Determination of protein structures from nuclear magnetic resonance data using a restrained molecular dynamics approach: The lac repressor DNA binding domain

Zuiderweg, E.R.P.; Scheek, Ruud M.; Boelens, Rolf; Vangunsteren, Wf; Kaptein, Robert

doi:10.1016/s0300-9084(85)80158-9

Cited by 49 publications

(23 citation statements)

References 19 publications

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“…Consequently the more precise structural characterisation of the repressor and of the repressor-operator complex relies on NMR experiments . From these studies it has been shown that the headpiece contains three a-helices: residues 6 to 13, 17 to 25,34 to 45 (17)(18)(19)(20)(21)(22)(23). The first two helices constitute a helix-tum-helix motif which has been previously predicted on the basis of sequence homology with other prokaryotic repressors (24,25).…”

Section: Lancelot Eta/mentioning

confidence: 92%

Two-Dimensional¹H,¹⁵N NMR investigation of uniformly¹⁵N-labeledLacRepressor Headpiece

Lancelot

Gervais

Maurizot

1992

Journal of Biomolecular Structure and Dynamics

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15N uniformly labeled lac repressor and lac repressor headpiece were prepared. 15N NMR spectra of lac repressor were shown resolution inadequate for detailed study while the data showed that the 15N labeled N-terminal part of the protein is quite suitable for this type of study allowing future investigation of the specific interaction of the lac repressor headpiece with the lac operator. We report here the total assignment of proton 1H and nitrogen 15NH backbone resonances of this headpiece in the free state. Assignments of the 15N resonances of the protein were obtained in a sequential manner using heteronuclear multiple quantum coherence (HMQC), relayed HMQC nuclear Overhauser and relayed HMQC-HOHAHA spectroscopy. More than 80 per cent of residues were assigned by their 15NH(i)-N1H(i + 1) and 15NH(i)-N1H(i - 1) connectivities. Values of the 3JNH alpha splitting for 39 of the 51 residues of the headpiece were extracted from HMQC and HMQC-J. The observed 15NH(i)-C beta H cross peaks and the 3JNH alpha coupling constants values are in agreement with the three alpha-helices previously described [Zuiderweg, E.R.P., Scheek, R.M., Boelens, R., van Gunsteren, W.F. and Kaptein, R., Biochimie 67, 707 (1985)]. The 3JNH alpha coupling constants can be now used for a more confident determination of the lac repressor headpiece. From these values it is shown that the geometry of the ends of the second and third alpha-helices exhibit deviation from the canonical alpha-helix structure. On the basis of NOEs and 3JNH alpha values, the geometry of the turn of the helix-turn-helix motif is discussed.

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Section: Lancelot Eta/mentioning

confidence: 92%

Two-Dimensional¹H,¹⁵N NMR investigation of uniformly¹⁵N-labeledLacRepressor Headpiece

Lancelot

Gervais

Maurizot

1992

Journal of Biomolecular Structure and Dynamics

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“…1) (7)(8)(9)(10). The N-terminal 60 amino acids in each monomer form a helix-turn-helix motif that directly contacts the operator DNA (7,(11)(12)(13)(14)(15)(16)(17). When isolated, this region exhibits site-specific DNA binding, albeit with significantly lower affinity than the intact protein (11,18,19).…”

mentioning

confidence: 99%

Glycine Insertion in the Hinge Region of Lactose Repressor Protein Alters DNA Binding

Falcon

Matthews

1999

Journal of Biological Chemistry

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Amino acid alterations were designed at the C terminus of the hinge segment (amino acids ϳ51-59 The lac repressor (LacI) negatively regulates lac mRNA synthesis by binding with high affinity to its target operator site and thereby precluding transcription by RNA polymerase (1-6). In response to the presence of inducer sugars, LacI undergoes a conformational change that results in diminished affinity for operator sequences without effect on nonspecific DNA binding (1, 2, 7). LacI is a protein of 150,000 Da composed of four identical subunits assembled as a dimer of dimers (see Fig.

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“…Various protocols are used for structure determination, but the initial step often utilizes distance geometry (DG) to generate a family of structures consistent with NOE distance constraints (5, 6). Some methods entail theoretical energy calculations, energy minimization, or restrained molecular dynamics, with pseudoenergy terms maintaining NOE-derived distances (7,8). Families of structures for several proteins have been produced that exhibit little internal variance and few distance violations.…”

mentioning

confidence: 99%

Protein solution structure determination using distances from two-dimensional nuclear Overhauser effect experiments: effect of approximations on the accuracy of derived structures.

Thomas

Basus

James

1991

Proc. Natl. Acad. Sci. U.S.A.

154

107

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Solution structures for many proteins have been determined to date utilizing interproton distance constraints estimated from two-dimensional nuclear Overhauser effect (2D NOE) spectra. Although the simple isolated spin pair approximation (ISPA) generally used can result in systematic errors in distances, the large number of constraints enables protein structure to be defmed with reasonably high resolution. Effects of these systematic errors on the resulting protein structure are examined. Iterative relaxation matrix calculations, which account for dipolar interactions between all protons in a molecule, can accurately determine internuclear distances with little or no a priori knowledge of the molecular structure. The value of this additional complexity is also addressed. To assess these distance determination methods, hypothetical "experimental" data, including random noise and peak overlap, are calculated for an arbitrary "true" protein structure. Three methods of obtaining distance constraints from 2D NOE peak intensities are examined: one entails a conservative use of ISPA, one assumes the ISPA to be fairly accurate, and one utilizes an iterative relaxation matrix method called MARDIGRAS (matrix analysis of relaxation for discerning the geometry of an aqueous structure), developed in this laboratory. A distance geometry algorithm was used to generate a family of structures for each distance set. The quality of the average structure from each family was good. The rootmean-square deviation of that average structure from the true structure was improved about 2-5% using the more restrictive rather than the more conservative ISPA approach. Use of MARDIGRAS in a conservative fashion-i.e., with a poor initial model-resulted in improvement in the root-mean-square deviation by 8-15%. With a better initial model, MARDIGRAS obtained even more accurate distances. MARDIGRAS also permits analysis of 2D NOE data at longer mixing times, yielding additional distances. Use of more restrictive ISPA distances did, however, result in a few systematically incorrect structural features in local regions of the protein, producing distortions of 2-3 A. Comparison between experimental data and spectra calculated for the structures correlates with root-mean-square deviation, offering a method of structure evaluation. An R factor for evaluating fit between experimental and calculated 2D NOE intensities is proposed.Interproton distances obtained from homonuclear proton two-dimensional nuclear Overhauser effect (2D NOE) experiments are used to determine three-dimensional protein structure in solution (1-4). Various protocols are used for structure determination, but the initial step often utilizes distance geometry (DG) to generate a family of structures consistent with NOE distance constraints (5, 6). Some methods entail theoretical energy calculations, energy minimization, or restrained molecular dynamics, with pseudoenergy terms maintaining NOE-derived distances (7,8). Families of structures for several proteins have been produc...

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Determination of protein structures from nuclear magnetic resonance data using a restrained molecular dynamics approach: The lac repressor DNA binding domain

Cited by 49 publications

References 19 publications

Two-Dimensional¹H,¹⁵N NMR investigation of uniformly¹⁵N-labeledLacRepressor Headpiece

Two-Dimensional¹H,¹⁵N NMR investigation of uniformly¹⁵N-labeledLacRepressor Headpiece

Glycine Insertion in the Hinge Region of Lactose Repressor Protein Alters DNA Binding

Protein solution structure determination using distances from two-dimensional nuclear Overhauser effect experiments: effect of approximations on the accuracy of derived structures.

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Determination of protein structures from nuclear magnetic resonance data using a restrained molecular dynamics approach: The lac repressor DNA binding domain

Cited by 49 publications

References 19 publications

Two-Dimensional1H,15N NMR investigation of uniformly15N-labeledLacRepressor Headpiece

Two-Dimensional1H,15N NMR investigation of uniformly15N-labeledLacRepressor Headpiece

Glycine Insertion in the Hinge Region of Lactose Repressor Protein Alters DNA Binding

Protein solution structure determination using distances from two-dimensional nuclear Overhauser effect experiments: effect of approximations on the accuracy of derived structures.

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Two-Dimensional¹H,¹⁵N NMR investigation of uniformly¹⁵N-labeledLacRepressor Headpiece

Two-Dimensional¹H,¹⁵N NMR investigation of uniformly¹⁵N-labeledLacRepressor Headpiece