bi7b00822_si_002.xlsx (33.71 kB)
Small Angle Neutron Scattering Studies of R67 Dihydrofolate Reductase, a Tetrameric Protein with Intrinsically Disordered N‑Termini
dataset
posted on 2017-10-11, 00:00 authored by Purva
P. Bhojane, Michael R. Duff, Khushboo Bafna, Pratul Agarwal, Christopher Stanley, Elizabeth E. HowellR67 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.
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Escherichia coli DHFRR 67 enzymeR 67 DHFR increasesR 67 DHFRPressure perturbation calorimetryprogram SASSIER 67 dimer crystal structureapoenzyme displayschromosomal DHFRsN-termini sampleSimilar studiesR glatter SANS dataglycine betaineangle neutronÅ.Small Angle Neutron Scattering Studies16 N-terminal residues results400 water molecules hydratingR g increases1250 water molecules hydratingosmolytes betaineTetrameric ProteinproteinR 67 Dihydrofolate ReductaseChymotrypsin cleavagesite-directed inhibitordimethyl sulfoxidesite pore
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