Investigation of protein structure by means of ¹⁹F‐NMR

Abstract

A 19F‐labeled derivative of hen egg‐white lysozyme, in which the six ɛ‐amino groups are trifluoroacetylated (LF,), was prepared by reaction of lysozyme with S‐ethyltrifluorothioacetate. The reaction mixture was fractionated by cation‐exchange chromatography at pH 7.3. A comparison of the circular dichroic spectra and the activity towards Micrococcus lysodeikticus of both LF6 and native lysozyme reveals that the labeling causes no major conformational changes of the polypeptide backbone. Assignment of the six resonances present in the 19F‐NMR spectrum of LF6 was accomplished by using a variety of techniques: specific chemical modifications, the effect of the inhibitor (GlcNAc)3, 19F‐shift/pH information and relaxation parameters.

Glucocorticoid receptor binding to calf thymus DNA. 1. Identification and characterization of a macromolecular factor involved in receptor-steroid complex binding to DNA

“…Among these are an -59K protein (Tai et ai., 1986;Rexin et al, 1988) as well as hsp90 (Bresnick et al, 1989). A low molecular weight inhibitor of activation (Bodine & Litwack, 1988a,b;Meshinchi et al, 1988), and possibly RNA (Webb et al, 1986;Sablonniere et al, 1988), may also be associated with the unactivated complex. Additional components have been found to influence the action of activated complexes (Simons et al, 1976;Schmidt & Litwack, 1982;Dahmer et al, 1985;Isohashi & Sakamoto, 1986;Tymoczko et al, 1988;Cavanaugh & Simons, 1990).…”

19F NMR as a Tool for Monitoring Individual Differentially Labeled Proteins in Complex Mixtures

“…Several previously reported fluorine tags for chemical modifications of proteins were investigated in this study, with a focus on maximizing labeling efficiency and obtaining a set of tags with a range of characteristic 19 F chemical shifts. Examples assessed included BTFA, 2,2,2-trifluoroethanthiol (TFET), , and S -ethyl-trifluorothioacetate (SETFA) . Primary amines were also investigated as sites for 19 F labeling, with reagents reported to target both the lysine side chains ( p -nitro trifluoroacetate, PNTFA) and the N-terminus of the protein ( N -succinimidyl trifluoroacetate, NSTFA) .…”

“…The alternative approach of targeting lysine side chains typically allows labeling at a greater number of sites on the protein, but compounds capable of this, such as p-nitrophenyl trifluoroacetate, often require a high pH (9−10) for efficient reaction, which may present problems for protein solubility and stability. 25 Modification of serine and threonine hydroxyl groups is another possibility. 20 Radical trifluoromethylation of tryptophan, tyrosine, and phenylalanine residues has also been recently reported.…”

“…Compounds for covalent linking to cysteine residues generally consist of a trifluoromethyl moiety linked to a group reactive to nucleophilic substitution, such as the commonly used 3-bromo-1,1,1-trifluoroacetone. − This approach does require that the protein has accessible cysteine sulfhydryl groups, so partial reduction may be needed in some cases to activate cysteines in disulfide bridges. The alternative approach of targeting lysine side chains typically allows labeling at a greater number of sites on the protein, but compounds capable of this, such as p -nitrophenyl trifluoroacetate, often require a high pH (9–10) for efficient reaction, which may present problems for protein solubility and stability . Modification of serine and threonine hydroxyl groups is another possibility .…”

Ribonuclease inhibits Ah receptor transformation in vitro

“…Numerous studies indicate that additional molecules associate with the ligand-binding subunit of the glucocorticoid receptor to mediate transformation and/or subsequent DNA binding of the receptor complex (reviewed by Bodine & Litwack, 1990). Results from several laboratories suggest that RNA may be one such component involved in the transformation of steroid hormone receptors (Kovacic-Milivojevic et al, 1985;Kovacic-Milivojevic & Vedeckis, 1986;Rowley et al, 1986;Kasayama et al, 1987;Ali & Vedeckis, 1987a,b;Sablonniere et al, 1988).…”