Design of an expression system for detecting folded protein domains and mapping macromolecular interactions by NMR

Abstract

Two protein expression vectors have been designed for the preparation of NMR samples. The vectors encode the immunoglobulin-binding domain of streptococcal protein G (GB1 domain) linked to the N-terminus of the desired proteins. This fusion strategy takes advantage of the small size, stable fold, and high bacterial expression capability of the GB 1 domain to allow direct NMR spectroscopic analysis of the fusion protein by ' H-l5N correlation spectroscopy.Using this system accelerates the initial assessment of protein NMR projects such that, in a matter of days, the solubility and stability of a protein can be determined. In addition, "N-labeling of peptides and their testing for DNA binding are facilitated. Several examples are presented that demonstrate the usefulness of this technique for screening protein/DNA complexes, as well as for probing ligand-receptor interactions, using iSN-labeled GBI-peptide fusions and unlabeled target.Keywords: domain structure; GBI-fusion protein; intermolecular interactions; NMR When investigating whether a protein or protein-DNA complex is suitable for NMR structural analysis, several technical goals must be met. First, the size, solubility, and stability of the protein domain must be optimized. Second, the protein should be expressed in a host (usually bacteria), allowing for easy and inexpensive labeling with "C and "N. A third challenge pertains to the preparation of protein-DNA complexes where both the protein and DNA sequences must be optimized to achieve a complex that is most amenable to structural investigation; that is, the system should not exhibit intermediate exchange on the NMR time scale. The practical size limit for structure determination by current solution NMR methodology is approximately 40 kDa, so that, for many proteins, subcloning of a smaller domain that retains functional qualities of the full-length protein is required. All too frequently, however, engineering of protein domains results in limited solubility and/or low stability, often to the point where many engineered proteins are partially or predominately unfolded. In practice, many variations of a protein sequence may be evaluated by NMR

The Hantavirus Glycoprotein G1 Tail Contains Dual CCHC-type Classical Zinc Fingers

“…GB1 is the B1 immunoglobulin-binding domain of Streptococcus protein G (19), and a GB1 expression plasmid (obtained from Peter E. Wright, Scripps Research Institute, La Jolla, California) was used in the subcloning. Isotopically ( 15 N or 15 N, 13 C) labeled protein was overexpressed in bacteria as follows: Freshly transformed Escherichia coli BL21(DE3) was grown in 1 liter M9 minimal media supplemented with 0.1 mM ZnSO 4 before and after induction.…”

Transcriptional Regulation inSaccharomyces cerevisiae: Transcription Factor Regulation and Function, Mechanisms of Initiation, and Roles of Activators and Coactivators

“…Most activation domains studied do not fold into well-ordered structures in the absence of a binding target, in contrast to the well-defined structure of DNAbinding motifs. For example, the activation domains of VP16, CREB, and Gcn4 all appear to be unstructured in the absence of a binding partner (Huth et al 1997;Radhakrishnan et al 1997;Uesugi et al 1997;Dames et al 2002;Freedman et al 2002;Brzovic et al 2011). In contrast to DNA-binding motifs, activation domain sequences are often not highly conserved (Martchenko et al 2007).…”

Slc26a9 Is Inhibited by the R-region of the Cystic Fibrosis Transmembrane Conductance Regulator via the STAS Domain

“…Construction of the vector (pGEVII) encodes the immunoglobin-binding domain of streptococcal protein G (GB1 domain) and was linked to the N terminus of the mouse Slc26a9-STAS domain (18). The mouse GB1-Slc26a9-STAS fusion was generated by using a similar PCR strategy for generating the mouse Slc26a9-STAS fragment with in-frame restriction sites, i.e.…”