2 research outputs found
Pulsed ESR Dipolar Spectroscopy for Distance Measurements in Immobilized Spin Labeled Proteins in Liquid Solution
Pulsed electron spin resonance (ESR) dipolar spectroscopy
(PDS)
in combination with site-directed spin labeling is unique in providing
nanometer-range distances and distributions in biological systems.
To date, most of the pulsed ESR techniques require frozen solutions
at cryogenic temperatures to reduce the rapid electron spin relaxation
rate and to prevent averaging of electron–electron dipolar
interaction due to the rapid molecular tumbling. To enable measurements
in liquid solution, we are exploring a triarylmethyl (TAM)-based spin
label with a relatively long relaxation time where the protein is
immobilized by attachment to a solid support. In this preliminary
study, TAM radicals were attached via disulfide linkages to substituted
cysteine residues at positions 65 and 80 or 65 and 76 in T4 lysozyme
immobilized on Sepharose. Interspin distances determined using double
quantum coherence (DQC) in solution are close to those expected from
models, and the narrow distance distribution in each case indicates
that the TAM-based spin label is relatively localized
Conformational Flexibility Enables the Function of a BECN1 Region Essential for Starvation-Mediated Autophagy
BECN1 is essential for autophagy,
a critical eukaryotic cellular
homeostasis pathway. Here we delineate a highly conserved BECN1 domain
located between previously characterized BH3 and coiled-coil domains
and elucidate its structure and role in autophagy. The 2.0 Ă…
sulfur-single-wavelength anomalous dispersion X-ray crystal structure
of this domain demonstrates that its N-terminal half is unstructured
while its C-terminal half is helical; hence, we name it the flexible
helical domain (FHD). Circular dichroism spectroscopy, double electron–electron
resonance–electron paramagnetic resonance, and small-angle
X-ray scattering (SAXS) analyses confirm that the FHD is partially
disordered, even in the context of adjacent BECN1 domains. Molecular
dynamic simulations fitted to SAXS data indicate that the FHD transiently
samples more helical conformations. FHD helicity increases in 2,2,2-trifluoroethanol,
suggesting it may become more helical upon binding. Lastly, cellular
studies show that conserved FHD residues are required for starvation-induced
autophagy. Thus, the FHD likely undergoes a binding-associated disorder-to-helix
transition, and conserved residues critical for this interaction are
essential for starvation-induced autophagy