3 years ago

Small Angle Neutron Scattering Studies of R67 Dihydrofolate Reductase, a Tetrameric Protein with Intrinsically Disordered N-Termini

Small Angle Neutron Scattering Studies of R67 Dihydrofolate Reductase, a Tetrameric Protein with Intrinsically Disordered N-Termini
Pratul Agarwal, Khushboo Bafna, Michael R. Duff, Purva P. Bhojane, Elizabeth E. Howell, Christopher Stanley
R67 dihydrofolate reductase (DHFR) is a homotetramer with a single active site pore and no sequence or structural homology with chromosomal DHFRs. The R67 enzyme provides resistance to trimethoprim, an active site-directed inhibitor of Escherichia coli DHFR. Sixteen to twenty N-terminal amino acids are intrinsically disordered in the R67 dimer crystal structure. Chymotrypsin cleavage of 16 N-terminal residues results in an active enzyme with a decreased stability. The space sampled by the disordered N-termini of R67 DHFR was investigated using small angle neutron scattering. From a combined analysis using molecular dynamics and the program SASSIE (http://www.smallangles.net/sassie/SASSIE_HOME.html), the apoenzyme displays a radius of gyration (Rg) of 21.46 ± 0.50 Å. Addition of glycine betaine, an osmolyte, does not result in folding of the termini as the Rg increases slightly to 22.78 ± 0.87 Å. SASSIE fits of the latter SANS data indicate that the disordered N-termini sample larger regions of space and remain disordered, suggesting they might function as entropic bristles. Pressure perturbation calorimetry also indicated that the volume of R67 DHFR increases upon addition of 10% betaine and decreased at 20% betaine because of the dehydration of the protein. Studies of the hydration of full-length R67 DHFR in the presence of the osmolytes betaine and dimethyl sulfoxide find around 1250 water molecules hydrating the protein. Similar studies with truncated R67 DHFR yield around 400 water molecules hydrating the protein in the presence of betaine. The difference of ∼900 waters indicates the N-termini are well-hydrated.

Publisher URL: http://dx.doi.org/10.1021/acs.biochem.7b00822

DOI: 10.1021/acs.biochem.7b00822

You might also like
Never Miss Important Research

Researcher is an app designed by academics, for academics. Create a personalised feed in two minutes.
Choose from over 15,000 academics journals covering ten research areas then let Researcher deliver you papers tailored to your interests each day.

  • Download from Google Play
  • Download from App Store
  • Download from AppInChina

Researcher displays publicly available abstracts and doesn’t host any full article content. If the content is open access, we will direct clicks from the abstracts to the publisher website and display the PDF copy on our platform. Clicks to view the full text will be directed to the publisher website, where only users with subscriptions or access through their institution are able to view the full article.