Long-range effects on dynamics in a temperature-sensitive mutant of trp repressor 1 1Edited by P. E. Wright

Jin, Lihua; Fukayama, June Wong; Pelczer, István; Carey, Jannette

doi:10.1006/jmbi.1998.2311

Cited by 13 publications

(55 citation statements)

References 89 publications

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“…The dark-state CD spectrum of LovTAP is close to the residue-weighted average of the dark-state AsLOV2 spectrum and the TrpR spectrum (Fig. 2D) (32,37). The difference of these two spectra indicates that some of the ␣-helix present in AsLOV2 or TrpR has been lost in the fusion and replaced with random coil (Fig.…”

Section: Discussionmentioning

confidence: 55%

“…TrpR, with its L-tryptophan cofactor, binds its operator DNA as a homodimer (30). Mutations throughout the protein, including those in the 21-residue aminoterminal helix, affect cofactor and operator binding, suggesting the presence of many allosterically sensitive sites (31)(32)(33)(34). Isolated TrpR domains occur widely in bacteria but are not known to participate in modular architectures.…”

Section: Discussionmentioning

confidence: 99%

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Light-activated DNA binding in a designed allosteric protein

Strickland

Moffat

Sosnick

2008

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

280

268

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An understanding of how allostery, the conformational coupling of distant functional sites, arises in highly evolvable systems is of considerable interest in areas ranging from cell biology to protein design and signaling networks. We reasoned that the rigidity and defined geometry of an ␣-helical domain linker would make it effective as a conduit for allosteric signals. To test this idea, we rationally designed 12 fusions between the naturally photoactive LOV2 domain from Avena sativa phototropin 1 and the Escherichia coli trp repressor. When illuminated, one of the fusions selectively binds operator DNA and protects it from nuclease digestion. The ready success of our rational design strategy suggests that the helical ''allosteric lever arm'' is a general scheme for coupling the function of two proteins.allostery ͉ LOV domain ͉ protein design ͉ trp repressor ͉ alpha helix

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Section: Discussionmentioning

confidence: 55%

Section: Discussionmentioning

confidence: 99%

Light-activated DNA binding in a designed allosteric protein

Strickland

Moffat

Sosnick

2008

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

280

268

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“…The destabilizing effect of the mutations R29Q and R34Q was surprising because the side chains of both, R29 and R34, are expected to be solvent exposed and not involved in interactions with other amino acids side chains. But for several other nucleic acid binding proteins, such as the Trp repressor, 42 Rop, 43 and the Cro repressor of bacteriophage , 44 it has been (---), and Cop⌬20 (--). Inset: Unfolding curves of wild-type protein (ࡗ), K10Q (OE), S28T (), R29Q (ƒ), R34Q (ᮀ), and Cop⌬20 (⌬).…”

Section: Exchanges Of Residues Predicted To Be Responsible For Sequenmentioning

confidence: 99%

Transcriptional repressor CopR: Structure model-based localization of the deoxyribonucleic acid binding motif

Steinmetzer

Hillisch²,

Behlke

et al. 2000

Proteins

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The plasmid pIP501 encoded transcriptional repressor CopR is one of the two regulators of plasmid copy number. CopR binds as a dimer to a nearly palindromic operator with the consensus sequence 5'-CGTG. Intermediate sequence searches revealed a significant structural relationship between CopR and the bacteriophage P22 c2 and the 434 c1 repressors. In this report we describe the experimental verification of a CopR homology model, which is based on a fairly low-sequence identity of 13.8% to P22 c2 repressor. A model for the complex of CopR with the deoxyribonucleic acid (DNA) target was built on the basis of experimental footprinting data, the above-mentioned CopR homology model, and the crystal structure of the 434 c1 repressor-DNA complex. Site-directed mutagenesis was used to test the function of amino acids involved in sequence and nonsequence-specific DNA recognition and amino acids important for correct protein folding. CD measurements were performed to detect structural changes caused by the mutations. Exchanges of residues responsible for sequence-specific DNA recognition reduced binding to a nonspecific level. Mutations of amino acids involved in nonspecific DNA binding lead to decreased binding affinity while maintaining selectivity. Substitution of amino acids necessary for proper folding caused dramatic structural changes. The experimental data support the model of CopR as a helix-turn-helix protein belonging to the lambda repressor superfamily.

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“…Binding studies have shown that the L75F-TrpR variant has a 10× lower L-Trp binding affinity than WT-TrpR (19). Our data suggest that the greater ps-ns internal motions of helix E and the “stiffening” of helix D of apo-L75F-TrpR contribute to the lower ligand-binding affinity of this ts TrpR variant.…”

Section: Discussionmentioning

confidence: 99%

“…The strains were grown in M9 minimal medium supplemented with [ 15 N]NH 4 Cl (99% 15 N-enriched, CIL, Cambridge, MA) or [ 15 N]NH 4 Cl/[ 13 C 6 ]-D-glucose (99% 13 C-enriched, CIL, Cambridge, MA) as the sole nitrogen and carbon sources, respectively. Protein purification procedures were carried out as described previously (19). The CY15071 cell cultures were supplemented with 4 mmoles/L of unlabeled threonine because this strain of E. coli cannot synthesize threonine de novo (25).…”

Section: Methodsmentioning

confidence: 99%

Internal Dynamics of the Tryptophan Repressor (TrpR) and Two Functionally Distinct TrpR Variants, L75F-TrpR and A77V-TrpR, in Their l-Trp-Bound Forms

2011

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Backbone amide dynamics of the Escherichia coli tryptophan repressor protein (WT-TrpR) and two functionally distinct variants, L75F-TrpR and A77V-TrpR, in their holo-(L-tryptophan (L-Trp) co-repressor-bound) form have been characterized using 15N NMR relaxation. The three proteins possess very similar structures ruling out major conformational differences as the source of their functional differences, and suggest that changes in protein flexibility are at the origin of their distinct functional properties. Comparison of site specific 15N-T1, 15N-T2, 15N-{1H}-nOes, reduced spectral density, and generalized order parameters (S2), indicates that backbone dynamics in the three holo-repressors are overall very similar with a few notable and significant exceptions for backbone atoms residing within the proteins’ DNA-binding domain. We find that flexibility is highly restricted for amides in core α-helices (i.e. helices A, B, C, and F), and a comparable “stiffening” is observed for residues in the DNA recognition helix (helix E) of the helix-D-turn-helix-E (HTH) DNA-binding domain of the three holo-repressors. Unexpectedly, amides located in helix D and in adjacent turn regions remain flexible. These data support the concept that residual flexibility in TrpR is essential for repressor function, DNA-binding, and molecular recognition of target operators. Comparison of the 15N NMR relaxation parameters of the holo-TrpRs with those of the apo-TrpRs indicate that the single point amino acid substitutions, L75F and A77V, perturb the flexibility of backbone amides of TrpR in very different ways, and are most pronounced in the apo-forms of the three repressors. Finally, we present these findings in the context of other DNA-binding proteins and the role of protein flexibility in molecular recognition.

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Long-range effects on dynamics in a temperature-sensitive mutant of trp repressor 1 1Edited by P. E. Wright

Cited by 13 publications

References 89 publications

Light-activated DNA binding in a designed allosteric protein

Light-activated DNA binding in a designed allosteric protein

Transcriptional repressor CopR: Structure model-based localization of the deoxyribonucleic acid binding motif

Internal Dynamics of the Tryptophan Repressor (TrpR) and Two Functionally Distinct TrpR Variants, L75F-TrpR and A77V-TrpR, in Their l-Trp-Bound Forms

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