Backbone and side chain dynamics of lac repressor headpiece (1-56) and its complex with DNA.

The dynamics of the backbone and (some of) the side chains of lac headpiece (1-56; lac HP56) have been studied for the free protein and for its complex with lac half-operator DNA by 15N T1 and T1p relaxation measurements combined with [1H-15N] NOE experiments. For the structurally well-defined part of the free lac HP56 (i.e., residues 3-49) a rigid backbone was found for residues in the three alpha-helices and for the turn of the helix-turn-helix motif. The loop between helices II and III of lac headpiece, which was characterized by slight disorder in the structure calculations, shows increased mobility. The detected side chains are very mobile. These data are in full agreement with the rms deviations in the structural data of free lac HP56. When lac HP56 is complexed with DNA, several changes in mobility take place. The most remarkable change was found for the loop region between helices II and III: residue His-29 within this loop interacts with Thy-3 of the operator DNA. As a result this mobile loop adapts itself to the DNA and becomes more rigid. Moreover, most DNA-contacting side chains show a significant decrease in flexibility, although these side chains do not become as rigid as the backbone. These results suggest that the mobility of the regions within lac HP56 important for complexation, i.e., the loop and the DNA-contacting side chains, is essential for a good fit onto the counterparts of the target DNA.

Refined structure of lac repressor headpiece (1-56) determined by relaxation matrix calculations from 2D and 3D NOE data: change of tertiary structure upon binding to the lac operator.

The solution structure of the DNA binding domain of lac repressor (headpiece 1-56; HP56) has been refined using data from 2D and 3D NMR spectroscopy. The structure was derived from 1546 restraints (giving an average of 27.6 per residue), comprising 389 intraresidual, 402 sequential, 385 medium range and 325 long range distance restraints and also 30 phi and 15 chi 1 dihedral angle restraints. The structures were determined by the method of direct refinement against nuclear Overhauser enhancement peak volumes with the program DINOSAUR. The final set of 32 selected structures displayed an r.m.s. deviation from the average of 0.43(+/-0.08) A angstroms (backbone) and 0.95(+/-0.08) angstroms (all heavy atoms) for the best defined region of the protein (residues 3 to 49). The ensemble R-factor was 0.35, which indicates close correspondence with the experimental data. The structures revealed good stereochemical qualities. The conformations of the NMR structures of free and DNA complexed lac repressor headpiece were compared. The regions comprising the secondary structure elements show close correspondence for both conformations. However, the conformation of the loop between helix II and III changes considerably upon complexation of the headpiece. This change in the conformation of the loop in lac HP56 is essential for binding of the side-chains of residues Asn25 and His29 to the lac operator DNA. Finally, the lac headpiece residues that are intolerant to mutations were analysed. Most of these mutation-sensitive residues are important for a correct folding of the headpiece region, and a number of these residues are also involved in contacting the operator DNA.

Improved HSQC experiments for the observation of exchange broadened signals.

We present here HSQC experiments with improved sensitivity for signals in the presence of exchange broadening. During periods of coherence transfer through scalar coupling the experiments employ CPMG-derived pulse trains to reduce loss of dephasing of spin coherence due to chemical exchange. (15)N-(1)H gradient CPMG-HSQC and SE-CPMG-HSQC experiments have been developed and applied to complexes of lac repressor headpiece with operator DNA. Improved sensitivity is demonstrated for many protein backbone and side-chain resonances in the complex, markedly for signals of protons located at the protein-DNA interface. In addition, a significant increase in intensity is observed for arginine guanidino groups undergoing conformational exchange.

Structure and dynamics of the lac repressor-operator complex as determined by NMR.

The structures of the lac repressor headpiece and of its complex with an 11 base-pair lac half-operator have been determined by NMR spectroscopy. By 15N relaxation studies the dynamic behavior of the free protein and of the protein in the complex could be established. In the three-helical lac headpiece local backbone mobility was detected in the N-terminal and C-terminal peptide regions and in the loop between helices II and III. Upon DNA binding this loop becomes more rigid and it changes its conformation considerably. The specificity of the protein-DNA interaction follows from a large number of hydrogen-bond and hydrophobic interactions between amino acid side chains and DNA backbone and bases. Restrained molecular dynamics calculations suggest that some of these interactions are dynamic in nature.

Graph-theoretical assignment of secondary structure in multidimensional protein NMR spectra: application to the lac repressor headpiece.

A novel procedure is presented for the automatic identification of secondary structures in proteins from their corresponding NOE data. The method uses a branch of mathematics known as graph theory to identify prescribed NOE connectivity patterns characteristic of the regular secondary structures. Resonance assignment is achieved by connecting these patterns of secondary structure together, thereby matching the connected spin systems to specific segments of the protein sequence. The method known as SERENDIPITY refers to a set of routines developed in a modular fashion, where each program has one or several well-defined tasks. NOE templates for several secondary structure motifs have been developed and the method has been successfully applied to data obtained from NOESY-type spectra. The present report describes the application of the SERENDIPITY protocol to a 3D NOESY-HMQC spectrum of the 15N-labelled lac repressor headpiece protein. The application demonstrates that, under favourable conditions, fully automated identification of secondary structures and semi-automated assignment are feasible.