2′-19F labelling of ribose in RNAs: a tool to analyse RNA/protein interactions by NMR in physiological conditions

Protein-RNA interactions are central to numerous cellular processes. In this work, we present an easy and straightforward NMR-based approach to determine the RNA binding site of RNA binding proteins and to evaluate the binding of pairs of proteins to a single-stranded RNA (ssRNA) under physiological conditions, in this case in nuclear extracts. By incorporation of a 19F atom on the ribose of different nucleotides along the ssRNA sequence, we show that, upon addition of an RNA binding protein, the intensity of the 19F NMR signal changes when the 19F atom is located near the protein binding site. Furthermore, we show that the addition of pairs of proteins to a ssRNA containing two 19F atoms at two different locations informs on their concurrent binding or competition. We demonstrate that such studies can be done in a nuclear extract that mimics the physiological environment in which these protein-ssRNA interactions occur. Finally, we demonstrate that a trifluoromethoxy group (-OCF3) incorporated in the 2′ribose position of ssRNA sequences increases the sensitivity of the NMR signal, leading to decreased measurement times, and reduces the issue of RNA degradation in cellular extracts

Surface passivation with a perfluoroalkane brush improves the precision of single-molecule measurements

Single-molecule imaging is invaluable for investigating the heterogeneous behavior and interactions of biological molecules. However, an impediment to precise sampling of single molecules is the irreversible adsorption of components onto the surfaces of cover glasses. This causes continuous changes in the concentrations of different molecules dissolved or suspended in the aqueous phase from the moment a sample is dispensed, which will shift, over time, the position of chemical equilibria between monomeric and multimeric components. Interferometric scattering microscopy (iSCAT) is a technique in the single-molecule toolkit that has the capability to detect unlabeled proteins and protein complexes both as they adsorb onto and desorb from a glass surface. Here, we examine the reversible and irreversible interactions between a number of different proteins and glass via analysis of the adsorption and desorption of protein at the single-molecule level. Furthermore, we present a method for surface passivation that virtually eliminates irreversible adsorption while still ensuring the residence time of molecules on surfaces is sufficient for detection of adsorption by iSCAT. By grafting high-density perfluoroalkane brushes on cover-glass surfaces, we observe approximately equal numbers of adsorption and desorption events for proteins at the measurement surface (±1%). The fluorous–aqueous interface also prevents the kinetic trapping of protein complexes and assists in establishing a thermodynamic equilibrium between monomeric and multimeric components. This surface passivation approach is valuable for in vitro single-molecule experiments using iSCAT microscopy because it allows for continuous monitoring of adsorption and desorption of protein without either a decline in detection events or a change in sample composition due to the irreversible binding of protein to surfaces

2′- 19 F labelling of ribose in RNAs: a tool to analyse RNA/protein interactions by NMR in physiological conditions

Protein-RNA interactions are central to numerous cellular processes. In this work, we present an easy and straightforward NMR-based approach to determine the RNA binding site of RNA binding proteins and to evaluate the binding of pairs of proteins to a single-stranded RNA (ssRNA) under physiological conditions, in this case in nuclear extracts. By incorporation of a 19F atom on the ribose of different nucleotides along the ssRNA sequence, we show that, upon addition of an RNA binding protein, the intensity of the 19F NMR signal changes when the 19F atom is located near the protein binding site. Furthermore, we show that the addition of pairs of proteins to a ssRNA containing two 19F atoms at two different locations informs on their concurrent binding or competition. We demonstrate that such studies can be done in a nuclear extract that mimics the physiological environment in which these protein-ssRNA interactions occur. Finally, we demonstrate that a trifluoromethoxy group (-OCF3) incorporated in the 2′ribose position of ssRNA sequences increases the sensitivity of the NMR signal, leading to decreased measurement times, and reduces the issue of RNA degradation in cellular extracts

A New Platform for Single Molecule Measurements Using the Fluorous Effect

Irreversible adsorption of biomolecules onto imaging substrates is an impediment to expand the applications of single molecule techniques. Traditional polyethylene glycol (PEG) surfaces are only effective at low concentrations of analytes and their structure prevents their use for interferometric scattering (iSCAT) microscopy. We propose a new platform that virtually eliminates non-specific binding thanks to the omniphobicity of perfluorinated compounds, also known as the fluorous effect. Here, we showcase the anti-fouling properties of these substrates at a single molecule level through iSCAT measurements of a protein mixture. We believe these novel engineered substrates show great promise to study biomachinery processes requiring large analyte concentrations, where other passivation methods are not effective, through iSCAT microscopy and other single molecule techniques

Nanoscale Confinement and Fluorescence Effects of Bacterial Light Harvesting Complex LH2 in Mesoporous Silicas

Many key chemical and biochemical reactions, particularly in living cells, take place in confined space at the mesoscopic scale. Toward understanding of physicochemical nature of biomacromolecules confined in nanoscale space, in this work we have elucidated fluorescence effects of a light harvesting complex LH2 in nanoscale chemical environments. Mesoporous silicas (SBA-15 family) with different shapes and pore sizes were synthesized and used to create nanoscale biomimetic environments for molecular confinement of LH2. A combination of UV-vis absorption, wide-field fluorescence microscopy, and in situ ellipsometry supports that the LH2 complexes are located inside the silica nanopores. Systematic fluorescence effects were observed and depend on degree of space confinement. In particular, the temperature dependence of the steady-state fluorescence spectra was analyzed in detail using condensed matter band shape theories. Systematic electronic-vibrational coupling differences in the LH2 transitions between the free and confined states are found, most likely responsible for the fluorescence effects experimentally observed

Multidimensional Fluorescence Polarization Imaging of Single Light Harvesting Complexes

This thesis presents my research journey in the Department of Chemical Physics,Lund University. Multidimensional fluorescence polarization imaging is used to study single LH2s. The method uses linearly polarized excitation light and the emission is detected through a polarizer. The multidimensionality is achieved by rotating the excitation and detection polarizations which enables the construction of 2-dimensional excitation-emission polarization maps of the fluorescence intensity. The method provides information about the molecular energy level structure and spatial orientation of the molecule. The method is particularly useful for multichromophoric systems like LH2 where it is capable of tracking the energy transfer from an excited chromophore to the emitting one. Simulations based on Redfield relaxation theory,are in good agreement with the experimental data. The simulations include the known structure of LH2, independent spectroscopic information about electron-phonon coupling and energetic disorder. The thesis is organized as a collection of the articles preceded by an overview of the light harvesting antenna system, the experimental technique and the results. Chapter I gives the general introduction to the light harvesting antenna systems and in particular the peripheral light harvesting antenna of purple bacterial, called LH2. The chapter II deals with the technical aspects of single complex multidimensional polarization imaging on LH2. The section also gives a short introduction to the relevant software and data analysis. Chapter III gives an overview of LH2 studies in the field of single molecule spectroscopy and bimolecular nano-technology. Last chapter IV summarizes the experimental results, simulations and main conclusions

Excitation-Emission Polarization Spectroscopy of Single Light Harvesting Complexes.

Excitation and emission polarization dependence of fluorescence intensity of single LH2 complexes from Rhodopseudomonas acidophila 10050 and Rhodobacter sphaeroides is reported. The results are presented as two-dimensional polarization plots and interpreted in terms of tilted light harvesting complexes indicating that sample preparation leads to partially oriented LH2 cylinders. An alternative explanation of the observation can be structural deformation. Fluorescence intensity of the complexes has four qualitatively distinct excitation-emission polarization dependencies. The differences in excitation polarization dependence are interpreted as due to the tilt of the complexes, whereas the emission polarization behavior is mainly determined by spectral inhomogeneity of the emitting B850 ring. Some complexes show abrupt reversible variations of the total emission intensity together with changes of the polarization properties which cannot be described by the simplest model of tilted LH2s with spectral disorder

Polarization single complex imaging of circular photosynthetic antenna.

Single complex fluorescence polarization spectroscopy is applied to study the peripheral light harvesting antenna (LH2) from photosynthetic purple bacterium Rhodopseudomonas (Rps.) acidophila. The measured two-dimensional excitation-emission polarization plots are used to construct geometric representation for the absorbing B800 and emitting B850 as ellipses. The shape and orientation of the ellipses is discussed in terms of tilted LH2 complexes where emission occurs from energetically disordered B850 excitons

Anchored LH2 Complexes in 2D Polarization Imaging.

Protein is a soft material with inherently large structural disorder. Consequently, the bulk spectroscopies of photosynthetic pigment protein complexes provide averaged information where many details are lost. Here we report spectroscopy of single light-harvesting complexes where fluorescence excitation and detection polarizations are both independently rotated. Two samples of peripheral antenna (LH2) complexes from Rhodopseudomonas acidophila were studied. In one, the complexes were embedded in polyvinyl alcohol (PVA) film; in the other, they were anchored on the glass surface and covered by the PVA film. LH2 contains two rings of pigment molecules-B800 and B850. The B800 excitation polarization properties of the two samples were found to be very similar, indicating that orientation statistics of LH2s are the same in these two very different preparations. At the same time, we found a significant difference in B850 emission polarization statistics. We conclude that the B850 band of the anchored sample is substantially more disordered. We argue that both B800 excitation and B850 emission polarization properties can be explained by the tilt of the anchored LH2s due to the spin-casting of the PVA film on top of the complexes and related shear forces. Due to the tilt, the orientation statistics of two samples become similar. Anchoring is expected to orient the LH2s so that B850 is closer to the substrate. Consequently, the tilt-related strain leads to larger deformation and disorder in B850 than in B800

Protein Configuration Landscape Fluctuations Revealed by Exciton Transition Polarizations in Single Light Harvesting Complexes.

Protein is a flexible material with broad distribution of conformations forming an energy landscape of quasi-stationary states. Disentangling the system dynamics along this landscape is the key for understanding the functioning of the protein. Here we studied a photosynthetic antenna pigment-protein complex LH2 with single molecule two-dimensional polarization imaging. Modeling based on the Redfield relaxation theory well describes the observed polarization properties of LH2 fluorescence and fluorescence excitation, strongly suggesting that at 77 K the conformational subspace of the LH2 is limited to about three configurations with relatively frequent switching among each other. At room temperature the next level of fluctuations determines the conformational dynamics. The results support the multitier model of the energy landscape of proteins and demonstrate the potential of the method for the studies of structural dynamics in proteins