Presentation Title

Purification and Preliminary Crystallization of a Bacterial Dihydromethanopterin Reductase (DmrA) Involved in Tetrahydromethanopterin Biosynthesis in Methylobacterium extorquens

Faculty Mentor

Madeline Rasche

Start Date

18-11-2017 2:15 PM

End Date

18-11-2017 3:15 PM

Location

BSC-Ursa Minor 93

Session

Poster 3

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

The one-carbon metabolism of methane-producing archaea and methylotrophic bacteria requires the cofactor tetrahydromethanopterin (H4MPT), a tetrahydrofolate analog. In archaea, the final step in H4MPT biosynthesis is the reduction of dihydromethanopterin to H4MPT by dihydromethanopterin reductase X (DmrX). However, no DmrX homolog exists in the genome of Methylobacterium extorquens AM1. Instead, the final reaction is catalyzed by DmrA, a homolog of bacterial dihydrofolate reductase (DHFR). Therefore, it is hypothesized that DmrA evolved from duplication and mutation of an ancestral dhfr gene. To study evolutionary changes that may have altered the substrate specificity from dihydrofolate to H2MPT, computational modeling and crystallography studies were initiated. Using DHFR as a template, ICM-Pro was able to predict the overall protein fold of DmrA and the NADPH binding site, but was unsuccessful in modeling a H2MPT binding site. Therefore, in preparation for crystallography, a four-histidine tag and cell storage at pH 8 allowed for purification of a single quaternary structure. Following nickel affinity chromatography and desalting, a protein concentration of 4.1 µg/µL was obtained. SDS-PAGE showed ~ 98% purity with a main band at 17 kDa, while a single band at ~400 kDa was obtained by native PAGE. A pre-crystallization trial resulted in light precipitation using a Tris hydrochloride, ammonium sulfate buffer at a 2:1 ratio of sample:buffer. However, a 288 condition crystallization screen resulted in no precipitate or crystals, indicating that a higher protein concentration may benefit crystallization. Future research will focus on identifying protein concentration, salt, and buffer conditions suitable for DmrA crystallization and structure determination.

Keywords: Methylobacterium extorquens, Tetrahydromethanopterin, Crystallography

This document is currently not available here.

Share

COinS
 
Nov 18th, 2:15 PM Nov 18th, 3:15 PM

Purification and Preliminary Crystallization of a Bacterial Dihydromethanopterin Reductase (DmrA) Involved in Tetrahydromethanopterin Biosynthesis in Methylobacterium extorquens

BSC-Ursa Minor 93

The one-carbon metabolism of methane-producing archaea and methylotrophic bacteria requires the cofactor tetrahydromethanopterin (H4MPT), a tetrahydrofolate analog. In archaea, the final step in H4MPT biosynthesis is the reduction of dihydromethanopterin to H4MPT by dihydromethanopterin reductase X (DmrX). However, no DmrX homolog exists in the genome of Methylobacterium extorquens AM1. Instead, the final reaction is catalyzed by DmrA, a homolog of bacterial dihydrofolate reductase (DHFR). Therefore, it is hypothesized that DmrA evolved from duplication and mutation of an ancestral dhfr gene. To study evolutionary changes that may have altered the substrate specificity from dihydrofolate to H2MPT, computational modeling and crystallography studies were initiated. Using DHFR as a template, ICM-Pro was able to predict the overall protein fold of DmrA and the NADPH binding site, but was unsuccessful in modeling a H2MPT binding site. Therefore, in preparation for crystallography, a four-histidine tag and cell storage at pH 8 allowed for purification of a single quaternary structure. Following nickel affinity chromatography and desalting, a protein concentration of 4.1 µg/µL was obtained. SDS-PAGE showed ~ 98% purity with a main band at 17 kDa, while a single band at ~400 kDa was obtained by native PAGE. A pre-crystallization trial resulted in light precipitation using a Tris hydrochloride, ammonium sulfate buffer at a 2:1 ratio of sample:buffer. However, a 288 condition crystallization screen resulted in no precipitate or crystals, indicating that a higher protein concentration may benefit crystallization. Future research will focus on identifying protein concentration, salt, and buffer conditions suitable for DmrA crystallization and structure determination.

Keywords: Methylobacterium extorquens, Tetrahydromethanopterin, Crystallography