models proposed for protein structure in the absence of high resolution crystallographic or NMR determinationsThe approaches taken for representing and interpreting experimental exchange data in terms of structural models have been largely qualitative Results of such analyses are often represented as twodimensional 2D “butterfly” “mirror” or colored “heat maps” where the deuteration level of peptides is represented by varying colors and arranged in register with the intact protein’s primary amino acid sequence If a highresolution 3D structure for a protein or a protein homolog is available the exchange data can be superimposed on such structures often by differential coloring Recent improvements in the comprehensiveness and resolution of DXMS experimentation 5 6 provide further impetus for the development of improved methods for applying experimental data to the testing of protein structure models 7 8 9 10 11 12We have developed a computational method DXCOREX by which amide hydrogen exchange rates for a crystallographically or NMRsolved protein structure or a hypothesized protein structure can be predicted and then transformed into a data set that precisely corresponds with the probe peptides and onexchange times of the available experimental data Correlation analysis of the experimental data for a protein and DXCOREX calculated datasets for a 3D structure proposed for the protein allows a quantitative evaluation of the degree to which the experimental data supports the proposed structural modelIn this work we have found that virtual DXMS data sets predicted by DXCOREX analysis of the known 3D structures of 13 proteins are highly correlated with the results of experimental DXMS analysis of these same proteins despite the data acquisition having been performed by multiple investigators employing varying exchange data acquisition methodologies We then demonstrate the use of this method in the quantitative assessment of the accuracy of a recently proposed 3D structure of a protein for which exchange data was availableThe COREX algorithm 13 14 15 allows efficient calculation of the insolution dynamic behavior of proteins whose structure had been previously determined as a static representation either crystallographically or by NMR experimentation COREX calculates residuespecific stability values for each amino acid in a protein from knowledge of the structural coordinates of the atoms in the protein’s deduced 3D structure and a partition function by which the relative energetic cost of exposing the various atoms in the structured protein to solvent water can be efficiently calculated At its inception a provisional peptide amide hydrogen exchange rate calculating capability based on the residuespecific stability values was incorporated into COREX to allow experimental estimation of the accuracy of the algorithm to calculate the residuespecific thermodynamic stability parameters Algorithmcalculated residuespecific exchange rates were calculated for several structurally solved proteins with available NMRobtained residuespecific exchange rate data It was found that the COREX calculated rates were in general agreement approximately 75 agreement with the available NMR data for the studied proteins 13 16 though it was recognized that the NMR exchange rate data sets available were incomplete most particularly in the measurements of the more rapidly exchanging amides in proteins With this validation in place the COREX algorithm had been subsequently applied to successfully describe protein features such as cold denaturation 17 18 allosteric binding effects 19 20 pHlinked structural transitions 21 and energetic profiling of protein folds 22These COREX calculations were based on the readily available coordinates of the heavy atoms typically represented in published structures that exclude representation of hydrogen and the competency for exchange of an amide hydrogen was estimated by measuring the solvent exposure of the entire amide nitrogen component of each peptide amide in the structure This approximation was accurate enough to allow validation of the COREX algorithm against multiple proteins 13 16 We have found that the accuracy of exchange rate calculation is improved if amide hydrogens are taken into account in the calculation In the present investigation COREX was modified to allow calculation of the exchangecompetent solvent exposure of explicitly represented amide hydrogens versus amide nitrogen termed HCOREX This modest change resulted in higher accuracy in calculating exchange rates than those obtained with unmodified COREX when evaluated against NMRderived rate measurements see Supplemental Material and Supplemental Material Figures S1 and S2

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