Research Mentor(s)
Smirnov, Serge
Description
In the realm of proteins, it is widely accepted that structure informs function. However, there are many proteins that contain intrinsically disordered regions (IDRs). These regions are areas in which the protein lacks defined structure, and IDPs are also often unstable, which complicates structural studies. NMR spectroscopy is an established method for probing protein structure and has been applied to that end in small IDRs. However, larger IDRs often have spectral overlap that makes data difficult to interpret. Furthermore, low-concentration samples limit spectral clarity. One method to address these difficulties is to use sortase ligation and segmental labeling, which increases protein yield and eliminates spectral overlap while maintaining the relevant polypeptide environment. We applied this method in order to investigate the conformational changes incurred by headpiece phosphorylation in dematin. Dematin is an important structural protein in red blood cells, which consists of a folded 68 residue headpiece domain and a large 315 residue IDR. To probe conformational changes induced by covalent modification of the headpiece, we created segmentally labeled samples for use in 15N-HSQC NMR spectroscopy. Fragments of the IDR were created with N-terminal sortase site (LPSTG), and both domains include FH8 and 6xHis tags for purification and a TEV cleavage site for removal of the tags after purification. A S381E headpiece mutation was used as a phosphorylated mimic. To create complete constructs, an isotopically labeled fragment is linked to the remaining domain via sortase-mediated ligation. This ongoing work shows the efficacy of this novel approach for preparing high-yields of segmentally labeled samples and promises to create new methodology for investigating large IDRs through heteronuclear NMR spectroscopy.
Document Type
Event
Start Date
18-5-2020 12:00 AM
End Date
22-5-2020 12:00 AM
Department
Biochemistry
Genre/Form
student projects, posters
Subjects – Topical (LCSH)
Microfilament proteins; Proteins--Chemical modification; Plant proteins--Analysis
Type
Image
Keywords
sortase, ligation, IDR, IDP, NMR, dematin, segmental labeling, intrinsically disordered regions, protein, proteins, chemistry, biochemistry
Rights
Copying of this document in whole or in part is allowable only for scholarly purposes. It is understood, however, that any copying or publication of this document for commercial purposes, or for financial gain, shall not be allowed without the author’s written permission.
Language
English
Format
application/pdf
Included in
Probing Large Intrinsically Disordered Regions through Novel Sortase-Mediated Ligation
In the realm of proteins, it is widely accepted that structure informs function. However, there are many proteins that contain intrinsically disordered regions (IDRs). These regions are areas in which the protein lacks defined structure, and IDPs are also often unstable, which complicates structural studies. NMR spectroscopy is an established method for probing protein structure and has been applied to that end in small IDRs. However, larger IDRs often have spectral overlap that makes data difficult to interpret. Furthermore, low-concentration samples limit spectral clarity. One method to address these difficulties is to use sortase ligation and segmental labeling, which increases protein yield and eliminates spectral overlap while maintaining the relevant polypeptide environment. We applied this method in order to investigate the conformational changes incurred by headpiece phosphorylation in dematin. Dematin is an important structural protein in red blood cells, which consists of a folded 68 residue headpiece domain and a large 315 residue IDR. To probe conformational changes induced by covalent modification of the headpiece, we created segmentally labeled samples for use in 15N-HSQC NMR spectroscopy. Fragments of the IDR were created with N-terminal sortase site (LPSTG), and both domains include FH8 and 6xHis tags for purification and a TEV cleavage site for removal of the tags after purification. A S381E headpiece mutation was used as a phosphorylated mimic. To create complete constructs, an isotopically labeled fragment is linked to the remaining domain via sortase-mediated ligation. This ongoing work shows the efficacy of this novel approach for preparing high-yields of segmentally labeled samples and promises to create new methodology for investigating large IDRs through heteronuclear NMR spectroscopy.