13 research outputs found
Direct Monitoring of β‑Sheet Formation in the Outer Membrane Protein TtoA Assisted by TtOmp85
Attenuated total
reflection Fourier-transform infrared (ATR-FTIR)
spectroscopy was applied to investigate the folding of an outer membrane
protein, TtoA, assisted by TtOmp85, both from the thermophilic eubacterium <i>Thermus thermophilus</i>. To directly monitor the formation
of β-sheet structure in TtoA and to analyze the function of
TtOmp85, we immobilized unfolded TtoA on an ATR crystal. Interaction
with TtOmp85 initiated TtoA folding as shown by time-dependent spectra
recorded during the folding process. Our ATR-FTIR experiments prove
that TtOmp85 possesses specific functionality to assist β-sheet
formation of TtoA. We demonstrate the potential of this spectroscopic
approach to study the interaction of outer membrane proteins in vitro
and in a time-resolved manner
Snapshot of a DNA Polymerase while Incorporating Two Consecutive C5-Modified Nucleotides
Functional nucleotides are important
in many cutting-edge biomolecular
techniques. Often several modified nucleotides have to be incorporated
consecutively. This structural study of KlenTaq DNA polymerase, a
truncated form of <i>Thermus aquaticus</i> DNA polymerase,
gives first insights how multiple modifications are processed by a
DNA polymerase and, therefore, contribute to the understanding of
these enzymes in their interplay with artificial substrates
Chorismatase Mechanisms Reveal Fundamentally Different Types of Reaction in a Single Conserved Protein Fold
Chorismatases are a class of chorismate-converting
enzymes involved
in the biosynthetic pathways of different natural products, many of
them with interesting pharmaceutical characteristics. So far, three
subfamilies of chorismatases are described that convert chorismate
into different (dihydro-)benzoate derivatives
(CH-FkbO, CH-Hyg5, and CH-XanB2). Until now, the detailed enzyme mechanism
and the molecular basis for the different reaction products were unknown.
Here we show that the CH-FkbO and CH-Hyg5 subfamilies share the same
protein fold, but employ fundamentally different reaction mechanisms.
While the FkbO reaction is a typical hydrolysis, the Hyg5 reaction
proceeds intramolecularly, most likely via an arene oxide intermediate.
Two nonconserved active site residues were identified that are responsible
for the different reaction mechanisms in CH-FkbO and CH-Hyg5. Further,
we propose an additional amino acid residue to be responsible for
the discrimination of the CH-XanB2 subfamily, which catalyzes the
formation of two different hydroxybenzoate regioisomers, likely in
a single active site. A multiple sequence alignment shows that these
three crucial amino acid positions are located in conserved motifs
and can therefore be used to assign unknown chorismatases to the corresponding
subfamily
Structural Insights into DNA Replication without Hydrogen Bonds
The genetic alphabet
is composed of two base pairs, and the development
of a third, unnatural base pair would increase the genetic and chemical
potential of DNA. d<b>5SICS</b>-d<b>NaM</b> is one of
the most efficiently replicated unnatural base pairs identified to
date, but its pairing is mediated by only hydrophobic and packing
forces, and in free duplex DNA it forms a cross-strand intercalated
structure that makes its efficient replication difficult to understand.
Recent studies of the KlenTaq DNA polymerase revealed that the insertion
of d<b>5SICS</b>TP opposite d<b>NaM</b> proceeds via a
mutually induced-fit mechanism, where the presence of the triphosphate
induces the polymerase to form the catalytically competent closed
structure, which in turn induces the pairing nucleotides of the developing
unnatural base pair to adopt a planar Watson–Crick-like structure.
To understand the remaining steps of replication, we now report the
characterization of the prechemistry complexes corresponding to the
insertion of d<b>NaM</b>TP opposite d<b>5SICS</b>, as
well as multiple postchemistry complexes in which the already formed
unnatural base pair is positioned at the postinsertion site. Unlike
with the insertion of d5<b>SICS</b>TP opposite d<b>NaM</b>, addition of d<b>NaM</b>TP does not fully induce the formation
of the catalytically competent closed state. The data also reveal
that once synthesized and translocated to the postinsertion position,
the unnatural nucleobases again intercalate. Two modes of intercalation
are observed, depending on the nature of the flanking nucleotides,
and are each stabilized by different interactions with the polymerase,
and each appear to reduce the affinity with which the next correct
triphosphate binds. Thus, continued primer extension is limited by
deintercalation and rearrangements with the polymerase active site
that are required to populate the catalytically active, triphosphate
bound conformation
Structures of <i>KlenTaq</i> DNA Polymerase Caught While Incorporating C5-Modified Pyrimidine and C7-Modified 7-Deazapurine Nucleoside Triphosphates
The capability of DNA polymerases to accept chemically
modified
nucleotides is of paramount importance for many biotechnological applications.
Although these analogues are widely used, the structural basis for
the acceptance of the unnatural nucleotide surrogates has been only
sparsely explored. Here we present in total six crystal structures
of modified 2′-deoxynucleoside-5′-<i>O</i>-triphosphates (dNTPs) carrying modifications at the C5 positions
of pyrimidines or C7 positions of 7-deazapurines in complex with a
DNA polymerase and a primer/template complex. The modified dNTPs are
in positions poised for catalysis leading to incorporation. These
structural data provide insight into the mechanism of incorporation
and acceptance of modified dNTPs. Our results open the door for rational
design of modified nucleotides, which should offer great opportunities
for future applications
Altered supercoiling response of dsDNA in presence of TrmBL2 for different forces.
<p>Arrows indicate the sense of rotation whereas the colour indicates the applied force. <b>(A)</b> Supercoiling curves in absence of TrmBL2 at different forces. The lighter and darker shade of a given color show curves that were obtained when inducing negative and positive supercoiling, respectively. Small cartoons of the plectoneme formation are shown. <b>(B)</b> Supercoiling curves taken at the same conditions in presence of 4.6 μM TrmBL2. The presented curves were all recorded using the same tether.</p
Kinetics of TrmBL2 binding to dsDNA at different concentrations of TrmBL2.
<p>The kinetics were measured by monitoring the change of the DNA length at a force of 0.4 pN, which linearly changes with the coverage of the DNA by the protein. The data shown were recorded from the same DNA molecule. At room temperature, the filament formation is completed (95%) within approximately 10 minutes and the final DNA length (dotted line) is observed.</p
Hairpin unzipping at constant force reveals discrete protein dissociation events.
<p><b>(A)</b> Time trajectory of DNA hairpin unzipping and rezipping in absence of TrmBL2. The force was alternated between 20.6 pN and 1.6 pN to induce the conformational changes of the hairpin. Hairpin unzipping occurred under these conditions in a single fast step. <b>(B)</b> Time trajectory of DNA hairpin unzipping and rezipping in presence of 30 μM TrmBL2. Different forces from 20.6 pN to 26.5 pN were used for unzipping (see force axis). Hairpin opening occurred in small steps obviously due to displacement of bound protein (see detail in the right panel). <b>(C)</b> Number of base pairs unzipped in single steps for the traces shown under (B). The heights of the steps were determined manually and converted into the number of opened base pairs with a force-dependent algorithm (unpublished).</p
A model for the TrmBL2 filament using the crystal structure of TrmBL2 with bound dsDNA.
<p>In the crystal structure of TrmBL2 from <i>Pyrococcus furiosus</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156098#pone.0156098.ref010" target="_blank">10</a>] four protein molecules bind to the 19 basepairs long TGM dsDNA. In the filament model shown in panel A, it comprises the first quarter with DNA (pink) and the protein tetramer (green). First an octameric complex was constructed from two copies of the crystal structure. In the uppermost panel it comprises the left halve with one pink, one red DNA piece, one green and one yellow tetramer. Two copies of the octameric complex were shifted in tandem to get a continuous dsDNA running through both as seen in panel A. The resulting dsDNA in the hexadecamer has zero overall curvature. The hexadecameric filament model is shown in ribbon representation in three orthogonal views (A-C). Note the almost full coverage of the DNA by TrmBL2 and that the DNA is in an almost perfectly linear conformation.</p
DNA unzipping in absence and presence of TrmBL2.
<p>A DNA hairpin is anchored through a dsDNA spacer at one end to the surface and at the other end to the magnetic bead (top). At sufficient force the hairpin is disrupted. When lowering the force, the hairpin spontaneously closes. <b>(B-E)</b> Unzipping (cyan) and rezipping (blue) cycles of the hairpin (see arrows for direction) in absence and presence of TrmBL2 as indicated in the figures. Hairpin rezipping is already affected at low TrmBL2 concentrations (19 nM) while hairpin unzipping is affected only when significant fractions of the DNA are covered by the protein (from 187 nM on). This shows that bound TrmBL2 shifts the unzipping to higher force and the rezipping to lower force indicating that the protein binds both, dsDNA and ssDNA.</p