ABSTRACT
Pseudocontact shifts (PCS) generated by paramagnetic lanthanide ions deliver powerful restraints for protein structure analysis by NMR spectroscopy. We present a new lanthanide tag that generates different PCSs than that of a related tag, which differs in structure by a single oxygen atom. It is highly reactive towards cysteine and performs well in turn-on luminescence and in EPR spectroscopy.
ABSTRACT
Proteins produced with leucine analogues, where CH2F groups substitute specific methyl groups, can readily be probed by 19F NMR spectroscopy. As CF and CH groups are similar in hydrophobicity and size, fluorinated leucines are expected to cause minimal structural perturbation, but the impact of fluorine on the rotational freedom of CH2F groups is unclear. We present high-resolution crystal structures of Escherichia coli peptidyl-prolyl cis-trans isomerase B (PpiB) prepared with uniform high-level substitution of leucine by (2S,4S)-5-fluoroleucine, (2S,4R)-5-fluoroleucine, or 5,5'-difluoroleucine. Apart from the fluorinated leucine residues, the structures show complete structural conservation of the protein backbone and the amino acid side chains except for a single isoleucine side chain located next to a fluorine atom in the hydrophobic core of the protein. The carbon skeletons of the fluorinated leucine side chains are also mostly conserved. The CH2F groups show a strong preference for staggered rotamers and often appear locked into single rotamers. Substitution of leucine CH3 groups for CH2F groups is thus readily tolerated in the three-dimensional (3D) structure of a protein, and the rotation of CH2F groups can be halted at cryogenic temperatures.
Subject(s)
Leucine , Leucine/chemistry , Escherichia coli/metabolism , Protein Conformation , Models, Molecular , Crystallography, X-Ray , Peptidylprolyl Isomerase/chemistry , Peptidylprolyl Isomerase/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolismABSTRACT
Global substitution of leucine for analogues containing CH2F instead of methyl groups delivers proteins with multiple sites for monitoring by 19F nuclear magnetic resonance (NMR) spectroscopy. The 19 kDa Escherichia coli peptidyl-prolyl cis-trans isomerase B (PpiB) was prepared with uniform high-level substitution of leucine by (2S,4S)-5-fluoroleucine, (2S,4R)-5-fluoroleucine, or 5,5'-difluoroleucine. The stability of the samples toward thermal denaturation was little altered compared to the wild-type protein. 19F nuclear magnetic resonance (NMR) spectra showed large chemical shift dispersions between 6 and 17 ppm. The 19F chemical shifts correlate with the three-bond 1H-19F couplings (3JHF), providing the first experimental verification of the γ-gauche effect predicted by [Feeney, J. J. Am. Chem. Soc. 1996, 118, 8700-8706] and establishing the effect as the predominant determinant of the 19F chemical shifts of CH2F groups. Individual CH2F groups can be confined to single rotameric states by the protein environment, but most CH2F groups exchange between different rotamers at a rate that is fast on the NMR chemical shift scale. Interactions between fluorine atoms in 5,5'-difluoroleucine bias the CH2F rotamers in agreement with results obtained previously for 1,3-difluoropropane. The sensitivity of the 19F chemical shift to the rotameric state of the CH2F groups potentially renders them particularly sensitive for detecting allosteric effects.
Subject(s)
Escherichia coli Proteins , Escherichia coli , Peptidylprolyl Isomerase , Peptidylprolyl Isomerase/metabolism , Peptidylprolyl Isomerase/chemistry , Escherichia coli/metabolism , Escherichia coli/genetics , Escherichia coli/enzymology , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Ligands , Nuclear Magnetic Resonance, Biomolecular/methods , Leucine/chemistry , Leucine/metabolism , Leucine/analogs & derivatives , Fluorine/chemistryABSTRACT
The substitution of a single hydrogen atom in a protein by fluorine yields a site-specific probe for sensitive detection by 19F nuclear magnetic resonance (NMR) spectroscopy, where the absence of background signal from the protein facilitates the detection of minor conformational species. We developed genetic encoding systems for the site-selective incorporation of 4-fluorotryptophan, 5-fluorotryptophan, 6-fluorotryptophan, and 7-fluorotryptophan in response to an amber stop codon and used them to investigate conformational heterogeneity in a designed amino acid binding protein and in flaviviral NS2B-NS3 proteases. These proteases have been shown to present variable conformations in X-ray crystal structures, including flips of the indole side chains of tryptophan residues. The 19F NMR spectra of different fluorotryptophan isomers installed at the conserved site of Trp83 indicate that the indole ring flip is common in flaviviral NS2B-NS3 proteases in the apo state and suppressed by an active-site inhibitor.