The stability and specificity of GCN4 leucine zipper mutants /
The leucine zipper is a mimetic coiled-coil structural motif. It consists of 4 to 6 heptad repeats, designated (abcdefg)[]. The dimer interface tends to involve buried hydrophobic interactions in the a and d positions and electrostatic interactions between e and g positions. In the GCN4 leucine zipp...
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
2000.
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| Subjects: | |
| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=731990521&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | The leucine zipper is a mimetic coiled-coil structural motif. It consists of 4 to 6 heptad repeats, designated (abcdefg)[]. The dimer interface tends to involve buried hydrophobic interactions in the a and d positions and electrostatic interactions between e and g positions. In the GCN4 leucine zipper, a position 16 is occupied by an Asn residue. An asymmetrical hydrogen bond was observed between the two Asn residues from different monomers in the crystal structure of the GCN4 leucine zippier dimer. To characterize the contributions of the buried Asn pairs to the stability of the GCN4 leucine zipper, a series of GCN4 leucine zipper variants was constructed and purified. These leucine zipper mutant proteins have lie in the a positions with buried Asn pairs replacing one or more of the a positions. The thermodynamic stabilities of the variants were measured using thermal denaturations monitored by circular dichroism. As expected, Ile is favored over Asn at these buried positions, but not as much as predicted by considering only the hydrophobic effect. It appears that interstrand hydrogen bonds form between the side chains of the buried Asn residues and contribute to the conformational stability of the coiled-coil reptiles. However, these contributions are highly dependent on the locations of the Asn pairs. An a position mutant containing a single Asn residue at position 9 has a three-state unfolding. The unfolding pathway of this mutant was determined by thermal denaturation, limited proteinase K digestion and sedimentation equilibrium analysis. The mutant first unfolds from its N-terminus and changes the oligomerization specificity of the mutant from a native dimer to a partially unfolded intermediate containing a mixture of dimers and primers. A pool of e and g mutants was constructed in our lab by random mutagenesis to evaluate the importance of the intersubunit charge-charge interactions to the specificity of the GCN4 leucine zipper. Two mutants were purified as lambda cI-fusion proteins. Their oligomerization states were determined by chemical cross-linking, sedimentation equilibrium and velocity, and gel filtration. Mutant s09 (e/g TEKK/AEKA), forms a betrayer. On the other hand, mutant n01 (e/g AAAT/AAAT) forms a complicated mixture of higher order oligomers. |
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| Item Description: | Vita. "Major Subject: Biochemistry". |
| Physical Description: | xii, 163 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilm Inc. |
| Bibliography: | Includes bibliographical references (leaves 121-137). |