Ligand-induced conformational changes in protein molecules detected by sum-frequency generation.

We present the first demonstration of ligand-induced conformational changes in a biological molecule, a protein, by sum-frequency generation (SFG). Constructs of KRas protein were prepared by selectively deuterating residues of a single amino acid type using isotope-labeled amino acids and cell-free protein synthesis. By attaching labeled protein to a supported bilayer membrane via a His-tag to Ni-NTA-bearing lipids, we ensured that single layers of ordered molecules were formed while preserving the protein's native structure.

Second harmonic generation detection of Ras conformational changes and discovery of a small molecule binder.

Second harmonic generation (SHG) is an emergent biophysical method that sensitively measures real-time conformational change of biomolecules in the presence of biological ligands and small molecules. This study describes the successful implementation of SHG as a primary screening platform to identify fragment ligands to oncogenic Kirsten rat sarcoma (KRas). KRas is the most frequently mutated driver of pancreatic, colon, and lung cancers; however, there are few well-characterized small molecule ligands due to a lack of deep binding pockets.

Angular Mapping of Protein Structure Using Nonlinear Optical Measurements.

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).

Second-Harmonic Generation (SHG) for Conformational Measurements: Assay Development, Optimization, and Screening.

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.

Identification of inactive conformation-selective interleukin-2-inducible T-cell kinase (ITK) inhibitors based on second-harmonic generation.

Many clinically approved protein kinase inhibitors stabilize an inactive conformation of their kinase target. Such inhibitors are generally highly selective compared to active conformation inhibitors, and consequently, general methods to identify inhibitors that stabilize an inactive conformation are much sought after. Here, we have applied a high-throughput, second-harmonic generation (SHG)-based conformational approach to identify small molecule stabilizers of the inactive conformation of interleukin-2-inducible T-cell kinase (ITK).

Engineering a Single-Agent Cytokine/Antibody Fusion That Selectively Expands Regulatory T Cells for Autoimmune Disease Therapy.

IL-2 has been used to treat diseases ranging from cancer to autoimmune disorders, but its concurrent immunostimulatory and immunosuppressive effects hinder efficacy. IL-2 orchestrates immune cell function through activation of a high-affinity heterotrimeric receptor (composed of IL-2Rα, IL-2Rβ, and common γ [γ]). IL-2Rα, which is highly expressed on regulatory T (T) cells, regulates IL-2 sensitivity.

Monomeric ephrinB2 binding induces allosteric changes in Nipah virus G that precede its full activation.

Nipah virus is an emergent paramyxovirus that causes deadly encephalitis and respiratory infections in humans. Two glycoproteins coordinate the infection of host cells, an attachment protein (G), which binds to cell surface receptors, and a fusion (F) protein, which carries out the process of virus-cell membrane fusion. The G protein binds to ephrin B2/3 receptors, inducing G conformational changes that trigger F protein refolding.

Evaluating the Contribution of Transition-State Destabilization to Changes in the Residence Time of Triazole-Based InhA Inhibitors.

A critical goal of lead compound selection and optimization is to maximize target engagement while minimizing off-target binding. Since target engagement is a function of both the thermodynamics and kinetics of drug-target interactions, it follows that the structures of both the ground states and transition states on the binding reaction coordinate are needed to rationally modulate the lifetime of the drug-target complex. Previously, we predicted the structure of the rate-limiting transition state that controlled the time-dependent inhibition of the enoyl-ACP reductase InhA.

Second-harmonic phase determination by real-time in situ interferometry.

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.

ATP-Competitive MLKL Binders Have No Functional Impact on Necroptosis.

MLKL is a pore forming pseudokinase involved in the final stage of necroptosis, a form of programmed cell death. Its phosphorylation by RIPK3 is necessary for triggering necroptosis but not for triggering apoptosis, which makes it a unique target for pharmacological inhibition to block necroptotic cell death. This mechanism has been described as playing a role in disease progression in neurodegenerative and inflammatory diseases.

Detection of Ligand-Induced Conformational Changes in Oligonucleotides by Second-Harmonic Generation at a Supported Lipid Bilayer Interface.

There is a high demand for characterizing oligonucleotide structural changes associated with binding interactions as well as identifying novel binders that modulate their structure and function. In this study, second-harmonic generation (SHG) was used to study RNA and DNA oligonucleotide conformational changes associated with ligand binding. For this purpose, we developed an avidin-based biotin capture surface based on a supported lipid bilayer membrane.

Small Molecules Detected by Second-Harmonic Generation Modulate the Conformation of Monomeric α-Synuclein and Reduce Its Aggregation in Cells.

Proteins are structurally dynamic molecules that perform specialized functions through unique conformational changes accessible in physiological environments. An ability to specifically and selectively control protein function via conformational modulation is an important goal for development of novel therapeutics and studies of protein mechanism in biological networks and disease. Here we applied a second-harmonic generation-based technique for studying protein conformation in solution and in real time to the intrinsically disordered, Parkinson disease related protein α-synuclein.

Protein Conformational Changes Are Detected and Resolved Site Specifically by Second-Harmonic Generation.

We present here a straightforward, broadly applicable technique for real-time detection and measurement of protein conformational changes in solution. This method is based on tethering proteins labeled with a second-harmonic generation (SHG) active dye to supported lipid bilayers. We demonstrate our method by measuring the conformational changes that occur upon ligand binding with three well-characterized proteins labeled at lysine residues: calmodulin (CaM), maltose-binding protein (MBP), and dihydrofolate reductase (DHFR).

A second-harmonic-active unnatural amino acid as a structural probe of biomolecules on surfaces.

Second-harmonic generation (SHG) is highly sensitive to the net, average orientation of SH-active molecules on surfaces and has recently emerged as a technique for detecting biomolecules and their conformational changes. As most biomolecules are not intrinsically SH-active, they must be labeled with probes to render them detectable. To date, exogenous probes have been used to do this, but second-harmonic-active unnatural amino acids offer important advantages for the long-range goal of precisely and directly determining structural changes in real time and may be used for both buried and surface sites.

Second-harmonic generation for studying structural motion of biological molecules in real time and space.

SHG and sum-frequency generation (SFG) are surface-selective, nonlinear optical techniques whose ability to measure the average tilt angle of molecules on surfaces is well known in non-biological systems. By labeling molecules with a second-harmonic-active dye probe, SHG detection is extended to any biological molecule. The method has been used in previous work to detect biomolecules at an interface and their ligand-induced conformational changes.

Detection of protein conformational change by optical second-harmonic generation.

An ability to detect and track conformational change in real time is essential to understanding the dynamic relationship between structure and function in biological molecules. Here I show that second-harmonic generation (SHG), a surface-selective technique, offers a new means to probe structural dynamics. A protein, calmodulin, was labeled with a second-harmonic-active dye and immobilized to a surface.