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L Swint-Kruse

Publications and source records attributed to L Swint-Kruse.

6 recordsLinked to original sources
PubMed2001

Plasticity of quaternary structure: twenty-two ways to form a LacI dimer.

The repressor proteins of the LacI/GalR family exhibit significant similarity in their secondary and tertiary structures despite less than 35% identity in their primary sequences. Furthermore, the core domains of these oligomeric repressors, which mediate dimerization, are homologous with the monomeric periplasmic binding proteins, extending the issue of plasticity to quaternary structure. To elucidate the determinants of assembly, a structure-based alignment has been created for three repressors and four periplasmic binding proteins. Contact maps have also been constructed for the three repressor interfaces to distinguish any conserved interactions. These analyses show few strict requirements for assembly of the core N-subdomain interface. The interfaces of repressor core C-subdomains are well conserved at the structural level, and their primary sequences differ significantly from the monomeric periplasmic binding proteins at positions equivalent to LacI 281 and 282. However, previous biochemical and phenotypic analyses indicate that LacI tolerates many mutations at 281. Mutations at LacI 282 were shown to abrogate assembly, but for Y282D this could be compensated by a second-site mutation in the core N-subdomain at K84 to L or A. Using the link between LacI assembly and function, we have further identified 22 second-site mutations that compensate the Y282D dimerization defect in vivo. The sites of these mutations fall into several structural regions, each of which may influence assembly by a different mechanism. Thus, the 360-amino acid scaffold of LacI allows plasticity of its quaternary structure. The periplasmic binding proteins may require only minimal changes to facilitate oligomerization similar to the repressor proteins.

Allosteric Site↗
PubMed1998

Comparison of simulated and experimentally determined dynamics for a variant of the Lacl DNA-binding domain, Nlac-P.

Recent advances in the experimentally determined structures and dynamics of the domains within LacI provide a rare context for evaluating dynamics calculations. A 1500-ps trajectory was simulated for a variant of the LacI DNA-binding domain, which consists of the first three helices in LacI and the hinge helix of the homologous PurR. Order parameters derived from dynamics simulations are compared to those obtained for the LacI DNA-binding domain with 15N relaxation NMR spectroscopy (Slijper et al., 1997. Biochemistry. 36:249-254). The MD simulations suggest that the unstructured loop between helices II and III does not exist in a discrete state under the conditions of no salt and neutral pH, but occupies a continuum of states between the DNA-bound and free structures. Simulations also indicate that the unstructured region between helix III and the hinge helix is very mobile, rendering motions of the hinge helix essentially independent of the rest of the protein. Finally, the alpha-helical hydrogen bonds in the hinge helix are broken after 1250 ps, perhaps as a prelude to helix unfolding.

Amino Acid Sequence↗
PubMed1997

Designed disulfide between N-terminal domains of lactose repressor disrupts allosteric linkage.

Substitution of Cys for Val at position 52 of the lac repressor was designed to permit disulfide bond formation between the two N-terminal DNA binding domains that comprise an operator DNA binding site. This position marks the closest approach of these domains based on the x-ray crystallographic structures of the homologous purine holorepressor-operator complex and lac repressor-operator complex (Schumacher, M. A., Choi, K. Y., Zalkin, H., and Brennan, R. G. (1994) Science 266, 763-770; Lewis, M., Chang, G., Horton, N.C., Kercher, M. A., Pace, H. C., Schumacher, M. A., Brennan, R. G., and Lu, P. (1996) Science 271, 1247-1254). The V52C mutation was generated by site-specific methods, and the mutant protein was purified and characterized. In the reduced form, V52C bound operator DNA with slightly increased affinity. Exposure to oxidizing conditions resulted in disulfide bond formation, and the oxidized protein bound operator DNA with approximately 6-fold higher affinity than wild-type protein. Inducer binding for both oxidized and reduced forms of V52C was comparable to wild-type lac repressor. In the presence of inducer, the reduced protein exhibited wild-type, diminished DNA binding. In contrast, DNA binding for the oxidized form was unaffected by inducer, even at 1 mM. Thus, the formation of the designed disulfide between Cys52 side chains within each dimer renders the protein-operator complex unresponsive to sugar binding, presumably by disrupting the allosteric linkage between operator and inducer binding.

Allosteric Regulation↗
PubMed1996

Temperature and pH dependences of hydrogen exchange and global stability for ovomucoid third domain.

Two-dimensional nuclear magnetic resonance spectroscopy has been used to monitor proton-deuterium exchange rates (kobs) for more than 30 residues in turkey ovomucoid third domain. To test whether exchange is governed by global unfolding, rates were measured over a wide range of pH and temperatures where the change in the free energy of unfolding (delta Gzerou) is known [Swint, L., & Robertson, A. D. (1993) Protein Sci. 2, 2037-2049; Swint-Kruse, L., & Robertson, A. D. (1995) Biochemistry 34, 4724-4732]. Under conditions where EX2 kinetics are observed, a subset of 6-11 residues exhibits a one-to-one correlation with global stability. These residues are all located in central regions of secondary structures. Many other sites show varied degrees of correlation with delta Gzerou, while some are slower than expected on the basis of delta Gzerou alone. Preliminary evidence suggests that the latter is due to deviation from EX2 kinetics, even though experimental conditions are relatively mild (pH* 3 and 40 degrees C) compared to those in which deviations were observed for bovine pancreatic trypsin inhibitor. These results, together with similar observations for hen egg white lysozyme and barnase, suggest that EX2 kinetics should not be assumed when interpreting exchange studies.

Amino Acid Sequence↗
PubMed1995

Hydrogen bonds and the pH dependence of ovomucoid third domain stability.

Thermal denaturation of turkey ovomucoid third domain (OMTKY3) has been monitored with differential scanning calorimetry (DSC) and circular dichroism in H2O and D2O, pH 1.5 to 5 and ionic strength 0.01 to 0.71. Results from DSC experiments are in good agreement with spectroscopic studies [Swint, L., & Robertson, A.D. (1993) Protein Sci. 2, 2037-2049] and fit well to the two-state unfolding model. The average ratio of calorimetric to van't Hoff enthalpies is 0.99 +/- 0.03 (n = 16), and the average value of delta Cp is 620 +/- 20 cal/(mol K) (n = 7). The free energy of unfolding (delta G(u)o) increases in the presence of salt at both pH 1.5 and 4.5. This stabilization is not due to ion binding and probably results from screened repulsive interactions between the cationic groups of OMTKY3. At very low ionic strength, the change in delta G(u)o from pH 1.5 to 4.5, delta delta GpHo, is 3.5 +/- 0.2 kcal/mol. Few interactions between ionizing groups are affected by the addition of 200 mM KCl; delta delta GpHo decreases by only 0.4 +/- 0.3 kcal/mol. Comparison of delta delta GpHo with values calculated from the pKas of all six carboxyl groups in OMTKY3 [Schaller, W.S., & Robertson, A.D. (1995) Biochemistry 34, 4714-4723] suggests that some pKas in the denatured state may be lower than those of model compounds. Moreover, calculated values of delta delta GpHo are very sensitive to modest changes in the cooperativity of proton binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗