Paul J. Rothwell

Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes.

By using single-molecule multiparameter fluorescence detection, fluorescence resonance energy transfer experiments, and newly developed data analysis methods, this study demonstrates directly the existence of three structurally distinct forms of reverse transcriptase (RT):nucleic acid complexes in solution. Single-molecule multiparameter fluorescence detection also provides first information on the structure of a complex not observed by x-ray crystallography. This species did not incorporate nucleotides and is structurally distinct from the other two observed species. We determined that the nucleic acid substrate is bound at a site far removed from the nucleic acid-binding tract observed by crystallography. In contrast, the other two states are identified as being similar to the x-ray crystal structure and represent distinct enzymatically productive stages in DNA polymerization. These species differ by only a 5-Å shift in the position of the nucleic acid. Addition of nucleoside triphosphate or of inorganic pyrophosphate allowed us to assign them as the educt and product state in the polymerization reaction cycle; i.e., the educt state is a complex in which the nucleic acid is positioned to allow nucleotide incorporation. The second RT:nucleic acid complex is the product state, which is formed immediately after nucleotide incorporation, but before RT translates to the next nucleotide.

Accurate single-molecule FRET studies using multiparameter fluorescence detection.

In the recent decade, single-molecule (sm) spectroscopy has come of age and is providing important insight into how biological molecules function. So far our view of protein function is formed, to a significant extent, by traditional structure determination showing many beautiful static protein structures. Recent experiments by single-molecule and other techniques have questioned the idea that proteins and other biomolecules are static structures. In particular, Förster resonance energy transfer (FRET) studies of single molecules have shown that biomolecules may adopt many conformations as they perform their function. Despite the success of sm-studies, interpretation of smFRET data are challenging since they can be complicated due to many artifacts arising from the complex photophysical behavior of fluorophores, dynamics, and motion of fluorophores, as well as from small amounts of contaminants. We demonstrate that the simultaneous acquisition of a maximum of fluorescence parameters by multiparameter fluorescence detection (MFD) allows for a robust assessment of all possible artifacts arising from smFRET and offers unsurpassed capabilities regarding the identification and analysis of individual species present in a population of molecules. After a short introduction, the data analysis procedure is described in detail together with some experimental considerations. The merits of MFD are highlighted further with the presentation of some applications to proteins and nucleic acids, including accurate structure determination based on FRET. A toolbox is introduced in order to demonstrate how complications originating from orientation, mobility, and position of fluorophores have to be taken into account when determining FRET-related distances with high accuracy. Furthermore, the broad time resolution (picoseconds to hours) of MFD allows for kinetic studies that resolve interconversion events between various subpopulations as a biomolecule of interest explores its structural energy landscape.

ACCURATE SINGLE-MOLECULE FRET STUDIES USING MULTIPARAMETER FLUORESCENCE DETECTION : SINGLE MOLECULE TOOLS, PT B

In the recent decade, single-molecule (sm) spectroscopy has come of age and is providing important insight into how biological molecules function. So far our view of protein function is formed, to a significant extent, by traditional structure determination showing many beautiful static protein structures. Recent experiments by single-molecule and other techniques have questioned the idea that proteins and other biomolecules are static structures. In particular, Förster resonance energy transfer (FRET) studies of single molecules have shown that biomolecules may adopt many conformations as they perform their function. Despite the success of sm-studies, interpretation of smFRET data are challenging since they can be complicated due to many artifacts arising from the complex photophysical behavior of fluorophores, dynamics, and motion of fluorophores, as well as from small amounts of contaminants. We demonstrate that the simultaneous acquisition of a maximum of fluorescence parameters by multiparameter fluorescence detection (MFD) allows for a robust assessment of all possible artifacts arising from smFRET and offers unsurpassed capabilities regarding the identification and analysis of individual species present in a population of molecules. After a short introduction, the data analysis procedure is described in detail together with some experimental considerations. The merits of MFD are highlighted further with the presentation of some applications to proteins and nucleic acids, including accurate structure determination based on FRET. A toolbox is introduced in order to demonstrate how complications originating from orientation, mobility, and position of fluorophores have to be taken into account when determining FRET-related distances with high accuracy. Furthermore, the broad time resolution (picoseconds to hours) of MFD allows for kinetic studies that resolve interconversion events between various subpopulations as a biomolecule of interest explores its structural energy landscape.

Polyinosinic acid is a ligand for toll-like receptor 3.

Innate immune responses are critical in controlling viral infections. Viral proteins and nucleic acids have been shown to be recognized by pattern recognition receptors of the Toll-like receptor (TLR) family, triggering downstream signaling cascades that lead to cellular activation and cytokine production. Viral DNA is sensed by TLR9, and TLRs 3, 7, and 8 have been implicated in innate responses to RNA viruses by virtue of their ability to sense double-stranded (ds) RNA (TLR3) or single-stranded RNA (murine TLR7 and human TLR8). Viral and synthetic dsRNAs have also been shown to be a potent adjuvant, promoting enhanced adaptive immune responses, and this property is also dependent on their recognition by TLR3. It has recently been shown that mRNA that is largely single-stranded is a ligand for TLR3. Here we have investigated the ability of single-stranded homopolymeric nucleic acids to induce innate responses by murine immune cells. We show for the first time that polyinosinic acid (poly(I)) activates B lymphocytes, dendritic cells, and macrophages and that these responses are dependent on the expression of both TLR3 and the adaptor molecule, Toll/IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF). We therefore conclude that TLR3 is able to sense both single-stranded RNA and dsRNA.

A Pre-equilibrium before Nucleotide Binding Limits Fingers Subdomain Closure by Klentaq1

Numerous studies have been undertaken to establish the mechanism of dNTP binding and template-directed incorporation by DNA polymerases. It has been established by kinetic experiments that a rate-limiting step, crucial for dNTP selection, occurs before chemical bond formation. Crystallographic studies indicated that this step may be due to a large open-to-closed conformational transition affecting the fingers subdomain. In previous studies, we established a fluorescence resonance energy transfer system to monitor the open-to-closed transition in the fingers subdomain of Klentaq1. By comparing the rates of the fingers subdomain closure with that of the rate-limiting step for Klentaq1, we showed that fingers subdomain motion was significantly faster than the rate-limiting step. We have now used this system to characterize DNA binding as well as to complete a more extensive characterization of incorporation of all four dNTPs. The data indicate that DNA binding occurs by a two-step association and that dissociation of the DNA is significantly slower in the case of the closed ternary complex. The data for nucleotide incorporation indicate a step occurring before dNTP binding, which differs for all four nucleotides. As the only difference between the (E·p/t) complexes is the templating base, it would suggest an important role for the templating base in initial ground state selection.

Single molecule FCS-based oxygen sensor (O2-FCSensor): a new intrinsically calibrated oxygen sensor utilizing fluorescence correlation spectroscopy (FCS) with single fluorescent molecule detection sensitivity

Abstract We report about a new intrinsically calibrated sensor technology exemplified by means of an O 2 -FCSensor. The O 2 -FCSensor is particularly suited for the quantitative determination of molecular oxygen within biological gases and fluids. The technology is based on the determination of the highly O 2 -dependent triplet transition rates of suitable fluorophores such as Rhodamine Green or Fluorescein. The method utilizes fluorescence correlation spectroscopy (FCS) with the ultimate detection sensitivity of a single fluorescent molecule. Calibration curves of the triplet fraction ( T ) and triplet relaxation time ( τ T ) as a dependence of the oxygen concentration have been determined using a thin (μm) sensor layer, and can be well approximated by hyperbolic sensor characteristics with oxygen sensitivities in the order of 0.1/(%O 2 ). Interestingly, the oxygen sensitivity of triplet fractions of O 2 -FCSensors are not influenced by changes in viscosity of the solution demonstrating that FCS-based O 2 measurements are not diffusion controlled (in contrast to the dynamic process of collisional fluorescence quenching by molecular oxygen). Due to the ultimate sensitivity of this technique it can be applied to nanomolar or even single molecule concentrations of fluorophores thus toxic or cancerogenic influences of the applied indicators are avoided or at least minimized. Furthermore, new fluorescent indicator classes with exceptional photophysical properties can be explored for FCS-based chemo- and biosensors enabling the measurement of other analytes such as, e.g. hydrogen ion activities and carbon dioxide. As a consequence, blood gas analysis (pH, pCO 2 and pO 2 ) may be realized on the basis of intrinsically calibrated FCS-Sensor technology comprising the potential of absolute measurement with unprecedented sensitivity.

dNTP-dependent Conformational Transitions in the Fingers Subdomain of Klentaq1 DNA Polymerase INSIGHTS INTO THE ROLE OF THE “NUCLEOTIDE-BINDING” STATE

Background: Conformational selection plays a key role in the polymerase cycle. Results: Klentaq1 exists in conformational equilibrium between three states (open, closed, and “nucleotide-binding”) whose level of occupancy is determined by the bound substrate. Conclusion: The “nucleotide-binding” state plays a pivotal role in the reaction pathway. Significance: Direct evidence is provided for the role of a conformationally distinct “nucleotide-binding” state during dNTP incorporation. DNA polymerases are responsible for the accurate replication of DNA. Kinetic, single-molecule, and x-ray studies show that multiple conformational states are important for DNA polymerase fidelity. Using high precision FRET measurements, we show that Klentaq1 (the Klenow fragment of Thermus aquaticus DNA polymerase 1) is in equilibrium between three structurally distinct states. In the absence of nucleotide, the enzyme is mostly open, whereas in the presence of DNA and a correctly base-pairing dNTP, it re-equilibrates to a closed state. In the presence of a dNTP alone, with DNA and an incorrect dNTP, or in elevated MgCl2 concentrations, an intermediate state termed the “nucleotide-binding” state predominates. Photon distribution and hidden Markov modeling revealed fast dynamic and slow conformational processes occurring between all three states in a complex energy landscape suggesting a mechanism in which dNTP delivery is mediated by the nucleotide-binding state. After nucleotide binding, correct dNTPs are transported to the closed state, whereas incorrect dNTPs are delivered to the open state.

dNTP-Dependent Conformational Transitions in the Fingers Subdomain of Klentaq1

DNA polymerases are responsible for the accurate replication of DNA. Kinetic studies indicate the requirement for multiple intermediate enzyme conformations in this process. Structural studies show a large conformational change in the fingers subdomain of DNA polymerase on binding of a correct dNTP. Using single molecule FRET we show that the conformational transition affecting the fingers subdomain also takes place in the apo and DNA-bound forms of the enzyme. In addition a third conformation is observed which is occupied in the presence of dNTP alone, and in the presence of a non base pairing dNTPs. The relative proportions of the identified states are altered dramatically depending on substrate. Binding of the correct nucleotide displaces this equilibrium dramatically towards the closed form, while binding of an incorrect nucleotide favors a more open conformation. The results suggest that the closed state is by design less energetically favored, providing a thermodynamic brake on incorrect nucleotide insertion.