3 research outputs found
Modulation of the Intrinsic Helix Propensity of an Intrinsically Disordered Protein Reveals Long-Range Helix–Helix Interactions
Intrinsically
disordered proteins (IDPs) are widespread and important
in biology but defy the classical protein structure–function
paradigm by being functional in the absence of a stable, folded conformation.
Here we investigate the coupling between transient secondary and tertiary
structure in the protein activator for thyroid hormone and retinoid
receptors (ACTR) by rationally modulating the helical propensity of
a partially formed α-helix via mutations. Eight mutations predicted
to affect the population of a transient helix were produced and investigated
by NMR spectroscopy. Chemical shift changes distant to the mutation
site are observed in regions containing other transient helices indicating
that distant helices are stabilized through long-range hydrophobic
helix–helix interactions and demonstrating the coupling of
transient secondary and tertiary structure. The long-range structure
of ACTR is also probed using paramagnetic relaxation enhancements
(PRE) and residual dipolar couplings, which reveal an additional long-range
contact between the N- and C-terminal segments. Compared to residual
dipolar couplings and PRE, modulation of the helical propensity by
mutagenesis thus reveals a different set of long-range interactions
that may be obscured by stronger interactions that dominate other
NMR measurements. This approach thus offers a complementary and generally
applicable strategy for probing long-range structure in disordered
proteins
Single-Molecule Measurements of Transient Biomolecular Complexes through Microfluidic Dilution
Single-molecule confocal microscopy
experiments require concentrations
which are low enough to guarantee that, on average, less than one
single molecule resides in the probe volume at any given time. Such
concentrations are, however, significantly lower than the dissociation
constants of many biological complexes which can therefore dissociate
under single-molecule conditions. To address the challenge of observing
weakly bound complexes in single-molecule experiments in solution,
we have designed a microfluidic device that rapidly dilutes samples
by up to one hundred thousand times, allowing the observation of unstable
complexes before they dissociate. The device can interface with standard
biochemistry laboratory experiments and generates a spatially uniform
dilution that is stable over time allowing the quantification of the
relative concentrations of different molecular species
Engineering a Prototypic P‑type ATPase Listeria monocytogenes Ca<sup>2+</sup>-ATPase 1 for Single-Molecule FRET Studies
Approximately 30% of the ATP generated
in the living cell is utilized
by P-type ATPase primary active transporters to generate and maintain
electrochemical gradients across biological membranes. P-type ATPases
undergo large conformational changes during their functional cycle
to couple ATP hydrolysis in the cytoplasmic domains to ion transport
across the membrane. The Ca<sup>2+</sup>-ATPase from Listeria monocytogenes, LMCA1, was found to be a
suitable model of P-type ATPases and was engineered to facilitate
single-molecule FRET studies of transport-related structural changes.
Mutational analyses of the endogenous cysteine residues in LMCA1 were
performed to reduce background labeling without compromising activity.
Pairs of cysteines were introduced into the optimized low-reactivity
background, and labeled with maleimide derivatives of Cy3 and Cy5
resulting in site-specifically double-labeled protein with moderate
activity. Ensemble and confocal single-molecule FRET studies revealed
changes in FRET distribution related to structural changes during
the transport cycle, consistent with those observed by X-ray crystallography
for the sarco/endoplasmic reticulum Ca<sup>2+</sup> ATPase (SERCA).
Notably, the cytosolic headpiece of LMCA1 was found to be distinctly
more compact in the E1 state than in the E2 state. Thus, the established
experimental system should allow future real-time FRET studies of
the structural dynamics of LMCA1 as a representative P-type ATPase