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Characterization of the DNA Binding Domains of Helix-Turn-Helix Proteins by Affinity Cleaving


Shin, Jumi Anne (1992) Characterization of the DNA Binding Domains of Helix-Turn-Helix Proteins by Affinity Cleaving. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/gmk8-2d12.


Protein recognition of specific DNA binding sites is critical for cellular processes; transcription, replication, and restriction are but a few of the biological activities regulated by DNA binding proteins. Although DNA can exist in any number of sequences and conformations, proteins have the exquisite capability of recognizing specific nucleotide sequences in an entire genome. Chapter One is a review that concentrates on sequence-specific DNA binding proteins primarily capable of regulating gene expression and also able to serve structural and catalytic roles in biological processes. The helix-turn-helix repressor proteins from phages λ and 434 and lac and trp repressors, leucine zipper proteins, zinc-finger proteins, the homeodomain, basic helixloop-helix proteins, arc and mnt repressors, and double helix-turn-helix proteins are discussed.

Affinity cleaving studies on mutants of the DNA binding domain of Hin recombinase, Hin(139-190), are discussed in Chapter Two. Arg140 has been previously found to be extremely important for sequence-specific binding of Hin(139-190) to the hixL operator. Mutants in which Arg140 of Hin(139-190) is replaced with Lys, Ala, β-Ala, Glu, Gly, and GIn were studied; a mutant in which Arg142 is replaced by Lys is also discussed. The binding affinities and sequence specificities of these mutants are very different from that of Hin(139- 190). Also Hin(139-184), in which the six carboxyl terminal residues are removed, was studied by affinity cleaving and shown to have a similar sequence specificity, albeit reduced binding affinity, compared to Hin(139-190). In Chapter Three, a procedure for quantitating thermodynamic parameters using the affinity cleaving technique is discussed. In this procedure, affinity cleaving reactions are run over a wide range of protein concentrations. A binding isotherm is generated and fit, using a least-squares program, and reproducible values for binding constants can be obtained. Binding constants were quantitated for Hin(39-190), two of the mutant proteins, and Hin(139-184). This methodology was developed in order to obtain thermodynamic parameters of complexation between these synthesized proteins and their DNA binding sites. Binding constants were measured only for the hixL IRL and IRR sites for each protein, although in some cases, strong binding was also exhibited at the secondary and even tertiary sites. This method can resolve binding curves for individual sites in a cooperative system. Typical binding constants ranged between 105 M-1 and 107 M-1 at 20mM NaCI for the hixL site.

Affinity cleaving studies were performed on the DNA binding domain of lac repressor in Chapter Four. Based upon sequence homology analysis between the binding domains of lac and cro repressors, the DNA binding interaction between the lac repressor and operator was assigned as a helix-turn-helix motif. NMR studies on the DNA binding domain of lac repressor have shown that the recognition helix of lac binds the major groove of DNA in an orientation opposite that of cro. Affinity cleaving studies on the lac repressor DNA binding domain support the NMR results and establish the orientation of the recognition helix of the lac repressor.

In Chapter Five are discussed affinity cleaving studies on the engrailed homeodomain, for which a high-resolution cocrystal structure was recently published. Until now, affinity cleaving has been performed on proteins for which no high-resolution structural data exist. Because the engrailed homeodomain contains sequence elements very similar to Hin(139-190), it was chosen for affinity cleaving studies in order to compare and to interpret structural data obtained by affinity cleaving with a protein of known structure.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Protein design, DNA, Hin recombinase, engrailed, homeodomain, helix-turn-helix, gene regulation, protein-DNA, protein synthesis
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Dervan, Peter B.
Thesis Committee:
  • Myers, Andrew G. (chair)
  • Dervan, Peter B.
  • Goddard, William A.
  • Rees, Douglas C.
  • Dougherty, Dennis A.
Defense Date:20 December 1991
Non-Caltech Author Email:jumi.shin (AT)
Record Number:CaltechTHESIS:09092011-135058263
Persistent URL:
Shin, Jumi Anne0000-0001-9939-2072
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:6660
Deposited On:09 Sep 2011 22:10
Last Modified:30 Oct 2020 22:06

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