Imitation of Escherichia coli aspartate receptor signaling in engineered dimers of the cytoplasmic domain

Science

Volumn 271, Issue 5252, 1996, Pages 1113-1116

  Cochran, Andrea G ^a   Kim, Peter S ^a  

^a MASSACHUSETTS INSTITUTE OF TECHNOLOGY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

AMINO ACID RECEPTOR; ASPARTIC ACID; PROTEIN KINASE;

ARTICLE; CHEMOTAXIS; CONFORMATIONAL TRANSITION; DIMERIZATION; ENZYME ACTIVATION; ESCHERICHIA COLI; LIGAND BINDING; NONHUMAN; PRIORITY JOURNAL; PROTEIN DOMAIN; PROTEIN FOLDING; SIGNAL TRANSDUCTION;

EID: 0029915153     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.271.5252.1113     Document Type: Article

References (67)

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37. DNA encoding the Tar c-fragment (residues 257 to 553) (Fig. 2B) (32, 33) was ligated into the T7 expression vector pAED4 [D. S Doering, thesis, Massachusetts Institute of Technology (1992)] according to standard molecular cloning protocols [J. Sambrook, E F. Fritsch, T. Maniatis, Molecular Cloning. A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, ed 2, 1989)]. The EEEE and QQQQ vanants were obtained by in vitro mutagenesis (Amersham kit RPN 1523) of the c-fragment construct. An expression vector (p4LZ) encoding residues 2 to 33 of the GCN4 dimerization domain has been described [K. J. Lumb, C. M. Carr, P. S. Kim, Biochemistry 33, 7361 (1994)]; the coding sequence was modified here to include two additional amino acids (Lys-Val) after the initiating methionine to yield plasmid pGCN4-pR (Fig. 2C). The encoded GCN4-pR peptide is fully folded as judged by its circular dichroism spectrum. The GCN4-Tar chimeras were obtained by joining the two constructs with short synthetic linkers encoding the sequences shown in Fig 2C. Proteins were expressed in E. coli strain RP3098, from which the che genes have been deleted [R. S. Smith and J S. Parkinson, Proc Natl. Acad. Sci U S A. 77, 5370 (1980)] The GCN4 fusion proteins were purified essentially as described for the c-fragment (34). Concentrations of dialyzed proteins were determined from their absorbance spectra in 6 M guanidine hydrochloride [H. Edelhoch, Biochemistry 6, 1948 (1967)]
38. DNA encoding the Tar c-fragment (residues 257 to 553) (Fig. 2B) (32, 33) was ligated into the T7 expression vector pAED4 [D. S Doering, thesis, Massachusetts Institute of Technology (1992)] according to standard molecular cloning protocols [J. Sambrook, E F. Fritsch, T. Maniatis, Molecular Cloning. A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, ed 2, 1989)]. The EEEE and QQQQ vanants were obtained by in vitro mutagenesis (Amersham kit RPN 1523) of the c-fragment construct. An expression vector (p4LZ) encoding residues 2 to 33 of the GCN4 dimerization domain has been described [K. J. Lumb, C. M. Carr, P. S. Kim, Biochemistry 33, 7361 (1994)]; the coding sequence was modified here to include two additional amino acids (Lys-Val) after the initiating methionine to yield plasmid pGCN4-pR (Fig. 2C). The encoded GCN4-pR peptide is fully folded as judged by its circular dichroism spectrum. The GCN4-Tar chimeras were obtained by joining the two constructs with short synthetic linkers encoding the sequences shown in Fig 2C. Proteins were expressed in E. coli strain RP3098, from which the che genes have been deleted [R. S. Smith and J S. Parkinson, Proc Natl. Acad. Sci U S A. 77, 5370 (1980)] The GCN4 fusion proteins were purified essentially as described for the c-fragment (34). Concentrations of dialyzed proteins were determined from their absorbance spectra in 6 M guanidine hydrochloride [H. Edelhoch, Biochemistry 6, 1948 (1967)]
39. DNA encoding the Tar c-fragment (residues 257 to 553) (Fig. 2B) (32, 33) was ligated into the T7 expression vector pAED4 [D. S Doering, thesis, Massachusetts Institute of Technology (1992)] according to standard molecular cloning protocols [J. Sambrook, E F. Fritsch, T. Maniatis, Molecular Cloning. A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, ed 2, 1989)]. The EEEE and QQQQ vanants were obtained by in vitro mutagenesis (Amersham kit RPN 1523) of the c-fragment construct. An expression vector (p4LZ) encoding residues 2 to 33 of the GCN4 dimerization domain has been described [K. J. Lumb, C. M. Carr, P. S. Kim, Biochemistry 33, 7361 (1994)]; the coding sequence was modified here to include two additional amino acids (Lys-Val) after the initiating methionine to yield plasmid pGCN4-pR (Fig. 2C). The encoded GCN4-pR peptide is fully folded as judged by its circular dichroism spectrum. The GCN4-Tar chimeras were obtained by joining the two constructs with short synthetic linkers encoding the sequences shown in Fig 2C. Proteins were expressed in E. coli strain RP3098, from which the che genes have been deleted [R. S. Smith and J S. Parkinson, Proc Natl. Acad. Sci U S A. 77, 5370 (1980)] The GCN4 fusion proteins were purified essentially as described for the c-fragment (34). Concentrations of dialyzed proteins were determined from their absorbance spectra in 6 M guanidine hydrochloride [H. Edelhoch, Biochemistry 6, 1948 (1967)]
40. DNA encoding the Tar c-fragment (residues 257 to 553) (Fig. 2B) (32, 33) was ligated into the T7 expression vector pAED4 [D. S Doering, thesis, Massachusetts Institute of Technology (1992)] according to standard molecular cloning protocols [J. Sambrook, E F. Fritsch, T. Maniatis, Molecular Cloning. A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, ed 2, 1989)]. The EEEE and QQQQ vanants were obtained by in vitro mutagenesis (Amersham kit RPN 1523) of the c-fragment construct. An expression vector (p4LZ) encoding residues 2 to 33 of the GCN4 dimerization domain has been described [K. J. Lumb, C. M. Carr, P. S. Kim, Biochemistry 33, 7361 (1994)]; the coding sequence was modified here to include two additional amino acids (Lys-Val) after the initiating methionine to yield plasmid pGCN4-pR (Fig. 2C). The encoded GCN4-pR peptide is fully folded as judged by its circular dichroism spectrum. The GCN4-Tar chimeras were obtained by joining the two constructs with short synthetic linkers encoding the sequences shown in Fig 2C. Proteins were expressed in E. coli strain RP3098, from which the che genes have been deleted [R. S. Smith and J S. Parkinson, Proc Natl. Acad. Sci U S A. 77, 5370 (1980)] The GCN4 fusion proteins were purified essentially as described for the c-fragment (34). Concentrations of dialyzed proteins were determined from their absorbance spectra in 6 M guanidine hydrochloride [H. Edelhoch, Biochemistry 6, 1948 (1967)]
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50. -1 at 0°C) } Thermal denaturation studies revealed a distinct transition midpoint for each interdomain linkage (with apparent midpoints of 52°, 57°, and 64°C, for the QEQE variants of 8, 5, and 9, respectively, at 1.3 μM receptor), consistent with the idea that the receptor conformation has been strained by shifting the dimer interface. These midpoints were unaffected by methylation site mutations
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67. We thank F W Dahlquist for providing expression plasmids for CheA, CheW, and CheY, J S. Parkinson for providing E. coli chemotaxis deletion strains; and K. M Shokat and J K. Judice for helpful comments on the manuscript. A G.C. was supported by a National Science Foundation postdoctoral fellowship (CHE-9102242) and a Public Health Service training grant (T32 AI07348-07). This research was supported by the Howard Hughes Medical Institute.