RESUMEN
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.
Asunto(s)
Dinámicas no Lineales , Imagen Óptica , Tetrahidrofolato Deshidrogenasa/química , Dominio Catalítico , Mutación/genética , Mutación Puntual/genética , Tetrahidrofolato Deshidrogenasa/genéticaRESUMEN
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.
RESUMEN
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.
Asunto(s)
Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Oligonucleótidos/metabolismo , Péptidos/metabolismo , Proteínas/metabolismo , Animales , Descubrimiento de Drogas/instrumentación , Diseño de Equipo , Ensayos Analíticos de Alto Rendimiento/instrumentación , Humanos , Ligandos , Conformación de Ácido Nucleico/efectos de los fármacos , Oligonucleótidos/química , Péptidos/química , Conformación Proteica/efectos de los fármacos , Proteínas/químicaRESUMEN
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.
Asunto(s)
Conductometría/métodos , ADN Viral/análisis , ADN Viral/genética , Nanoporos/ultraestructura , Compuestos de Silicona/química , Espectrometría de Fluorescencia/métodos , Técnicas Biosensibles/métodos , ADN Viral/química , Conductividad EléctricaRESUMEN
Kinesin-1 is an ATP-driven, processive motor that transports cargo along microtubules in a tightly regulated stepping cycle. Efficient gating mechanisms ensure that the sequence of kinetic events proceeds in the proper order, generating a large number of successive reaction cycles. To study gating, we created two mutant constructs with extended neck-linkers and measured their properties using single-molecule optical trapping and ensemble fluorescence techniques. Owing to a reduction in the inter-head tension, the constructs access an otherwise rarely populated conformational state in which both motor heads remain bound to the microtubule. ATP-dependent, processive backstepping and futile hydrolysis were observed under moderate hindering loads. On the basis of measurements, we formulated a comprehensive model for kinesin motion that incorporates reaction pathways for both forward and backward stepping. In addition to inter-head tension, we found that neck-linker orientation is also responsible for ensuring gating in kinesin.
Asunto(s)
Cinesinas/química , Adenosina Trifosfato/metabolismo , Sitios de Unión , Fenómenos Biomecánicos , Humanos , Hidrólisis , Cinesinas/metabolismo , Cinesinas/fisiología , Cinética , Microtúbulos/química , Microtúbulos/metabolismo , Modelos Moleculares , Pinzas ÓpticasRESUMEN
We study the hydrodynamic expansion of a rotating strongly interacting Fermi gas by releasing a cigar-shaped cloud with a known angular momentum from an optical trap. As the aspect ratio of the expanding cloud approaches unity, the angular velocity increases, indicating quenching of the moment of inertia I to as low as 0.05 of the rigid body value I(rig). Remarkably, we observe this behavior in both the superfluid and collisional normal fluid regimes, which obey nearly identical zero-viscosity irrotational hydrodynamics. We attribute irrotational flow in the normal fluid to a decay of the rotational part of the stream velocity during expansion, which occurs when the shear viscosity is negligible. Using conservation of angular momentum, we directly observe a fundamental result of irrotational hydrodynamics, I/I(rig) = delta2, where delta is the deformation parameter of the cloud.