A microscale protein NMR sample screening pipeline

As part of efforts to develop improved methods for NMR protein sample preparation and structure determination, the Northeast Structural Genomics Consortium (NESG) has implemented an NMR screening pipeline for protein target selection, construct optimization, and buffer optimization, incorporating efficient microscale NMR screening of proteins using a micro-cryoprobe. The process is feasible because the newest generation probe requires only small amounts of protein, typically 30–200 μg in 8–35 μl volume. Extensive automation has been made possible by the combination of database tools, mechanization of key process steps, and the use of a micro-cryoprobe that gives excellent data while requiring little optimization and manual setup. In this perspective, we describe the overall process used by the NESG for screening NMR samples as part of a sample optimization process, assessing optimal construct design and solution conditions, as well as for determining protein rotational correlation times in order to assess protein oligomerization states. Database infrastructure has been developed to allow for flexible implementation of new screening protocols and harvesting of the resulting output. The NESG micro NMR screening pipeline has also been used for detergent screening of membrane proteins. Descriptions of the individual steps in the NESG NMR sample design, production, and screening pipeline are presented in the format of a standard operating procedure

SpecDB: A relational database for archiving biomolecular NMR spectral data

NMR is a valuable experimental tool in the structural biologist's toolkit to elucidate the structures, functions, and motions of biomolecules. The progress of machine learning, particularly in structural biology, reveals the critical importance of large, diverse, and reliable datasets in developing new methods and understanding in structural biology and science more broadly. Biomolecular NMR research groups produce large amounts of data, and there is renewed interest in organizing these data to train new, sophisticated machine learning architectures and to improve biomolecular NMR analysis pipelines. The foundational data type in NMR is the free-induction decay (FID). There are opportunities to build sophisticated machine learning methods to tackle long-standing problems in NMR data processing, resonance assignment, dynamics analysis, and structure determination using NMR FIDs. Our goal in this study is to provide a lightweight, broadly available tool for archiving FID data as it is generated at the spectrometer, and grow a new resource of FID data and associated metadata. This study presents a relational schema for storing and organizing the metadata items that describe an NMR sample and FID data, which we call Spectral Database (SpecDB). SpecDB is implemented in SQLite and includes a Python software library providing a command-line application to create, organize, query, backup, share, and maintain the database. This set of software tools and database schema allow users to store, organize, share, and learn from NMR time domain data. SpecDB is freely available under an open source license at https://github.rpi.edu/RPIBioinformatics/SpecDB

ATPase Active-Site Electrostatic Interactions Control the Global Conformation of the 100 kDa SecA Translocase

SecA is an intensively studied mechanoenzyme that uses ATP hydrolysis to drive processive extrusion of secreted proteins through a protein-conducting channel in the cytoplasmic membrane of eubacteria. The ATPase motor of SecA is strongly homologous to that in DEAD-box RNA helicases. It remains unclear how local chemical events in its ATPase active site control the overall conformation of an ∼100 kDa multidomain enzyme and drive protein transport. In this paper, we use biophysical methods to establish that a single electrostatic charge in the ATPase active site controls the global conformation of SecA. The enzyme undergoes an ATP-modulated endothermic conformational transition (ECT) believed to involve similar structural mechanics to the protein transport reaction. We have characterized the effects of an isosteric glutamate-to-glutamine mutation in the catalytic base, a mutation which mimics the immediate electrostatic consequences of ATP hydrolysis in the active site. Calorimetric studies demonstrate that this mutation facilitates the ECT in Escherichia coli SecA and triggers it completely in Bacillus subtilis SecA. Consistent with the substantial increase in entropy observed in the course of the ECT, hydrogen–deuterium exchange mass spectrometry demonstrates that it increases protein backbone dynamics in domain–domain interfaces at remote locations from the ATPase active site. The catalytic glutamate is one of ∼250 charged amino acids in SecA, and yet neutralization of its side chain charge is sufficient to trigger a global order–disorder transition in this 100 kDa enzyme. The intricate network of structural interactions mediating this effect couples local electrostatic changes during ATP hydrolysis to global conformational and dynamic changes in SecA. This network forms the foundation of the allosteric mechanochemistry that efficiently harnesses the chemical energy stored in ATP to drive complex mechanical processes

The RAS-Binding Domain of Human BRAF Protein Serine/Threonine Kinase Exhibits Allosteric Conformational Changes upon Binding HRAS

SummaryRAS binding is a critical step in the activation of BRAF protein serine/threonine kinase and stimulation of the mitogen-activated protein kinase signaling pathway. Mutations in both RAS and BRAF are associated with many human cancers. Here, we report the solution nuclear magnetic resonance (NMR) and X-ray crystal structures of the RAS-binding domain (RBD) from human BRAF. We further studied the complex between BRAF RBD and the GppNHp bound form of HRAS in solution. Backbone, side-chain, and 19F NMR chemical shift perturbations reveal unexpected changes distal to the RAS-binding face that extend through the core of the RBD structure. Moreover, backbone amide hydrogen/deuterium exchange NMR data demonstrate conformational ensemble changes in the RBD core structure upon complex formation. These changes in BRAF RBD reveal a basis for allosteric regulation of BRAF structure and function, and suggest a mechanism by which RAS binding can signal the drastic domain rearrangements required for activation of BRAF kinase