New approaches for chemical cross-linking in structural proteomics

Author(s)

Mariana Fioramonte

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Química
• Defense date: 0000-00-00

Examining board members

Fábio Cesar Gozzo; Adriana Franco Paes Leme; Richard Hemmi Valente; Ricardo Aparicio; Alessandra Sussulini

• Advisor: Fábio Cesar Gozzo

Abstract

Studying structure and conformation of proteins and proteins complexes is fundamental in biochemistry, since the vast majority of biological processes is regulated by these macromolecules and the knowledge about their structure may lead to their molecular mechanism of action. Chemical cross-linking coupled to mass spectrometry methodology has been shown to be a powerful technique to obtain structure information of proteins and protein complexes, especially when the target protein is not amenable to high resolution techniques such as protein crystallography and nuclear magnetic resonance. The present work is composed by two chapters, both bringing new developments to chemical cross-linking coupled to mass spectrometry methodology in structural proteomics. In chapter I, entitled "The use of chemical cross-linking to study protein secondary structure", we employed molecular dynamics of hypothetical peptides to study the distance distribution between amino acid residues over time and used these distributions to identify which amino acid sites could be bound by a cross-linking reagent based on its spacer arm distance. As a result, it is possible to correlate cross-linked amino acid residues to their secondary structure, showing that the technique is capable to provide information about protein secondary structure. Chapter II, entitled "Development of a new cross-linking reagent", focused on development of new cross-linkers as the vast majority of commercially available cross-linkers are reactive toward amino groups on lysine residues, preventing a more widespread use. Thus, a new reaction for chemical cross-linking was developed that reacts with acidic amino acids, glutamic acid and aspartic acid, as well as serine and lysine, adding new specificities to the methodology, increasing the numbers of cross-links and expanding the technique applicability (AU)

• FAPESP's process: 12/10862-7 - Structural Proteomics by Chemical Cross-linking Coupled to Mass Spectrometry: Development, Fundamental Studies and Applications.
• Grantee: Mariana Fioramonte
• Support Opportunities: Scholarships in Brazil - Doctorate