Development and application of NMR methods to study biomolecular dynamics

Structural biology has generated profound insights into biomolecular machines. The molecular basis of processes like binding, folding, catalysis and regulation, which underlie the inner working of living organisms would have largely remained unexplored without the thousands of structures that have been solved over the years. But these machines, formed by proteins and nucleic acids, are inherently dynamic, and information about this fourth dimension, the modulation of their structure with time, is often lacking. Nuclear magnetic resonance (NMR) is exquisitely suited to characterize dynamics over a wide timescale, from picoseconds, where amplitudes and correlation times can be extracted, to microsecond, milliseconds and seconds, where in favourable cases information about the kinetics, the thermodynamics and the structure of an excited state can be retrieved. With increasing size of the molecular system under consideration, however, this characterization is progressively challenging for NMR, and the analysis often focuses on 13CH3 spin systems in a perdeuterated background. As an alternative approach, fluorine NMR has grown in popularity. The 19F isotope can be introduced site-specifically, it gives rise to background-free one-dimensional spectra and the technique bypasses the need for perdeuteration. In my disseration, I expanded the existing toolkit of 19F NMR, applied 19F experiments that report on dynamics to high-molecular weight systems and combined their advantages with established methyl group NMR techniques. Development of 19F relaxation dispersion experiments To develop 19F relaxation dispersion (RD) experiments, I used a 7.5 kDa cold shock protein from the thermophilic organism Thermotoga maritima as a protein folding/unfolding model system. The global analysis of three RD experiments showed consistent results for the two-state exchange process. Our new rotating frame relaxation pulse sequences allowed to extract the absolute chemical shift of the unfolded state and significantly extended the range of timescales that can be assessed experimentally. Employing a 360 kDa double heptameric complex, I validated the applicability of the experiments on a highly challenging assembly. Conformational changes in the exoribonuclease Xrn2 The 5'-3' exoribonuclease Xrn2 operates in the nucleus in RNA processing and RNA turn-over pathways. Static structures of its cytoplasmic homologue Xrn1 in the presence of substrates implicate that the enzymes undergo conformational changes to progress through the catalytic cycle. Here, I solved the X-ray structure of Xrn2 from the thermophilic organism Chaetomium thermophilum to 3 Å resolution and combined methyl group and fluorine relaxation dispersion to characterize the exchange in a 100 kDa apo protein core construct in solution. Upon binding of a substrate, the conformational equilibrium is substantially shifted towards the active state. Importantly, the 19F experiments allowed to characterize dynamics in these unstable samples and I could show that the exchange of the enzyme:substrate complex are largely suppressed. Multi-site exchange in a neomycin-sensing riboswitch The existence of multiple sparsely populated states complicates the characterization of an exchanging system. Using a synthetic neomycin-binding riboswitch bound to different aminoglycoside ligands, I demonstrated that fluorine NMR can be employed to study exchange topologies with up to four states. To this end, I take advantage of an additional off-resonance technique, 19F chemical exchange saturation transfer. Combined with 19F RD and longitudinal exchange experiments, the results support the notion of a modular impact of aminoglycoside functional groups on the riboswitch dynamics. Taken together, these results expand and complement the NMR toolbox to study exchanging systems, with an emphasis on high-molecular weight systems and intricate exchange topologies involving more than two states. Furthermore, they elucidate the molecular dynamics in the 5'-3' exoribonuclease Xrn2 and provide a conceptional framework to study dynamics in related systems such as Xrn1

In-situ strain tuning in hBN-encapsulated graphene electronic devices

Using a simple setup to bend a flexible substrate, we demonstrate deterministic and reproducible in-situ strain tuning of graphene electronic devices. Central to this method is the full hBN encapsulation of graphene, which preserves the exceptional quality of pristine graphene for transport experiments. In addition, the on-substrate approach allows one to exploit strain effects in the full range of possible sample geometries and at the same time guarantees that changes in the gate capacitance remain negligible during the deformation process. We use Raman spectroscopy to spatially map the strain magnitude in devices with two different geometries and demonstrate the possibility to engineer a strain gradient, which is relevant for accessing the valley degree of freedom with pseudo-magnetic fields. Comparing the transport characteristics of a suspended device with those of an on-substrate device, we demonstrate that our new approach does not suffer from the ambiguities encountered in suspended devices

Foscarnet Decreases Human Immunodeficiency Virus RNA

Foscarnet inhibits human immunodeficiency virus (HIV) replication in vitro and decreases p24 antigenemia in patients with cytomegalovirus (CMV) retinitis. To evaluate the effect of foscarnet on HIV replication, HIV RNA was quantitated in 17 patients before and during foscarnet therapy. Fifteen patients had CMV retinitis, 1 had CMV encephalitis, and 1 had intractable zoster. A decrease in HIV RNA was observed in 16 of 17 patients. Before the introduction of foscarnet, mean HIV RNA was 5.82 ± 0.24 log RNA/mL and, after a median of 13 days of therapy, mean HIV RNA was 5.30 ± 0.27 log RNA/mL (P < .001). Among patients with detectable p24 antigen at baseline, a significant decrease was observed (P = .017). This decrease in HIV RNA demonstrates that foscarnet is a potent antiretroviral dru

Observation of conformational changes that underlie the catalytic cycle of Xrn2

Nuclear magnetic resonance (NMR) methods that quantitatively probe motions on molecular and atomic levels have propelled the understanding of biomolecular processes for which static structures cannot provide a satisfactory description. In this work, we studied the structure and dynamics of the essential 100-kDa eukaryotic 5′→3′ exoribonuclease Xrn2. A combination of complementary fluorine and methyl-TROSY NMR spectroscopy reveals that the apo enzyme is highly dynamic around the catalytic center. These observed dynamics are in agreement with a transition of the enzyme from the ground state into a catalytically competent state. We show that the conformational equilibrium in Xrn2 shifts substantially toward the active state in the presence of substrate and magnesium. Finally, our data reveal that the dynamics in Xrn2 correlate with the RNA degradation rate, as a mutation that attenuates motions also affects catalytic activity. In that light, our results stress the importance of studies that go beyond static structural information

Cytomegalovirus Retinitis: Decreased Risk of Bilaterality with Increased Use of Systemic Treatment

Cytomegalovirus (CMV) retinitis may be treated systemically or intravitreally. We reviewed retrospectively patients with CMV retinitis, in order to determine whether systemic treatment was associated with less spread of CMV retinitis from one eye to the other. Of 222 cases, 92 patients had bilateral disease at onset of CMV retinitis, leaving 130 for analysis. Bilaterality occurred in 10 patients during 12,687 days of systemic treatment and in 34 during 14,791 days without systemic treatment (odds ratio [OR] = 2.92; confidence interval [CI], 1.44-5.90). Patients who had received systemic treatment for <50% of the follow-up period had a greater risk of bilaterality (OR = 3.7; CI, 2.79-4.54) than did the more intensively treated patients. CD4 cell levels also contributed to increased risk, but multivariate analysis showed that CD4 cell counts and treatment intensity were independent risk factors. CMV retinitis was more likely to become bilateral in patients who received less intravenous therapy. Local treatment can complete but does not replace systemically administered therap

Once-Weekly Administration of Dapsone/Pyrimethamine vs. Aerosolized Pentamidine as Combined Prophylaxis for Pneumocystis carinii Pneumonia and Toxoplasmic Encephalitis in Human Immunodeficiency Virus-Infected Patients

To evaluate combined prophylaxis for Pneumocystis carinii pneumonia (PCP) and toxoplasmic encephalitis, 533 patients with symptomatic human immunodeficiency virus infection and/or CD4 lymphocyte counts of <200/µL were randomized to receive dapsone/pyrimethamine (200/75 mg once weekly) or aerosolized pentamidine (300 mg every 4 weeks). The median CD4 lymphocyte count was 110/µL; 47.5% were seropositive for toxoplasma antibodies. The median duration of follow-up was 483 days. In the intent-to-treat analysis, 12 cases of PCP and 14 of toxoplasmic encephalitis occurred in the dapsone/pyrimethamine group and 13 and 20 cases, respectively, in the aerosolized pentamidine group (adjusted relative risk for toxoplasmosis, 0.56; P = .10). However, only two of the 14 cases of toxoplasmic encephalitis in the dapsone/pyrimethamine group developed during actual treatment. The mortality among the two groups was similar. Dapsone/pyrimethamine was not tolerated by 30% of participants. A subanalysis of 240 matched, tolerant patients yielded a relative risk for toxoplasmosis of 0.21 (P = .014), a result favoring the use of dapsone/pyrimethamine. Dapsone/pyrimethamine was as effective as aerosolized pentamidine as prophylaxis for PCP and significantly reduced the incidence of toxoplasmic encephalitis among those participants who tolerated i

Multi‐Site Conformational Exchange in the Synthetic Neomycin‐Sensing Riboswitch Studied by 19 F NMR

The synthetic neomycin-sensing riboswitch interacts with its cognate ligand neomycin as well as with the related antibiotics ribostamycin and paromomycin. Binding of these aminoglycosides induces a very similar ground state structure in the RNA, however, only neomycin can efficiently repress translation initiation. The molecular origin of these differences has been traced back to differences in the dynamics of the ligand:riboswitch complexes. Here, we combine five complementary fluorine based NMR methods to accurately quantify seconds to microseconds dynamics in the three riboswitch complexes. Our data reveal complex exchange processes with up to four structurally different states. We interpret our findings in a model that shows an interplay between different chemical groups in the antibiotics and specific bases in the riboswitch. More generally, our data underscore the potential of 19F NMR methods to characterize complex exchange processes with multiple excited states

A non-oxidizing fabrication method for lithographic break junctions of sensitive metals

Electrochemically active metals offer advanced functionalities with respect to the well-established gold electrode arrangements in various electronic transport experiments on atomic scale objects. Such functionalities can arise from stronger interactions with the leads which provide better coupling to specific molecules and may also facilitate metallic filament formation in atomic switches. However, the higher reactivity of the electrode metal also imposes challenges in the fabrication and reliability of nanometer scale platforms, limiting the number of reported applications. Here we present a high-yield lithographic fabrication procedure suitable to extend the experimental toolkit with mechanically controllable break junctions of oxygen sensitive metallic electrodes. We fabricate and characterize silver break junctions exhibiting single-atomic conductance and superior mechanical and electrical stability at room temperature. As a proof-of-principle application, we demonstrate resistive switching between metastable few-atom configurations at finite voltage bias