Structural Analysis of Gelsolin Using Synchrotron Protein Footprinting

Protein footprinting provides detailed structural information on protein structure in solution by directly identifying accessible and hydroxyl radical-reactive side chain residues. Radiolytic generation of hydroxyl radicals using millisecond pulses of a synchrotron white beam results in the formation of stable side chain oxidation products, which can be digested with proteases for mass spectrometry (MS) analysis. Liquid chromatography-coupled MS and tandem MS methods allow for the quantitation of the ratio of modified and unmodified peptides and identify the specific side chain probes that are oxidized, respectively. The ability to monitor the changes in accessibility of multiple side chain probes by monitoring increases or decreases in their oxidation rates as a function of ligand binding provides an efficient and powerful tool for analyzing protein structure and dynamics. In this study, we probe the detailed structural features of gelsolin in its inactive and Ca2+-activated state. Oxidation rate data for 81 peptides derived from the trypsin digestion of gelsolin are presented; 60 of these peptides were observed not to be oxidized, and 21 had detectable oxidation rates. We also report the Ca2+-dependent changes in oxidation for all 81 peptides. Fifty-nine remained unoxidized, five increased their oxidation rate, and two experienced protections. Tandem mass spectrometry was used to identify the specific side chain probes responsible for the Ca2+-insensitive and Ca2+-dependent responses. These data are consistent with crystallographic data for the inactive form of gelsolin in terms of the surface accessibility of reactive residues within the protein. The results demonstrate that radiolytic protein footprinting can provide detailed structural information on the conformational dynamics of ligand-induced structural changes, and the data provide a detailed model for gelsolin activation

Protein Footprinting: Auxiliary Engine to Power the Structural Biology Revolution

Structural biology is entering an exciting time where many new high-resolution structures of large complexes and membrane proteins are determined regularly. These advances have been driven by over fifteen years of technology advancements, first in macromolecular crystallography, and recently in Cryo-electron microscopy. These structures are allowing detailed questions about functional mechanisms of the structures, and the biology enabled by these structures, to be addressed for the first time. At the same time, mass spectrometry technologies for protein structure analysis, “footprinting” studies, have improved their sensitivity and resolution dramatically and can provide detailed sub-peptide and residue level information for validating structures and interactions or understanding the dynamics of structures in the context of ligand binding or assembly. In this perspective, we review the use of protein footprinting to extend our understanding of macromolecular systems, particularly for systems challenging for analysis by other techniques, such as intrinsically disordered proteins, amyloidogenic proteins, and other proteins/complexes so far recalcitrant to existing methods. We also illustrate how the availability of high-resolution structural information can be a foundation for a suite of hybrid approaches to divine structure-function relationships beyond what individual techniques can deliver

Structurally Distinct External Solvent-Exposed Domains Drive Replication of Major Human Prions

There is a limited understanding of structural attributes that encode the iatrogenic transmissibility and various phenotypes of prions causing the most common human prion disease, sporadic Creutzfeldt-Jakob disease (sCJD). Here we report the detailed structural differences between major sCJD MM1, MM2, and VV2 prions determined with two complementary synchrotron hydroxyl radical footprinting techniques—mass spectrometry (MS) and conformation dependent immunoassay (CDI) with a panel of Europium-labeled antibodies. Both approaches clearly demonstrate that the phenotypically distant prions differ in a major way with regard to their structural organization, and synchrotron-generated hydroxyl radicals progressively inhibit their seeding potency in a strain and structure-specific manner. Moreover, the seeding rate of sCJD prions is primarily determined by strain-specific structural organization of solvent-exposed external domains of human prion particles that control the seeding activity. Structural characteristics of human prion strains suggest that subtle changes in the organization of surface domains play a critical role as a determinant of human prion infectivity, propagation rate, and targeting of specific brain structures

Small Molecule Inhibitors of 15-PGDH Exploit a Physiologic Induced-Fit Closing System

15-prostaglandin dehydrogenase (15-PGDH) is a negative regulator of tissue stem cells that acts via enzymatic activity of oxidizing and degrading PGE2, and related eicosanoids, that support stem cells during tissue repair. Indeed, inhibiting 15-PGDH markedly accelerates tissue repair in multiple organs. Here we have used cryo-electron microscopy to solve the solution structure of native 15-PGDH and of 15-PGDH individually complexed with two distinct chemical inhibitors. These structures identify key 15-PGDH residues that mediate binding to both classes of inhibitors. Moreover, we identify a dynamic 15-PGDH lid domain that closes around the inhibitors, and that is likely fundamental to the physiologic 15-PGDH enzymatic mechanism. We furthermore identify two key residues, F185 and Y217, that act as hinges to regulate lid closing, and which both inhibitors exploit to capture the lid in the closed conformation, thus explaining their sub-nanomolar binding affinities. These findings provide the basis for further development of 15-PGDH targeted drugs as therapeutics for regenerative medicine

Maintainable systems with a business object approach

The concept of Business Objects (BOs) has been recently promoted as a new way of exploiting object-orientation for achieving large-grain reuse. In this paper, we address the issue of how to effectively re-engineer business software applications using BOs as a reuse technique. To this end, we first identify the reuse features of business objects and then compare them with other reuse techniques. In addition, we show that software re-engineering can be more economical when business objects are used. Our work also provides guidance on how to develop and use a Business Object Architecture (BOA), which is shared by a group of interrelated and interdependent software applications. We argue that such architecture allows for more efficient reuse and better maintainability and it is illustrated by means of a case study in a realistic manufacturing environment

MGO-adducted rhBD-2 shows a concentration-dependent reduction of bactericidal activity, shown as a reduction in CFU vs unadducted peptide.

<p>CFU within a defined area were counted following radial diffusion assays performed with gram-negative, facultative anaerobes <i>Escherichia coli</i> (<i>e</i>.<i>c</i>.) and <i>Pseudomonas aeruginosa</i> (<i>p</i>.<i>a</i>.), and with the gram-positive, facultative anaerobe <i>Staphylococcus aureus</i> (<i>s</i>.<i>a</i>.) exposed to 0.5 μg/5 μl with or without MGO. Wild-type hBD-2 is highly bactericidal against most gram-negative bacterial strains, including <i>E</i>. <i>coli</i>, but this function is dramatically reduced following 72 h incubation of rhBD-2 (0.5 μg/5 μl) with 100 μM MGO at 37°C (inset). MGO (100 <i>μ</i>M) also reduces rhBD-2 bactericidal function against the gram-positive <i>S</i>. <i>aureus</i> strain. Data is presented as the Mean ± S.D. for N = 5 (<i>e</i>.<i>c</i>.) or N = 6 experiments (<i>p</i>.<i>a</i>., <i>s</i>.<i>a</i>.). Graph line for <i>e</i>.<i>c</i>. is offset slightly for clarity. (♦, ◊: p = 0.01; _*: p = 0.05).</p

Comparison of deconvoluted tandom MS/MS spectra of untreated (a) vs modified (b) rhBD-2 peptide RYKQIGTCGLPGTK (23–36) after trypsin digestion of the rhBD-2 protein.

<p>The modified hBD-2 peptide was previously incubated in 100 μM MGO at 37°C for 72 h. The presence of the b1 ion with a +54 Da mass shift and unmodified doubly protonated y13 shows that modification of this peptide occurred at Arg²³.</p

Effect of MGO adduction to rhBD-2 on chemoattraction for CEM cells.

<p>Inset, untreated rhBD-2 shows a concentration-dependent optimum in chemoattraction. Fluorescence units are proportional to migrated cell number due to labeling of migrating cell DNA by the CyQuant probe. Lower graph, chemoattractive function is reduced by incubating rhBD-2 with 100 uM MGO for 72 h at 37ଌ. SDF-1 is positive control for chemotaxis of CEM cells. Data is presented as the Mean ± S.E.M. for N₁ = 3, N₂ = 3 experiments _{* *}: p = 0.05).</p