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...

To understand high-density lipoprotein (HDL) structure at the molecular level, the location and stability of α-helical segments in human apolipoprotein (apo) A-I in large (9.6 nm) and small (7.8 nm) discoidal HDL particles were determined by hydrogen-deuterium exchange (HX) and mass spectrometry methods. The measured HX kinetics of some 100 apoA-I peptides specify, at close to amino acid resolution, the structural condition of segments throughout the protein sequence and changes in structure and stability that occur on incorporation into lipoprotein particles. When incorporated into the large HDL particle, the nonhelical regions in lipid-free apoA-I (residues 45-53, 66-69, 116-146, and 179-236) change conformation from random coil to α-helix so that nearly the entire apoA-I molecule adopts helical structure (except for the terminal residues 1-6 and 237-243). The amphipathic α-helices have relatively low stability, in the range 3-5 kcal∕mol, indicating high flexibility and dynamic unfolding and refolding in seconds or less. A segment encompassed by residues 125-158 exhibits bimodal HX labeling indicating co-existing helical and disordered loop conformations that interchange on a time scale of minutes. When incorporated around the edge of the smaller HDL particle, the increase in packing density of the two apoA-I molecules forces about 20% more residues out of direct contact with the phospholipid molecules to form disordered loops, and these are the same segments that form loops in the lipid-free state. The region of disc-associated apoA-I that binds the lecithin-cholesterol acyltransferase enzyme is well structured and not a protruding unstructured loop as reported by others.atherosclerosis | amphipathic α-helix | protein secondary structure T here is great interest in understanding the structure-function relationships of high-density lipoprotein (HDL) because of its important antiatherogenic properties. Because high-resolution structures of HDL microemulsion particles cannot be obtained by current X-ray crystallography and NMR methods, alternative biophysical approaches have been used to characterize various subspecies of HDL. Structural models that show the general lipid and protein organization in HDL particles are now available (for reviews, see refs. 1-6). To derive detailed understanding at the molecular level of how HDL functions in cholesterol transport (7) and in reducing the incidence of premature cardiovascular disease (8), higher-resolution structural information is required.Reconstituted discoidal HDL particles that are models of nascent HDL (9) created by the interaction of apolipoprotein (apo) A-I (3, 10), the principal protein of HDL, with the cellsurface ATP binding cassette transporter (ABCA1) (11, 12) have received a great deal of attention. A major advantage of model particles is the possibility of obtaining preparations that are sufficiently homogeneous for detailed structural investigation. The structure of a discoidal HDL particle (approximately 10 nm hydrodynamic diameter) comprising a 16...

Measurement of the naturally occurring hydrogen exchange (HX) behavior of proteins can in principle provide highly resolved thermodynamic and kinetic information on protein structure, dynamics, and interactions. The HX fragment separation-mass spectrometry method (HX-MS) is able to measure hydrogen exchange in biologically important protein systems that are not accessible to NMR methods. In order to achieve high structural resolution in HX-MS experiments, it will be necessary to obtain many sequentially overlapping peptide fragments and be able to identify and analyze them efficiently and accurately by mass spectrometry. This paper describes operations which, when applied to four different proteins ranging in size from 140 to 908 residues, routinely provides hundreds of useful unique peptides, covering the entire protein length many times over. Coverage in terms of the average number of peptide fragments that span each amino acid exceeds 10. The ability to achieve these results required the integrated application of experimental methods that are described here and a computer analysis program, called ExMS, described in a following paper.

A previous paper considered the problems that presently limit the hydrogen exchange - mass spectrometry (HX-MS) method for studying the biophysical and functional properties of proteins. Many of these problems can be overcome by obtaining and analyzing hundreds of sequentially overlapping peptide fragments that cover the protein many times over (Mayne et al. J. Am. Soc. Mass Spectrom. 2011: 10.1007/s13361-011-0235-4). This paper describes a computer program called ExMS that furthers this advance by making it possible to efficiently process crowded mass spectra and definitively identify and characterize these many peptide fragments. ExMS automatically scans through high resolution MS data to find the individual isotopic peaks and isotopic envelopes of a list of peptides previously identified by MS/MS. It performs a number of tests to ensure correct identification in spite of peptide overlap in both chromatographic and mass spectrometric dimensions and possible multi-modal envelopes due to static or dynamic structural heterogeneity or HX EX1 behavior. The program can automatically process data from many sequential HX time points with no operator intervention at the rate of ~2 sec per peptide per HX time point using desktop computer equipment, but it also provides for rapid manual checking and decision when ambiguity exists. Additional subroutines can provide a step by step report of performance at each test along the way and parameter adjustment, deconvolute isotopic envelopes, and plot the time course of single and multi-modal H-D exchange. The program will be available on an open source basis at: http://HX2.med.upenn.edu/download.html

• HNPs inhibit proteolytic cleavage of VWF by ADAMTS13 by physically blocking VWF-ADAMTS13 interactions.• Plasma levels of HNP1, HNP2, and HNP3 are markedly increased in patients with acquired autoimmune TTP.Infection or inflammation may precede and trigger formation of microvascular thrombosis in patients with acquired thrombotic thrombocytopenic purpura (TTP). However, the mechanism underlying this clinical observation is not fully understood.Here, we show that human neutrophil peptides (HNPs) released from activated and degranulated neutrophils inhibit proteolytic cleavage of von Willebrand factor (VWF) by ADAMTS13 in a concentration-dependent manner. Half-maximal inhibitory concentrations of native HNPs toward ADAMTS13-mediated proteolysis of peptidyl VWF73 and multimeric VWF are 3.5 mM and 45 mM, respectively. Inhibitory activity of HNPs depends on the RRY motif that is shared by the spacer domain of ADAMTS13. Native HNPs bind to VWF73 (K D 5 0.72 mM), soluble VWF (K D 5 0.58 mM), and ultra-large VWF on endothelial cells. Enzyme-linked immunosorbent assay (ELISA) demonstrates markedly increased plasma HNPs1-3 in most patients with acquired autoimmune TTP at presentation (median, ∼170 ng/mL; range, 58-3570; n 5 19) compared with healthy controls (median, ∼23 ng/mL; range, 6-44; n 5 18) (P < .0001). Liquid chromatography plus tandem mass spectrometry (LC-MS/MS) reveals statistically significant increases of HNP1, HNP2, and HNP3 in patient samples (all P values <.001).There is a good correlation between measurement of HNPs1-3 by ELISA and by LC-MS/MS (Spearman r 5 0.7932, P < .0001).Together, these results demonstrate that HNPs1-3 may be potent inhibitors of ADAMTS13 activity, likely by binding to the central A2 domain of VWF and physically blocking ADAMTS13 binding. Our findings may provide a novel link between inflammation/ infection and the onset of microvascular thrombosis in acquired TTP and potentially other immune thrombotic disorders. (Blood. 2016;128(1):110-119)

Elucidation of apoA-I secondary structure in spherical plasma HDL particles is essential for understanding HDL structure and function at the molecular level. To provide this information, we have applied hydrogen exchange (HX) and mass spectrometry methods to compare apoA-I secondary structure in discoidal (two apoA-I molecules/particle) and spherical (five apoA-I molecules/particle) HDL particles. The HX kinetics indicate that the locations of helical segments within the apoA-I molecules are the same in both discoidal and spherical HDL particles (approximately 10 nm hydrodynamic diameter). Helix stabilities in both types of particles are 3–5 kcal/mol, consistent with the apoA-I molecules being in a highly dynamic state with helical segments unfolding and refolding in seconds. For the spherical HDL, apoA-I fragments corresponding to residues 115–158 exhibit bimodal HX kinetics consistent with this segment adopting an inter-converting (on the timescale of tens of minutes) helix-loop configuration. The segment adopting this configuration in the 10 nm disc is shorter because the surface area available to each apoA-I molecule is apparently larger. Loop formation in the central region of the apoA-I molecule contributes to the ability of the protein to adapt to changes in available space on the HDL particle surface. Overall, apoA-I secondary structure is largely unaffected by a change in HDL particle shape from disc to sphere.

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Background: The molecular basis for heterogeneity in nascent HDL particle cholesterol content is unknown.Results: Apolipoprotein A-I-mediated solubilization of phospholipid/cholesterol membranes creates large cholesterol-rich and small cholesterol-poor HDL particles.Conclusion: Variations in cholesterol distribution between nanoscale HDL discs are due to particle size-dependent molecular packing constraints.Significance: This explains the origin of HDL particles with different cholesterol contents.