Apolipoprotein A-I (apoA-I) stabilizes anti-atherogenic high density lipoprotein particles (HDL) in the circulation and governs their biogenesis, metabolism, and functional interactions. To decipher these important structure-function relationships, it will be necessary to understand the structure, stability, and plasticity of the apoA-I molecule. Biophysical studies show that lipid-free apoA-I contains a large amount of ␣-helical structure but the location of this structure and its properties are not established. We used hydrogen-deuterium exchange coupled with a fragmentation-separation method and mass spectrometric analysis to study human lipid-free apoA-I in its physiologically pertinent monomeric form. The acquisition of Ϸ100 overlapping peptide fragments that redundantly cover the 243-residue apoA-I polypeptide made it possible to define the positions and stabilities of helical segments and to draw inferences about their interactions and dynamic properties. Residues 7-44, 54 -65, 70 -78, 81-115, and 147-178 form ␣-helices, accounting for a helical content of 48 ؎ 3%, in agreement with circular dichroism measurements (49%). At 3 to 5 kcal/mol in free energy of stabilization, the helices are far more stable than could be achieved in isolation, indicating mutually stabilizing helix bundle interactions. However the helical structure is dynamic, unfolding and refolding in seconds, allowing facile apoA-I reorganization during HDL particle formation and remodeling.high density lipoprotein ͉ cholesterol ͉ protein secondary structure ͉ amphipathic alpha-helix T he incidence of coronary artery disease is inversely related to the plasma level of HDL, which mediates the reverse transport of cholesterol from peripheral cells to the liver for excretion (1, 2). The biogenesis, metabolism, and transport of antiatherogenic HDL particles and their functional interactions are governed by the principal protein component, apolipoprotein A-I (apoA-I) (3, 4). Lipid-free apoA-I can interact with a cell surface lipid transporter (ABCA1) to mediate the efflux of cellular phospholipid and cholesterol and the creation of nascent HDL particles (5). ApoA-I is then able to reorganize to accommodate and solubilize the lipids in different configurations of HDL particles as they mature. In the lipid-bound state, apoA-I governs lipid transport, receptor recognition, and other functions including the activation of lecithin-cholesterol acyltransferase, which converts cholesterol to cholesteryl ester (6).Widespread efforts to dissect these important functional relationships are presently restricted by our limited understanding of lipoprotein and apolipoprotein structure (3, 7). Crystallographic study of the microemulsion-like HDL complexes appears to be impossible. Secondary structure prediction suggests 11 amphipathic ␣-helical segments in human apoA-I (8). Biophysical studies distinguish an N-terminal highly helical domain (residues 1-189) and a more flexible C-terminal domain (residues 190-243) (for reviews see refs. 3, 4, 9). A crystallogra...

An automated high-throughput, high-resolution deuterium exchange HPLC-MS method (DXMS) was used to extend previous hydrogen exchange studies on the position and energetic role of regulatory structure changes in hemoglobin. The results match earlier highly accurate but much more limited tritium exchange results, extend the analysis to the entire sequence of both hemoglobin subunits, and identify some energetically important changes. Allosterically sensitive amide hydrogens located at near amino acid resolution help to confirm the reality of local unfolding reactions and their use to evaluate resolved structure changes in terms of allosteric free energy.H ydrogen exchange (HX) measurements can, in principle, locate protein-binding sites and structure changes and can quantify otherwise unavailable dynamic and energetic parameters (1-4). For relatively small proteins, HX can be measured at an amino acid resolved level by NMR methods. For larger, functionally more interesting proteins, other strategies are required. Earlier work (5, 6) developed a ''functional-labeling'' approach that can selectively label, by hydrogen-tritium (H-T) or hydrogen-deuterium (H-D) exchange, just those sites that change in any functional process. In favorable cases, the label can then be located at medium resolution by a proteolytic fragmentation method in which the fragments are quickly produced and then separated by HPLC under conditions where the loss of isotopic label is slow (6-9).To move toward higher resolution and more comprehensive coverage of target proteins, recent work in many laboratories has coupled the HPLC separation to a second dimension of fragment resolution by online MS (10, 11). These methods tend to be labor intensive and time consuming, with limitations in throughput and comprehensiveness and in the structural resolution of functionally important changes. This article merges previous HX functional labeling and fragment separation methods with an automated MS approach termed deuterium exchange MS (DXMS) (12-18).We are using Hb as a model system to study how protein molecules manage intramolecular signal transduction processes. Hb functions by transducing a part of the binding energy of its initially bound O 2 ligands into structure-change energy. The energy is carried through the protein to distant heme sites in the form of energetic structure changes, and there transduced back into binding energy. The initial reduced binding energy and the later enhanced binding produces the physiologically important sigmoid binding curve. In short, the currency of allosteric interactions is free energy. Trying to understand allostery without measuring free energy is like trying to understand an economic system without measuring money. A great deal of information on regulatory structure change in many proteins is now available, but mainly in a qualitative pictorial sense from ''before and after'' crystallographic or NMR views. How these changes participate in energy transduction and translocation has been little explored (19)(20)(21...

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The structure of an AKAP docked to the dimerization/docking (D/D) domain of the type II (RIIa) isoform of protein kinase A (PKA) has been well characterized, but there currently is no detailed structural information of an AKAP docked to the type I (RIa) isoform. Dual-specific AKAP2 (D-AKAP2) binds in the nanomolar range to both isoforms and provided us with an opportunity to characterize the isoform-selective nature of AKAP binding using a common docked ligand. Hydrogen/ deuterium (H/D) exchange combined with mass spectrometry (DXMS) was used to probe backbone structural changes of an a-helical A-kinase binding (AKB) motif from D-AKAP2 docked to both RIa and RIIa D/D domains. The region of protection upon complex formation and the magnitude of protection from H/D exchange were determined for both interacting partners in each complex. The backbone of the AKB ligand was more protected when bound to RIa compared to RIIa, suggesting an increased helical stabilization of the docked AKB ligand. This combined with a broader region of backbone protection induced by the AKAP on the docking surface of RIa indicated that there were more binding constraints for the AKB ligand when bound to RIa. This was in contrast to RIIa, which has a preformed, localized binding surface. These distinct modes of AKAP binding may contribute to the more discriminating nature of the RIa AKAP-docking surface. DXMS provides valuable structural information for understanding binding specificity in the absence of a high-resolution structure, and can readily be applied to other protein-ligand and protein-protein interactions.