3 research outputs found
Mapping Molecular Flexibility of Proteins with Site-Directed Spin Labeling: A Case Study of Myoglobin
Site-directed spin labeling (SDSL) has potential for
mapping protein
flexibility under physiological conditions. The purpose of the present
study was to explore this potential using 38 singly spin-labeled mutants
of myoglobin distributed throughout the sequence. Correlation of the
EPR spectra with protein structure provides new evidence that the
site-dependent variation in line shape, and hence motion of the spin
label, is due largely to differences in mobility of the helical backbone
in the ns time range. Fluctuations between conformational substates,
typically in the Ī¼sāms time range, are slow on the EPR
time scale, and the spectra provide a snapshot of conformational equilibria
frozen in time as revealed by multiple components in the spectra.
A recent study showed that osmolyte perturbation can positively identify
conformational exchange as the origin of multicomponent spectra (LoĢpez et al. (2009), Protein Sci. 18, 1637). In the present study, this new strategy is employed
in combination with line shape analysis and pulsed-EPR interspin distance
measurements to investigate the conformation and flexibility of myoglobin
in three folded and partially folded states. The regions identified
to be in conformational exchange in the three forms agree remarkably
well with those assigned by NMR, but the faster time scale of EPR
allows characterization of localized states not detected in NMR. Collectively,
the results suggest that SDSL-EPR and osmolyte perturbation provide
a facile means for mapping the amplitude of fast backbone fluctuations
and for detecting sequences in slow conformational exchange in folded
and partially folded protein sequences
Stationary-Phase EPR for Exploring Protein Structure, Conformation, and Dynamics in Spin-Labeled Proteins
Proteins tethered to solid supports
are of increasing interest
in bioanalytical chemistry and protein science in general. However,
the extent to which tethering modifies the energy landscape and dynamics
of the protein is most often unknown because there are few biophysical
methods that can determine secondary and tertiary structures and explore
conformational equilibria and dynamics of a tethered protein with
site-specific resolution. Site-directed spin labeling (SDSL) combined
with electron paramagnetic resonance (EPR) offers a unique opportunity
for this purpose. Here, we employ a general strategy using unnatural
amino acids that enables efficient and site-specific tethering of
a spin-labeled protein to a Sepharose solid support. Remarkably, EPR
spectra of spin-labeled T4 lysozyme (T4L) reveal that a single site-specific
attachment suppresses rotational motion of the protein sufficiently
to allow interpretation of the spectral line shape in terms of protein
internal dynamics. Importantly, line shape analysis and distance mapping
using double electronāelectron resonance reveal that internal
dynamics, the tertiary fold, conformational equilibria, and ligand
binding of the tethered proteins were similar to those in solution,
in contrast to random attachment via native lysine residues. The results
of this study set the stage for the development of an EPR-based flow
system that will house soluble and membrane proteins immobilized site-specifically,
thereby enabling facile screening of structural and dynamical effects
of binding partners
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 electronāelectron 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