Knowledge about protein structure is critical for understanding biological mechanisms. To gain insight into protein structural changes at the residue level we have developed a new benchtop nonlinear optical technique where second-harmonic generation (SHG) and two-photon fluorescence (TPF) are used together in a polarization-dependent manner to determine the mean angle and angular distribution of probes bound to protein molecules. SHG and TPF signals are generated by a monolayer of protein attached to a supported lipid bilayer and the signal intensities are highly dependent on the angle between the probe and surface normal, rendering the technique exceptionally sensitive to probe angular changes resulting from modifications in protein structure. We applied the technique to a protein, E. coli dihydrofolate reductase (DHFR), by attaching a probe at specific cysteines engineered on the protein through mutagenesis. Conformational states resulting from either binding ligands, or between wild-type and mutant proteins, were mapped using angular information from multiple probe attachment sites. To validate the technique, we benchmarked our angular data of DHFR ligand-complexes against previous X-ray and nuclear magnetic resonance (NMR) studies. We also applied the technique to investigate a catalytically impaired point mutant, DHFR-G121V, which is unamenable to crystallography. Our work reveals structural differences between DHFR and DHFR-G121V and suggests a previously unknown mechanism for impaired activity is both an increase in flexibility of the F-G loop in the vicinity of the mutation, as well as a reduced range of motion for the Met20 active site loop.

A number of experimental and computational approaches can determine a conformational change of a structure in response to a perturbation. However, many of these techniques suffer from limitations on the size of the system, operate under non-physiological conditions, are difficult to perform, or are low-throughput. Here, we develop a computational method for modeling rigid body conformational changes using primarily Second Harmonic Generation (SHG) data, a high-throughput technique that informs relative orientations of structural components in ensembles of multiple states under nearly native conditions. We benchmark the technique using simulated and experimentally determined data. For example, we estimate the achievable accuracy and precision of models inferred using SHG data by varying the relative density of simulated data and degrees of freedom of modeled systems. Finally, future experiments and computational advances to improve the accuracy and precision are proposed. The proposed modeling method is in principle applicable to many different protein systems and is available through the open-source Integrative Modeling Platform (IMP) software package.

Proteins are inherently dynamic, flexible molecules that execute precise conformational changes to perform their functions, but existing techniques to directly measure relevant structural changes in solution at room temperature remain limited. Here, we demonstrate a structural technique using second-harmonic generation and two-photon fluorescence under single-laser excitation to map both the mean angular orientation and the distribution width of a probe at various sites throughout the protein with high sensitivity. Our work resolves distinct dihydrofolate reductase (DHFR) ligand-protein conformations, allows interrogation of regions unresolvable by other techniques, and reveals structural differences between DHFR and a point mutant (DHFR-G121V). The technique, angular mapping of protein structure, enables direct and rapid determination of previously unseen aspects of protein structure in a benchtop optical system.

Second-harmonic generation (SHG) has recently emerged as a biophysical tool for conformational sensing of a target biomolecule upon binding to ligands such as small molecules, fragments, proteins, peptides, and oligonucleotides. To date, SHG has been used to measure conformational changes of targets such as soluble proteins, protein complexes, intrinsically disordered proteins, peripheral and integral membrane proteins, peptides, and oligonucleotides upon binding of ligands over a wide range of affinities. In this chapter, we will provide a technology overview, detailed protocols for optimizing assays and screening, practical considerations, and an example case study to guide the reader in developing robust and informative measurements using the Biodesy Delta SHG platform.

Second Harmonic Generation (SHG) has emerged as a highly sensitive probe of protein conformation. SHG can also be used to determine the tilt angle of an SHG-active moiety bound to a surface-adsorbed protein through polarization-dependent measurements. However, due to the coherent nature of SHG, interference occurs between the SHG produced by the SHG-active moieties and background sources at a solid-liquid interface, obscuring the signal of interest. In order to separate the protein-specific signal from the background signal, the phase difference between these two different sources of SHG must be determined. Although the phase difference can be obtained through a conventional interferometric approach involving a phase-modulated SHG source external to the sample, it can be sensitive to drift and other instabilities. We present here a simple, convenient, and crucially, model-independent method to determine the phase difference for any system in which the intensity of SHG-active moieties can be varied. We demonstrate the approach with time-resolved measurements of an SHG-active labeled protein binding to a supported lipid bilayer surface using a total internal reflection (TIR) geometry. This approach requires no additional optics beyond what is required to measure SHG and is highly stable since the interferometry occurs in situ, within the sample over a nanometer length scale, rather than external to it. To validate our measurements and the general approach, we constructed a dual-beam, external SHG interferometer in a TIR geometry. We also validated our approach by applying the in situ method to previously published measurements of the phase difference, obtaining the same values without recourse to a specific adsorption model.

While conventional solid-state nanopore measurements utilize ionic current, there is a growing interest in alternative sensing paradigms, including optical detection. However, a limiting factor in the application of optical schemes in particular is the inherent background fluorescence created by the solid-state membrane itself, which can interfere with the desired signal and place restrictions on the fluorophores that can be employed. An ideal device would incorporate a localized reduction in membrane fluorescence using a method that can be integrated easily with the nanopore fabrication process. Here, we demonstrate that in addition to forming nanopores and nanopore arrays, a focused helium ion beam can be used to reduce the fluorescence of a conventional silicon nitride membrane controllably. The reduction in background produces low-fluorescence devices that can be used for optical detection of double-strand DNA, as well as for conventional resistive pulse sensing. This approach is used to identify the translocation of short single-strand DNA through individual nanopores within an array, creating potential for a massively-parallel detection scheme.

Solid-state nanopores are an emerging technology for detection and analysis at the single-molecule level. Here, we discuss fabrication and characterization of nanopores and nanopore arrays using Helium Ion Milling. We investigate nanopore dimensions using atomic force microscopy and transmission electron microscopy. We demonstrate that nanopore diameter and local membrane thickness are controlled precisely through ion beam dose, achieving pore diameters below 3 nm and device thickness below 5 nm. The technique is high-throughput and lithographic patterning can be used to form arrays of arbitrary size. The resulting devices are compatible with both electrokinetic and optical detection schemes for biodetection applications.

2668-Pos Board B687 Parallel and Automated Formation of Lipid Bilayers on Microstructured Chips for Ion Channel and Nanopore Recordings Gerhard Baaken1, Mohamed Kreir2, Astrid Seifert2, Matthias Beckler2, Juan M. Del Rio-Martinez1, Marcel Hoffmann1, Soenke Petersen1, Jan C. Behrends1, Niels Fertig2. University of Freiburg, Institute of Physiology, Freiburg, Germany, Nanion Technologies, München, Germany. Bilayer recording is a well-established technique for in-depth studies of biophysical properties of ion channels and is particularly suited for functional studies on proteins residing in intracellular membranes. Moreover, this technique supports a host of powerful emerging analytical techniques using biological nanopores as molecular sensors. Despite its proven value, bilayer recording can be frustrating due to the capricious nature of lipid bilayers, which have to be formed manually one by one and which often lack stability. We here show a new approach and device, which speeds up the entire process by the rapid and simultaneous formation of 16, highly stable micrometer-sized bilayers using Micro-Electrode-Cavity-Array (MECA)-Chips. A study will be presented