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