25 research outputs found
Stabilization of i-motif structures by 2'-β-fluorination of DNA
i-Motifs are four-stranded DNA structures consisting of two parallel DNA duplexes held together by hemi-protonated and intercalated cytosine base pairs (C:CH(+)). They have attracted considerable research interest for their potential role in gene regulation and their use as pH responsive switches and building blocks in macromolecular assemblies. At neutral and basic pH values, the cytosine bases deprotonate and the structure unfolds into single strands. To avoid this limitation and expand the range of environmental conditions supporting i-motif folding, we replaced the sugar in DNA by 2-deoxy-2-fluoroarabinose. We demonstrate that such a modification significantly stabilizes i-motif formation over a wide pH range, including pH 7. Nuclear magnetic resonance experiments reveal that 2-deoxy-2-fluoroarabinose adopts a C2'-endo conformation, instead of the C3'-endo conformation usually found in unmodified i-motifs. Nevertheless, this substitution does not alter the overall i-motif structure. This conformational change, together with the changes in charge distribution in the sugar caused by the electronegative fluorine atoms, leads to a number of favorable sequential and inter-strand electrostatic interactions. The availability of folded i-motifs at neutral pH will aid investigations into the biological function of i-motifs in vitro, and will expand i-motif applications in nanotechnology.This work is dedicated to the Memory of Alfredo Villasante,
valuable collaborator and friend.
FUNDING
Funding for open access charge: NSERC Discovery grant
(to M.J.D., A.K.M.); CIHR DDTP Training Grant (to
H.A., R.H.V.); MINECO [BFU2014-52864-R to C.G.];
CSIC-JAE contract (to N.M.P.).
Conflict of interest statement.
None declaredS
Local Folding and Misfolding in the PBX Homeodomain from a Three-State Analysis of CPMG Relaxation Dispersion NMR Data
NMR Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments represent a powerful approach for characterizing protein internal motions and for gaining insight into fundamental biological processes such as protein folding, catalysis, and allostery. In most cases, CPMG data are analyzed assuming that the protein exchanges between two different conformational states. Systems exchanging among more than two states are far more challenging to characterize by CPMG NMR. For example, in the case of three-state exchange in the fast time scale regime, it is difficult to uniquely connect the parameters extracted from CPMG analyses with the physical parameters of most interest, intercoversion rates, populations, and chemical shift differences for exchanging states. We have developed a grid search selection procedure that allows these physical parameters to be uniquely determined from CPMG data, based on additional information, which in this study comprises ligand-induced chemical shift perturbations. We applied this approach to the PBX homeodomain (PBX-HD), a three-helix protein with a C-terminal extension that folds into a fourth helix upon binding to DNA. We recently showed that the C-terminal extension transiently folds, even in the absence DNA, in a process that is likely tied to the cooperative binding of PBX-HD to DNA and other homeodomains. Using the grid search selection procedure, we found that PBX-HD undergoes exchange between three different conformational states, a major form in which the C-terminal extension is unfolded, the previously identified state in which the C-terminal extension forms a fourth helix, and an additional state in which the C-terminal extension is misfolded
Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms
Rapid characterization of folding and binding interactions with thermolabile ligands by DSC
Differential scanning calorimetry (DSC) is a powerful technique for measuring tight biomolecular interactions. However, many pharma- ceutically relevant ligands are chemically unstable at the high temperatures used in DSC analyses. Thus, measuring binding inter- actions is challenging because the concentrations of ligands and thermally-converted products are constantly changing within the calorimeter cell. Using experimental data for two DNA aptamers that bind to the thermolabile ligand cocaine, we present a new global fitting analysis that yields the complete set of folding and binding parameters for the initial and final forms of the ligand from a pair of DSC experiments, while accounting for the thermal conversion. Furthermore, we show that the rate constant for thermolabile ligand conversion may be obtained with only one additional DSC dataset
Local Folding and Misfolding in the PBX Homeodomain from a Three-State Analysis of CPMG Relaxation Dispersion NMR Data
NMR Carr–Purcell–Meiboom–Gill (CPMG)
relaxation
dispersion experiments represent a powerful approach for characterizing
protein internal motions and for gaining insight into fundamental
biological processes such as protein folding, catalysis, and allostery.
In most cases, CPMG data are analyzed assuming that the protein exchanges
between two different conformational states. Systems exchanging among
more than two states are far more challenging to characterize by CPMG
NMR. For example, in the case of three-state exchange in the fast
time scale regime, it is difficult to uniquely connect the parameters
extracted from CPMG analyses with the physical parameters of most
interest, intercoversion rates, populations, and chemical shift differences
for exchanging states. We have developed a grid search selection procedure
that allows these physical parameters to be uniquely determined from
CPMG data, based on additional information, which in this study comprises
ligand-induced chemical shift perturbations. We applied this approach
to the PBX homeodomain (PBX-HD), a three-helix protein with a C-terminal
extension that folds into a fourth helix upon binding to DNA. We recently
showed that the C-terminal extension transiently folds, even in the
absence DNA, in a process that is likely tied to the cooperative binding
of PBX-HD to DNA and other homeodomains. Using the grid search selection
procedure, we found that PBX-HD undergoes exchange between three different
conformational states, a major form in which the C-terminal extension
is unfolded, the previously identified state in which the C-terminal
extension forms a fourth helix, and an additional state in which the
C-terminal extension is misfolded
Measuring Rapid Time-Scale Reaction Kinetics Using Isothermal Titration Calorimetry
Isothermal titration
calorimetry (ITC) is a powerful tool for acquiring
both thermodynamic and kinetic data for biological interactions including
molecular recognition and enzymatic catalysis. ITC-based kinetics
measurements typically focus on reactions taking place over long time
scales (tens of minutes or hours) in order to avoid complications
due to the finite length of time needed detect heat flow in the calorimeter
cell. While progress has been made toward analyzing more rapid reaction
kinetics by ITC, the capabilities and limitations of this approach
have not been thoroughly tested to date. Here, we report that the
time resolution of commercial instruments is on the order of 0.2 s
or less. We successfully performed rapid ITC kinetics assays with
durations of just tens of seconds using the enzyme trypsin. This is
substantially shorter than previous ITC enzyme measurements. However,
we noticed that for short reaction durations, standard assumptions
regarding the ITC instrument response led to significant deviations
between calculated and measured ITC peak shapes. To address this issue,
we developed an ITC empirical response model (ITC-ERM) that quantitatively
reproduces ITC peak shapes for all reaction durations. Applying the
ITC-ERM approach to another enzyme (prolyl oligopeptidase), we unexpectedly
discovered non-Michaelis–Menten kinetics in short time-scale
measurements that are absent in more typical long time-scale experiments
and are obscured in short time-scale experiments when standard assumptions
regarding the instrument response are made. This highlights the potential
of ITC measurements of rapid time scale kinetics in conjunction with
the ITC-ERM approach to shed new light on biological dynamics
Complete Kinetic Characterization of Enzyme Inhibition in a Single Isothermal Titration Calorimetric Experiment
Techniques for rapidly
measuring both the strength and mode of
enzyme inhibitors are crucial to lead generation and optimization
in drug development. Isothermal titration calorimetry (ITC) is emerging
as a powerful tool for measuring enzyme kinetics with distinct advantages
over traditional techniques. ITC measures heat flow, a feature of
nearly all chemical reactions, and gives an instantaneous readout
of enzyme velocity, eliminating the need for artificial substrates
or postreaction processing. In principle, ITC is an ideal method for
characterizing enzyme inhibition. However, existing ITC experiments
are not well-suited to rapid throughput and few studies to date have
employed this approach. We have developed a new ITC experiment, in
which substrate and inhibitor are premixed in the injection syringe,
that yields complete kinetic characterization of an enzyme inhibitor
in an hour or less. This corresponds to savings in time and material
of 5-fold or greater compared to previous ITC methods. We validated
the approach using the trypsin inhibitor benzamidine as a model system,
recapitulating both its competitive inhibition mode and binding constant.
Our approach combines the rapid throughput of optimized spectroscopic
assays with the universality and precision of ITC-based methods, providing
substantially improved inhibitor characterization for biochemistry
and drug development applications