Kinesin-1 is an ATP-driven, processive motor that transports cargo along microtubules in a tightly regulated stepping cycle. Efficient gating mechanisms ensure that the sequence of kinetic events proceeds in the proper order, generating a large number of successive reaction cycles. To study gating, we created two mutant constructs with extended neck-linkers and measured their properties using single-molecule optical trapping and ensemble fluorescence techniques. Owing to a reduction in the inter-head tension, the constructs access an otherwise rarely populated conformational state in which both motor heads remain bound to the microtubule. ATP-dependent, processive backstepping and futile hydrolysis were observed under moderate hindering loads. On the basis of measurements, we formulated a comprehensive model for kinesin motion that incorporates reaction pathways for both forward and backward stepping. In addition to inter-head tension, we found that neck-linker orientation is also responsible for ensuring gating in kinesin. 
Kai Wang, Ethan Schonbrun, Paul Steinvurzel, and Kenneth B. Crozier
Nucleosomes sterically occlude their wrapped DNA from interacting with many large protein complexes. How proteins gain access to nucleosomal DNA target sites in vivo is not known. Outer stretches of nucleosomal DNA spontaneously unwrap and rewrap with high frequency, providing rapid and efficient access to regulatory DNA target sites located there; however, rates for access to the nucleosome interior have not been measured. Here we show that for a selected high-affinity nucleosome positioning sequence, the spontaneous DNA unwrapping rate decreases dramatically with distance inside the nucleosome. The rewrapping rate also decreases, but only slightly. Our results explain the previously known strong position dependence on the equilibrium accessibility of nucleosomal DNA, which is characteristic of both selected and natural sequences. Our results point to slow nucleosome conformational fluctuations as a potential source of cell–cell variability in gene activation dynamics, and they reveal the dominant kinetic path by which multiple DNA binding proteins cooperatively invade a nucleosome.
Ligands that stabilize the formation of telomeric DNA
G-quadruplexes have potential as cancer treatments, because the G-quadruplex
structure cannot be extended by telomerase, an enzyme over-expressed in many
cancer cells. Understanding the kinetic, thermodynamic and mechanical
properties of small-molecule binding to these structures is therefore
important, but classical ensemble assays are unable to measure these
simultaneously. Here, we have used a laser tweezers method to investigate such
interactions. With a force jump approach, we observe that pyridostatin promotes
the folding of telomeric G-quadruplexes. The increased mechanical stability
of pyridostatin-bound G-quadruplex permits the determination of a
dissociation constant Kd of
490 ± 80 nM. The free-energy change of binding obtained from a
Hess-like process provides an identical Kd for pyridostatin and
a Kd of
42 ± 3 µM for a weaker ligand RR110. We anticipate that this
single-molecule platform can provide detailed insights into the mechanical,
kinetic and thermodynamic properties of liganded bio-macromolecules, which have
biological relevance.
Zinc (Zn) is one of the most abundant metals and is
essential for life. Through ligand interactions, often with thiolate from
cysteine residues in proteins, Zn can play important structural roles in
organizing protein structure and augmenting protein folding and stability.
However, it is difficult to separate the contributions of Zn-ligand
interactions from those originating from intrinsic protein folding in
experimental studies of Zn-containing metalloproteins, which makes the study of
Zn-ligand interactions in proteins challenging. Here, we used single-molecule
force spectroscopy to directly measure the mechanical rupture force of the
Zn-thiolate bond in Zn-rubredoxin. Our results show that considerable force is
needed to rupture Zn-thiolate bonds (∼170 pN, which is
significantly higher than the force necessary to rupture the coordination bond
between Zn and histidines). To our knowledge, our study not only provides new
information about Zn-thiolate bonds in rubredoxin, it also opens a new avenue
for studying metal-ligand bonds in proteins using single-molecule force
spectroscopy.
DNA melting under torsion plays an important role in a wide variety of cellular processes. In the present Letter, we have investigated DNA melting at the single-molecule level using an angular optical trap. By directly measuring force, extension, torque, and angle of DNA, we determined the structural and elastic parameters of torsionally melted DNA. Our data reveal that under moderate forces, the melted DNA assumes a left-handed structure as opposed to an open bubble conformation and is highly torsionally compliant. We have also discovered that at low forces melted DNA properties are highly dependent on DNA sequence. These results provide a more comprehensive picture of the global DNA force-torque phase diagram.
The crucial early stages of amyloid
growth, in which normally soluble proteins are converted into fibrillar
nanostructures, are challenging to study using conventional techniques yet are
critical to the protein aggregation phenomena implicated in many common
pathologies. As with all nucleation and growth phenomena, it is difficult to
track individual nuclei in traditional macroscopic experiments, which probe the
overall temporal evolution of the sample, but do not yield detailed information
on the primary nucleation step as they mix independent stochastic events into
an ensemble measurement. To overcome this limitation, we have developed
microdroplet assays enabling us to detect single primary nucleation events and
to monitor their subsequent spatial as well as temporal evolution, both of
which we find to be determined by secondary nucleation phenomena. By deforming
the droplets to high aspect ratio, we visualize in real-time propagating waves
of protein assembly emanating from discrete primary nucleation sites. We show
that, in contrast to classical gelation phenomena, the primary nucleation step
is characterized by a striking dependence on system size, and the filamentous
protein self-assembly process involves a highly nonuniform spatial distribution
of aggregates. These findings deviate markedly from the current picture of
amyloid growth and uncover a general driving force, originating from
confinement, which, together with biological quality control mechanisms, helps
proteins remain soluble and therefore functional in nature.
Transcript elongation by RNA polymerase involves the sequential
appearance of several alternative and off-pathway states of the transcript
elongation complex (TEC), and this complicates modeling of the kinetics of the
transcription elongation process. Based on solutions of the chemical master
equation for such transcription systems as a function of time, we here develop
a modular scheme for simulating such kinetic transcription data. This scheme
deals explicitly with the problem of TEC desynchronization as transcript
synthesis proceeds, and develops kinetic modules to permit the various
alternative states of the TECs (paused states, backtracked states, arrested
states, and terminated states) to be introduced one-by-one as needed. In this
way, we can set up a comprehensive kinetic model of appropriate complexity to
fit the known transcriptional properties of any given DNA template and set of
experimental conditions, including regulatory cofactors. In the companion
article, this modular scheme is successfully used to model kinetic
transcription elongation data obtained by bulk-gel electrophoresis quenching
procedures and real-time surface plasmon resonance methods from a template of
known sequence that contains defined pause, stall, and termination sites.
Prompted by recent reports suggesting that interaction of filamin A (FLNa) with its binding partners is regulated by mechanical force, we examined mechanical properties of FLNa domains using magnetic tweezers. FLNa, an actin cross-linking protein, consists of two subunits that dimerize through a C-terminal self-association domain. Each subunit contains an N-terminal spectrin-related actin-binding domain followed by 24 immunoglobulinlike (Ig) repeats. The Ig repeats in the rod 1 segment (repeats 1–15) are arranged as a linear array, whereas rod 2 (repeats 16–23) is more compact due to interdomain interactions. In the rod 1 segment, repeats 9–15 augment F-actin binding to a much greater extent than do repeats 1–8. Here, we report that the three segments are unfolded at different forces under the same loading rate. Remarkably, we found that repeats 16–23 are susceptible to forces of ∼10 pN or even less, whereas the repeats in the rod 1 segment can withstand significantly higher forces. The differential force response of FLNa Ig domains has broad implications, since these domains not only support the tension of actin network but also interact with many transmembrane and signaling proteins, mostly in the rod 2 segment. In particular, our finding of unfolding of repeats 16–23 at ∼10 pN or less is consistent with the hypothesized force-sensing function of the rod 2 segment in FLNa.
Tetrahydrofolate
(THF), a biologically active form of the vitamin folate (B9), is an essential cofactor in one-carbon transfer
reactions. In bacteria, expression of folate-related genes is controlled by
feedback modulation in response to specific binding of THF and related
compounds to a riboswitch. Here, we present the X-ray structures of the
THF-sensing domain from the Eubacterium siraeumriboswitch
in the ligand-bound and unbound states. The structure reveals an “inverted”
three-way junctional architecture, most unusual for riboswitches, with the
junction located far from the regulatory helix P1 and not directly
participating in helix P1 formation. Instead, the three-way junction,
stabilized by binding to the ligand, aligns the riboswitch stems for long-range
tertiary pseudoknot interactions that contribute to the organization of helix
P1 and therefore stipulate the regulatory response of the riboswitch. The
pterin moiety of the ligand docks in a semiopen pocket adjacent to the
junction, where it forms specific hydrogen bonds with two moderately conserved
pyrimidines. The aminobenzoate moiety stacks on a guanine base, whereas the
glutamate moiety does not appear to make strong interactions with the RNA. In
contrast to other riboswitches, these findings demonstrate that the THF
riboswitch uses a limited number of available determinants for ligand
recognition. Given that modern antibiotics target folate metabolism, the THF
riboswitch structure provides insights on mechanistic aspects of riboswitch
function and may help in manipulating THF levels in pathogenic bacteria.
