scholarly journals Diversity in prion protein oligomerization pathways results from domain expansion as revealed by hydrogen/deuterium exchange and disulfide linkage

2007 ◽  
Vol 104 (18) ◽  
pp. 7414-7419 ◽  
Author(s):  
Frederic Eghiaian ◽  
Thorsten Daubenfeld ◽  
Yann Quenet ◽  
Marieke van Audenhaege ◽  
Anne-Pascale Bouin ◽  
...  
Keyword(s):  
Prion Protein ◽  
Conformational Changes ◽  
Present Report ◽  
Deuterium Exchange ◽  
Amyloid Formation ◽  
Protein Oligomerization ◽  
Hydrogen Deuterium Exchange ◽  
High Concentration ◽  
Disulfide Linkage ◽  
Hydrogen Deuterium

The prion protein (PrP) propensity to adopt different structures is a clue to its biological role. PrP oligomers have been previously reported to bear prion infectivity or toxicity and were also found along the pathway of in vitro amyloid formation. In the present report, kinetic and structural analysis of ovine PrP (OvPrP) oligomerization showed that three distinct oligomeric species were formed in parallel, independent kinetic pathways. Only the largest oligomer gave rise to fibrillar structures at high concentration. The refolding of OvPrP into these different oligomers was investigated by analysis of hydrogen/deuterium exchange and introduction of disulfide bonds. These experiments revealed that, before oligomerization, separation of contacts in the globular part (residues 127–234) occurred between the S1–H1–S2 domain (residues 132–167) and the H2–H3 bundle (residues 174–230), implying a conformational change of the S2–H2 loop (residues 168–173). The type of oligomer to be formed depended on the site where the expansion of the OvPrP monomer was initiated. Our data bring a detailed insight into the earlier conformational changes during PrP oligomerization and account for the diversity of oligomeric entities. The kinetic and structural mechanisms proposed here might constitute a physicochemical basis of prion strain genesis.

scholarly journals Hydrogen Exchange Mass Spectrometry Reveals Allosteric Interactions Between the Distal and the Proximal Domains of Human ADAMTS13

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2535-2535 ◽  
Author(s):  
Catherine B. Zander ◽  
Leland Mayne ◽  
S. Walter Englander ◽  
X. Long Zheng
Keyword(s):  
Mass Spectrometry ◽  
Conformational Changes ◽  
Hydrogen Exchange ◽  
Deuterium Exchange ◽  
Hydrogen Deuterium Exchange ◽  
Allosteric Interaction ◽  
Allosteric Interactions ◽  
Hydrogen Deuterium ◽  
Direct Binding ◽  
Terminal Domains

Abstract Background: Deletion of the distal C-terminal domains, or a decrease in pH, or the binding of a monoclonal antibody to the C-terminal domains of ADAMTS13 increases its proteolytic activity towards VWF peptide substrate, VWF73, by approximately 2-fold; this suggests an allosteric interaction between the distal and proximal domains of ADAMTS13. However, neither the underlying mechanism of the allosteric interaction nor the exact sites of interaction between the proximal domains and the distal C-terminal domain are fully understood. Objective: To determine the binding sites and kinetic interactions between the proximal and distal domains of ADAMTS13 and to gain insight into the mechanism of allosteric regulation of ADAMTS13 function. Methods: Hydrogen deuterium exchange plus LC-mass spectrometry (HX-MS) and surface plasmon resonance (SPR) were employed to determine conformational changes and direct binding interactions between the proximal and distal domains of ADAMTS13. Results: Recombinant full-length (rFL-A13) and an ADAMTS13 variant truncated after the spacer domain (rMDTCS) were purified to homogeneity. After incubation with deuterium for various times (0-6 hours), both rFL-A13 and rMDTCS were digested with pepsin and fungal protease-13 and then partially resolved by liquid chromatography before being injected into a LTQ ObitrapXL mass spectrometer for peptide identification. Overall, 188 of the total 338 peptides in rFL-A13 were identified, spanning the metalloprotease, cysteine-rich, and spacer domain. Only 16 peptides were found to be less accessible (or protected from deuterium-hydrogen exchange) in rFL-A13 than in rMDTCS. Two of these regions reside in the metalloprotease domain (residue 78-127 and 282-304). The next region is at a loop on the cysteine-rich domain (residue 446-473 and 495-501). The next peptides are in the adjacent loops of the spacer domain with one peptide 596-603, and five overlapping peptides spanning from 632-642, and then finally one peptide from the C-terminal of the MDTCS variant (residue 676 to 688). These results indicate that the distal C-terminal domains interact with multiple discrete sites in the proximal domains of ADAMTS13, resulting in protection from hydrogen-deuterium exchange. Such an interaction was further demonstrated by direct binding experiments with SPR. The distal domains including CUB, T2-CUB, and T5-CUB bound directly to M, MDT, and MDTCS under flow with high affinity. Conclusion: Our findings demonstrate the allosteric interactions between the distal domains and the proximal domains at multiple discrete sites. Such an interaction may regulate catalytic efficiency and substrate specificity of the metalloprotease domain of ADAMTS13 under various conditions. Disclosures Zheng: Ablynx: Consultancy; Alexion: Research Funding.

scholarly journals Conformational Changes in Active and Inactive States of Human PP2Cα Characterized by Hydrogen/Deuterium Exchange–Mass Spectrometry

Biochemistry ◽  
2017 ◽  
Vol 56 (21) ◽  
pp. 2676-2689 ◽  
Author(s):  
Sharlyn J. Mazur ◽  
Elyssia S. Gallagher ◽  
Subrata Debnath ◽  
Stewart R. Durell ◽  
Kyle W. Anderson ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Conformational Changes ◽  
Deuterium Exchange ◽  
Hydrogen Deuterium Exchange ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry

scholarly journals Conformational changes in gastric H+/K+-ATPase monitored by difference Fourier-transform infrared spectroscopy and hydrogen/deuterium exchange

2004 ◽  
Vol 382 (1) ◽  
pp. 121-129 ◽  
Author(s):  
Frantz SCHEIRLINCKX ◽  
Vincent RAUSSENS ◽  
Jean-Marie RUYSSCHAERT ◽  
Erik GOORMAGHTIGH
Keyword(s):  
Fourier Transform ◽  
Secondary Structure ◽  
Conformational Changes ◽  
Tertiary Structure ◽  
Structural Information ◽  
Deuterium Exchange ◽  
Attenuated Total Reflection ◽  
Hydrogen Deuterium Exchange ◽  
Hydrogen Deuterium ◽  
Difference Fourier

Gastric H+/K+-ATPase is a P-type ATPase responsible for acid secretion in the stomach. This protein adopts mainly two conformations called E1 and E2. Even though two high-resolution structures for a P-ATPase in these conformations are available, little structural information is available about the transition between these two conformations. In the present study, we used two experimental approaches to investigate the structural differences that occur when gastric ATPase is placed in the presence of various ligands and ligand combinations. We used attenuated total reflection–Fourier-transform IR experiments under a flowing buffer to modify the environment of the protein inside the measurement cell. The high accuracy of the results allowed us to demonstrate that the E1–E2 transition induces a net change in the secondary structure that concerns 10–15 amino acid residues of a total of 1324 in the proteins. The E2.K+ structure is characterized by a decreased β-sheet content and an increase in the disordered structure content with respect to the E1 form of the enzyme. Modifications in the absorption of the side chain of amino acids are also suggested. By using hydrogen/deuterium-exchange kinetics, we show that tertiary-structure modifications occurred in the presence of the same ligands, but these changes involved several hundreds of residues. The present study suggests that conformational changes in the catalytic cycle imply secondary-structure rearrangements of small hinge regions that have an impact on large domain re-organizations.

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