Biomedical subjects
Publications and source records attributed to P Lu.
A common feature that appears in a number of DNA sites where proteins interact is the sequence GTG/CAC. In the lac operator this sequence leads to a region with a higher imino proton exchange rate well below the optical melting temperature. It is suggested that this reflects a structural feature recognized by proteins that bind specific sites on the DNA molecule.
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We show here the changes in the NMR spectra of the Escherichia coli lac repressor when bound to isolated lac operator DNA. The observations focus on the aromatic residues--four tyrosines and a single histidine--in the amino-terminal DNA binding domain of the lac repressor. There is a good correlation between chemical shift changes seen by 19F NMR when compared with 1 H NMR of otherwise identical repressor--DNA complexes. The results suggest that the tyrosines do not intercalate in the DNA. The NMR spectral changes with similarly sized DNA fragments, not containing the lac operator DNA sequence, are different. Thus, the amino-terminal domain of the lac repressor is independently capable of discriminating between lac operator and nonspecific DNA sequences. There can be two amino-terminal fragments per operator in the specific complex.
The effects of binding inducer and anti-inducer of the Escherichia coli lac operon to the lac repressor were examined by taking advantage of fluorine-19 NMR. The fluorine nucleus was biosynthetically incorporated into the lac repressor with either 5-fluorotryptophan or 3-fluorotyrosine. It is suggested that these small effector molecules influence the operator-binding properties of the tetrameric lac repressor by altering the intersubunit relationships in the protein.
The DNA binding fragment from Escherichia coli lac repressor, the N-terminal 56 amino acid residue "headpiece", has been examined by high-resolution 1H NMR spectroscopy at 360 MHz. The aromatic region has been examined in detail along with the four headpieces of altered repressors that are each missing one of the tyrosines, respectively. The spectra here show more resolved resonances and correct errors in the resonance assignments that have been published by Ribeiro et al. (1981b) Ribeiro, A. A., Wemmer, D., Bray, R. P., Wade-Jardetzky, N. G., & Jardetzky, O. (1981) Biochemistry 20, 818-823]. These corrections allow an interpretation of the spectroscopic observations that is now consistent with the extensive genetic analysis that has been carried out with the lac repressor gene. In addition, nuclear Overhauser enhancement measurements give a guide to the interresidue distances among the aromatic residues in this protein fragment.
A general strategy for the insertion of nuclear spin labels throughout the sequence of a protein is illustrated with the Escherichia coli lac repressor. Examples are shown where the 19F nucleus is incorporated using 3-fluorotyrosine, as well as the selective insertion of additional protons. These selectively inserted nuclei give additional resonances in the respective NMR spectra that can be used to probe the structure and function of the proteins.
By using a systematic genetic approach, the resonances in the 19F NMR spectrum of 3-fluorotyrosine-substituted lac repressor protein have been assigned. The NMR data indicate that each monomer of the repressor consists of two distinct and independent domains. One domain, the NH2-terminal sixth of the primary sequence, which has been shown to be very important for DNA binding, is very mobile. The remaining COOH-terminal sequence is more rigid. Ligands of the repressor, which affect its DNA binding capability, lead to conformational changes in the COOH-terminal domain. The approach to the assignment of spectral features taken here can be extended to other systems.
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By comparing the fluorescence emission properties of the wild type lac repressor with two lac repressors altered at tryptophan 190 and 209, respectively, we show that tryptophan residue 209 has its environment changed, either by its own motion, or that of the surrounding amino acids when an inducer molecule is bound. Substitution of this tryptophan with other amino acids results in lac respressor molecules with reduced affinity for inducer molecules, indicating that the geometry at residue 209 affects the geometry of the inducer-binding site. From the results of potassium iodide quenching of fluorescence from the tryptophans, and from attempts to react the native lac repressor with dimethyl(2-hydroxy-5-nitrobenzyl) sulfonium bromide and 2-hydroxy-5-nitrobenzyl bromide, we propose that tryptophan residue 209 is involved in a conformational change of the protein upon binding of inducer, but does not come in direct contact with inducer.
This paper describes the isolation of 3-fluorotyrosine-substituted lac repressor, and its 19F nuclear magnetic resonance spectrum. From the spectrum, one can conclude that for each of the four identical subunits of the repressor there are four or five surface tyrosines, two buried or internal tyrosines, and one tyrosine with an phenolic group ionized or involved in a hydrogen bond. Conditions are described that can be used for the 3-fluorotyrosine substitution of a variety of Escherichia coli proteins for 19F nuclear magnetic resonance studies.
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