Favorite Techniques
X-ray Crystallography
The Horton lab uses x-ray crystallography to determine the three dimensional structures of proteins and nucleic acids of interest. The macromolecules must first be purified to 99% purity or greater, then are subjected to crystallization trials to find the best conditions for crystals to form. The crystals are then harvested and flash frozen in liquid nitrogen, and transported to synchrotron labs such as SSRL for x-ray data collection. The data are then processed to produce electron density maps, which allow the atomic model of the macromolecule to be built.
CryoEM
The Horton lab also uses single particle and helical reconstruction cryoEM to determine structures of proteins and nucleic acids resistant to crystallization. This is especially important for filament forming proteins and large, complex and dynamic complexes which are very difficult to crystallize.
Recent advances in cryoEM have allowed structures to be determined to resolutions comparable to those of x-ray crystallography.
The Horton lab currently collaborates with the laboratory of Prof. Dmitry Lyumkis of the Salk Institute for Biological Research in CryoEM structural studies.
Enzyme Kinetics
Enzyme kinetic measurements are used to understand the steps between intermediates which is then combined with the structures of those intermediates determined using x-ray crystallography and/or CryoEM to provide a complete picture of the enzyme reaction. Global fitting of the kinetic data to computational models of the reaction pathway provides information on the rate constants for each step, which is then used to understand the advantages or biological role of the reaction mechanism. The Horton lab is equipped with a Kintek Rapid Kinetic Quench Flow instrument, a PC1 fluorimeter, capacity for radiolabeled measurements, and optical spectrometry.
Analytical Ultracentrifugation
Analytical ultracentrifugation (AUC) is a technique to determine the native molecular weight of a macromolecular species. Currently, only two AUC instruments are located in Arizona, both affiliated with the Horton lab. An NSF MRI grant allowed the purchase of a state-of-the art Optima AUC with rapid scanning of four independent wavelengths. This allows the measurement sedimentation of four independent species to give information such as binding affinities, stoichiometry of binding, and total molecular weight.
Fluorescence
Fluorescence spectroscopy is used in the Horton lab to measure binding affinities (using fluorescence quenching or fluorescence anisotropy) or reaction kinetics (using FRET). This information is useful in global modeling of reaction pathways (described above), and understanding determinants of binding specificity. The Horton lab uses an ISS PCI T format fluorimeter with a rapid mixing attachment to perform these measurements.
Computatonal Modeling: Enzyme Reaction Mechanisms
As described above, computational modeling using programs such as Kintek Global Kinetic Explorer or NFsim allow for predictions to be made of enzyme behavior in a biological context.
Computatonal Modeling: Molecular Dynamics Simulations
The Horton lab uses the software Maestro (Schrodinger) to perform simulations of molecular structures. These simulations provide insight into enzyme mechanisms, as structural methods provide only a fixed snapshot of enzyme conformation. Further, methods used to trap enzyme complexes in the crystal and for CryoEM can be "reversed" to an untrapped state to identify changes relevant to enzyme activity.