Presentation Title

Characterization of C terminus Cu2+ binding in Prion Protein by EPR

Faculty Mentor

Dr. Glenn L. Millhauser

Start Date

23-11-2019 8:00 AM

End Date

23-11-2019 8:45 AM

Location

263

Session

poster 1

Type of Presentation

Poster

Subject Area

physical_mathematical_sciences

Abstract

The Prion Protein (PrP) consists of a globular C terminus and an unstructured N terminus. Histidines on the C and N terminus of PrP bind divalent metal ions tethering the two termini together. This cis-interaction prevents toxic action by PrP. Disruption of the cis-interaction is linked to transmissible spongiform encephalopathies (TSEs). TSEs are a class of diseases with no known treatments or cures.

While the role of N terminus histidine residues in the cis-interaction has been identified. The role of C terminus histidine residues remains unclear. To characterize how C terminus histidine residues bind copper, three PrP constructs were labelled with a nitroxide tag and probed using copper-nitroxide electron paramagnetic resonance (EPR) spectroscopy.

Protein constructs were prepared by expressing PrP mutants with an extra cysteine near the expected copper binding site. Protein constructs were then reduced with tris(2-carboxyethyl)phosphine, and different folds were separated by high performance liquid chromatography. To determine which fraction contained the proper fold, the fractions were reacted with N-ethyl-maleimide and trypsinized. The constructs were peptide mapped using liquid chromatography mass spectrometry. The correctly folded fractions were then selected and a nitroxide spin label, S-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate (MTSL), was ligated to the free cystine. The MTSL labeled protein was then lyophilized. The protein was reconstituted in copper containing buffer. These samples were subjected to double electron electron resonance (DEER) and relaxation-induced dipolar modulation enhancement (RIDME) EPR to determine the distance between bound copper and the spin label.

This document is currently not available here.

Share

COinS
 
Nov 23rd, 8:00 AM Nov 23rd, 8:45 AM

Characterization of C terminus Cu2+ binding in Prion Protein by EPR

263

The Prion Protein (PrP) consists of a globular C terminus and an unstructured N terminus. Histidines on the C and N terminus of PrP bind divalent metal ions tethering the two termini together. This cis-interaction prevents toxic action by PrP. Disruption of the cis-interaction is linked to transmissible spongiform encephalopathies (TSEs). TSEs are a class of diseases with no known treatments or cures.

While the role of N terminus histidine residues in the cis-interaction has been identified. The role of C terminus histidine residues remains unclear. To characterize how C terminus histidine residues bind copper, three PrP constructs were labelled with a nitroxide tag and probed using copper-nitroxide electron paramagnetic resonance (EPR) spectroscopy.

Protein constructs were prepared by expressing PrP mutants with an extra cysteine near the expected copper binding site. Protein constructs were then reduced with tris(2-carboxyethyl)phosphine, and different folds were separated by high performance liquid chromatography. To determine which fraction contained the proper fold, the fractions were reacted with N-ethyl-maleimide and trypsinized. The constructs were peptide mapped using liquid chromatography mass spectrometry. The correctly folded fractions were then selected and a nitroxide spin label, S-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate (MTSL), was ligated to the free cystine. The MTSL labeled protein was then lyophilized. The protein was reconstituted in copper containing buffer. These samples were subjected to double electron electron resonance (DEER) and relaxation-induced dipolar modulation enhancement (RIDME) EPR to determine the distance between bound copper and the spin label.