1,605 research outputs found
Multiple barriers in forced rupture of protein complexes
Curvatures in the most probable rupture force () versus log-loading rate
() observed in dynamic force spectroscopy (DFS) on biomolecular
complexes are interpreted using a one-dimensional free energy profile with
multiple barriers or a single barrier with force-dependent transition state.
Here, we provide a criterion to select one scenario over another. If the
rupture dynamics occurs by crossing a single barrier in a physical free energy
profile describing unbinding, the exponent , from with being a critical force in the
absence of force, is restricted to . For biotin-ligand
complexes and leukocyte-associated antigen-1 bound to intercellular adhesion
molecules, which display large curvature in the DFS data, fits to experimental
data yield , suggesting that ligand unbinding is associated with
multiple-barrier crossing.Comment: 8 pages, 5 figure
Directly measuring single molecule heterogeneity using force spectroscopy
One of the most intriguing results of single molecule experiments on proteins
and nucleic acids is the discovery of functional heterogeneity: the observation
that complex cellular machines exhibit multiple, biologically active
conformations. The structural differences between these conformations may be
subtle, but each distinct state can be remarkably long-lived, with random
interconversions between states occurring only at macroscopic timescales,
fractions of a second or longer. Though we now have proof of functional
heterogeneity in a handful of systems---enzymes, motors, adhesion
complexes---identifying and measuring it remains a formidable challenge. Here
we show that evidence of this phenomenon is more widespread than previously
known, encoded in data collected from some of the most well-established single
molecule techniques: AFM or optical tweezer pulling experiments. We present a
theoretical procedure for analyzing distributions of rupture/unfolding forces
recorded at different pulling speeds. This results in a single parameter,
quantifying the degree of heterogeneity, and also leads to bounds on the
equilibration and conformational interconversion timescales. Surveying ten
published datasets, we find heterogeneity in five of them, all with
interconversion rates slower than 10 s. Moreover, we identify two
systems where additional data at realizable pulling velocities is likely to
find a theoretically predicted, but so far unobserved cross-over regime between
heterogeneous and non-heterogeneous behavior. The significance of this regime
is that it will allow far more precise estimates of the slow conformational
switching times, one of the least understood aspects of functional
heterogeneity.Comment: Main text: 13 pages, 6 figures; SI: 9 pages, 6 figure
Urea-induced denaturation of PreQ1-riboswitch
Urea, a polar molecule with a large dipole moment, not only destabilizes the
folded RNA structures, but can also enhance the folding rates of large
ribozymes. Unlike the mechanism of urea-induced unfolding of proteins, which is
well understood, the action of urea on RNA has barely been explored. We
performed extensive all atom molecular dynamics (MD) simulations to determine
the molecular underpinnings of urea-induced RNA denaturation. Urea displays its
denaturing power in both secondary and tertiary motifs of the riboswitch (RS)
structure. Our simulations reveal that the denaturation of RNA structures is
mainly driven by the hydrogen bonds and stacking interactions of urea with the
bases. Through detailed studies of the simulation trajectories, we found that
geminate pairs between urea and bases due to hydrogen bonds and stacks persist
only ~ (0.1-1) ns, which suggests that urea-base interaction is highly dynamic.
Most importantly, the early stage of base pair disruption is triggered by
penetration of water molecules into the hydrophobic domain between the RNA
bases. The infiltration of water into the narrow space between base pairs is
critical in increasing the accessibility of urea to transiently disrupted
bases, thus allowing urea to displace inter base hydrogen bonds. This
mechanism, water-induced disruption of base-pairs resulting in the formation of
a "wet" destabilized RNA followed by solvation by urea, is the exact opposite
of the two-stage denaturation of proteins by urea. In the latter case, initial
urea penetration creates a dry-globule, which is subsequently solvated by water
penetration leading to global protein unfolding. Our work shows that the
ability to interact with both water and polar, non-polar components of
nucleotides makes urea a powerful chemical denaturant for nucleic acids.Comment: 41 pages, 18 figure
Pathways and kinetic barriers in mechanical unfolding and refolding of RNA and proteins
Using self-organized polymer models, we predict mechanical unfolding and
refolding pathways of ribo-zymes, and the green fluorescent protein. In
agreement with experiments, there are between six and eight unfolding
transitions in the Tetrahymena ribozyme. Depending on the loading rate, the
number of rips in the force-ramp unfolding of the Azoarcus ribozymes is between
two and four. Force-quench refolding of the P4-P6 subdomain of the Tetrahymena
ribozyme occurs through a compact intermediate. Subsequent formation of
tertiary contacts between helices P5b-P6a and P5a/P5c-P4 leads to the native
state. The force-quench refolding pathways agree with ensemble experiments. In
the dominant unfolding route, the N-terminal a helix of GFP unravels first,
followed by disruption of the N terminus b strand. There is a third
intermediate that involves disruption of three other strands. In accord with
experiments, the force-quench refolding pathway of GFP is hierarchic, with the
rate-limiting step being the closure of the barrel.Comment: 33 pages 7 figure
- β¦