A microscale protein NMR sample screening pipeline

Abstract

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 lg in 8-35 ll 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.

The dynamics of interleukin‐8 and its interaction with human CXC receptor I peptide

“…In folded proteins the effective correlation time relates well to the molecular weight of the protein up to 25 to 30 kDa. 25,26 The evaluation of correlation times allows for relatively accurate estimation of protein molecular weights. 24,27 Therefore, this method can readily be used to monitor protein dimerization and protein interactions in general, based on changes that alter molecular weight.…”

“…3(C), black dots], which were acquired at the same temperature. 25,26 From these measurements, we estimated the molecular weight of free CXCL8 and CXCL8M to be 14.7 kDa and 9.4 kDa, respectively [ Fig. 3(C), blue dots].…”

Rapid, robotic, small‐scale protein production for NMR screening and structure determination

“…Distance constraints were obtained from 3D 15 N-edited NOESY-HSQC and 13 Cedited NOESY-HSQC spectra (s mix ¼ 80 ms). Backbone w and / dihedral angle constraints were generated from secondary shifts of the 1 H a , 13 C a , 13 C b , 13 C 0 , and 15 N nuclei shifts by the program TALOS. 24 Initial structures were generated using the NOEASSIGN module of the torsion angle dynamics program CYANA, 25,26 followed by iterative manual refinement to eliminate consistently violated restraints.…”

“…Production of 15 N-labeled proteins for NMR is typically a secondary screen requiring large-scale expression and purification at a substantial cost per target. For abundantly expressed, soluble target proteins, a large-scale (0.5-1 L) culture of 15 N-labeled protein is subjected to immobilized metal affinity chromatography (IMAC) purification and the dispersion, number, and intensity of signals in the 2D 1 H- 15 N HSQC spectrum are evaluated before proceeding to production of the 13 C/ 15 N-enriched material needed to solve the structure. Production of 15 Nlabeled proteins is not easily parallelized and the entire screening process often requires two weeks to complete.…”

“…Improved mass sensitivity can be obtained with RF coils optimized for sample volumes smaller than the typical 5-mm diameter format. 11 Microcoil probes using 1 or 1.7 mm sample cells have been used for 1D 12 or 2D NMR screening, 13 and this format is particularly valuable when protein yields are severely limiting. However, in our testing, the signal-to-noise ratio of an HSQC spectrum acquired on a 1.7-mm cryoprobe was 10-fold lower than one acquired on a 3-mm sample with a 5-mm cryoprobe using identical sample and acquisition parameters.…”