Exploitation of proteomics strategies in protein structure-function studies

Marino, Gennaro; Pucci, Piero; Birolo, Leila; Ruoppolo, Margherita

doi:10.1351/pac200375020309

Cited by 5 publications

(3 citation statements)

References 13 publications

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“…In the assumption that the distribution of the preferential proteolytic sites is strictly dependent on the protein conformation, limited proteolysis experiments, causing single cleavage events in the protein structure (primary site) and originating complementary peptide fragments (‘complementary proteolysis’), seems well suited to investigate conformational changes in protein structure upon formation of protein complexes . Based on this approach, very few primary, complementary cleavage sites are detected for highly structured proteins that remain largely undigested, thus providing an instrumental, low‐resolution picture of the protein topology.…”

Section: Resultsmentioning

confidence: 99%

Regulating levels of the neuromodulator d‐serine in human brain: structural insight into pLG72 and d‐amino acid oxidase interaction

et al. 2016

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The human flavoenzyme D-amino acid oxidase (hDAAO) degrades the NMDA-receptor modulator D-serine in the brain. Although hDAAO has been extensively characterized, little is known about its main modulator pLG72, a small protein encoded by the primate-specific gene G72 that has been associated with schizophrenia susceptibility. pLG72 interacts with neosynthesized hDAAO, promoting its inactivation and degradation. In this work, we used low-resolution techniques to characterize the surface topology of the hDAAO-pLG72 complex. By using limited proteolysis coupled to mass spectrometry, we could map the exposed regions in the two proteins after complex formation and highlighted an increased sensitivity to proteolysis of hDAAO in complex with pLG72. Cross-linking experiments by using bis(sulfosuccinimidyl)suberate identified the single covalent bond between T182 in hDAAO and K62 in pLG72. In order to validate the designed mode of interaction, three pLG72 variants incrementally truncated at the C terminus, in addition to a form lacking the 71 N-terminal residues, were produced. All variants were dimeric, folded, and interacted with hDAAO. The strongest decrease in affinity for hDAAO (as well as for the hydrophobic drug chlorpromazine) was apparent for the N-terminally deleted pLG72 form, which lacked K62. On the other hand, eliminating the disordered C-terminal tail yielded a more stable pLG72 protein, improved the binding to hDAAO, although giving lower enzyme inhibition. Elucidation of the mode of hDAAO-pLG72 interaction now makes it possible to design novel molecules that, by targeting the protein complex, can be therapeutically advantageous for diseases related to impairment in D-serine metabolism.Abbreviations BS 3 , bis(sulfosuccinimidyl)suberate; CPZ, chlorpromazine; DAAO, D-amino acid oxidase; DTT, dithiothreitol; FAD, flavin adenine dinucleotide; Glu-C, endoprotease Glu-C; hDAAO, human D-amino acid oxidase; NLS, N-lauroylsarcosine; NMDAR, N-methyl-D-aspartate receptor; RP-HPLC, reverse-phase high-performance liquid chromatography; SPR, surface plasmon resonance; TFA, trifluoroacetic acid.

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Section: Resultsmentioning

confidence: 99%

Regulating levels of the neuromodulator d‐serine in human brain: structural insight into pLG72 and d‐amino acid oxidase interaction

et al. 2016

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“…Limited Proteolysis. Limited proteolysis experiments were designed to study the surface topology of (IF 1 ) 2 on the grounds that the portions of the protein that are involved in highly structured regions or are located at the intersubunits interface are protected from proteases (29).…”

Section: Materials Recombinant If 1 Was Provided By Mrc Medicalmentioning

confidence: 99%

“…Fragments released from the dimer were identified on the basis of their unique mass values and the specificity of the proteolytic enzyme. Identification of the two complementary peptides following a single proteolytic event led to the assignment of the preferential cleavage sites (29). In Supporting Information, an example of the MALDI-TOF analysis of the time course experiments with Glu-C endoprotease is reported.…”

Section: Structuralmentioning

confidence: 99%

Stoichiometry and Topology of the Complex of the Endogenous ATP Synthase Inhibitor Protein IF₁ with Calmodulin

et al. 2010

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IF(1), the natural inhibitor protein of F(O)F(1)ATP synthase able to regulate the ATP hydrolytic activity of both mitochondrial and cell surface enzyme, exists in two oligomeric states depending on pH: an inactive, highly helical, tetrameric form above pH 6.7 and an active, inhibitory, dimeric form below pH 6.7 [ Cabezon , E. , Butler , P. J. , Runswick , M. J. , and Walker , J. E. ( 2000 ) J. Biol. Chem. 275 , 25460 -25464 ]. IF(1) is known to interact in vitro with the archetypal EF-hand calcium sensor calmodulin (CaM), as well to colocalize with CaM on the plasma membrane of cultured cells. Low resolution structural data were herein obtained in order to get insights into the molecular interaction between IF(1) and CaM. A combined structural proteomic strategy was used which integrates limited proteolysis and chemical cross-linking with mass spectrometric analysis. Specifically, chemical cross-linking data clearly indicate that the C-terminal lobe of CaM molecule contacts IF(1) within the inhibitory, flexible N-terminal region that is not involved in the dimeric interface in IF(1). Nevertheless, native mass spectrometry analysis demonstrated that in the micromolar range the stoichiometry of the IF(1)-CaM complex is 1:1, thereby indicating that binding to CaM promotes IF(1) dimer dissociation without directly interfering with the intersubunit contacts of the IF(1) dimer. The relevance of the finding that only the C-terminal lobe of CaM is involved in the interaction is two fold: (i) the IF(1)-CaM complex can be included in the category of noncanonical structures of CaM complexes; (ii) it can be inferred that the N-terminal region of CaM might have the opportunity to bind to a second target.

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Introduction to Marine Biotechnology

Kornprobst

2014

Encyclopedia of Marine Natural Products

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The article contains sections titled: Introduction Recent Developments in the Culture of Invertebrates Lipids and Pigments: Examples of Nutritional Applications New Substances and Biomaterials: Examples of Biomedical Applications Bioremediation and Biofouling: Examples of Environmental Applications Dialkylphthalates in Marine Organisms Another Type of Application: Bioadhesives Some Examples of Biotechnology in Prokaryotes

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Exploitation of proteomics strategies in protein structure-function studies

Cited by 5 publications

References 13 publications

Regulating levels of the neuromodulator d‐serine in human brain: structural insight into pLG72 and d‐amino acid oxidase interaction

Regulating levels of the neuromodulator d‐serine in human brain: structural insight into pLG72 and d‐amino acid oxidase interaction

Stoichiometry and Topology of the Complex of the Endogenous ATP Synthase Inhibitor Protein IF₁ with Calmodulin

Introduction to Marine Biotechnology

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Exploitation of proteomics strategies in protein structure-function studies

Cited by 5 publications

References 13 publications

Regulating levels of the neuromodulator d‐serine in human brain: structural insight into pLG72 and d‐amino acid oxidase interaction

Regulating levels of the neuromodulator d‐serine in human brain: structural insight into pLG72 and d‐amino acid oxidase interaction

Stoichiometry and Topology of the Complex of the Endogenous ATP Synthase Inhibitor Protein IF1 with Calmodulin

Introduction to Marine Biotechnology

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Stoichiometry and Topology of the Complex of the Endogenous ATP Synthase Inhibitor Protein IF₁ with Calmodulin