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. 2012;7(4):e35984.
doi: 10.1371/journal.pone.0035984. Epub 2012 Apr 23.

The proline rich homeodomain protein PRH/Hhex forms stable oligomers that are highly resistant to denaturation

Anshuman Shukla  1 , Nicholas M Burton, Padma-Sheela Jayaraman, Kevin Gaston
Affiliations

The proline rich homeodomain protein PRH/Hhex forms stable oligomers that are highly resistant to denaturation

Anshuman Shukla et al. PLoS One. 2012.

Abstract

Background: Many transcription factors control gene expression by binding to specific DNA sequences at or near the genes that they regulate. However, some transcription factors play more global roles in the control of gene expression by altering the architecture of sections of chromatin or even the whole genome. The ability to form oligomeric protein assemblies allows many of these proteins to manipulate extensive segments of DNA or chromatin via the formation of structures such as DNA loops or protein-DNA fibres.

Principal findings: Here we show that the proline rich homeodomain protein PRH/Hhex forms predominantly octameric and/or hexadecameric species in solution as well as larger assemblies. We show that these assemblies are highly stable resisting denaturation by temperature and chemical denaturants.

Conclusion: These data indicate that PRH is functionally and structurally related to the Lrp/AsnC family of proteins, a group of proteins that are known to act globally to control gene expression in bacteria and archaea.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. PRH oligomers predominate in solution.
(A) A schematic representation of the PRH protein and its functional domains. Proline Rich indicates the transcriptional repression domain, HD indicates the homeodomain and C indicates the C-terminal domain. The PRH HDC protein lacks the N-terminal domain. The intensity size distribution of PRH particles (B) and PRH HDC particles (C) in solution determined using DLS.
Figure 2
Figure 2. PRH oligomers are thermostable.
(A) Circular dichroism was used to examine the secondary structure of the full length PRH protein at the temperatures shown. The insert shows the near-UV circular dichroism spectra at the temperatures shown. (B) The far-UV circular dichroism signal at 200 nm measured while increasing (black line) and then decreasing (grey line) the temperature. (C) The mean diameter of PRH particles at increasing temperature determined using DLS.
Figure 3
Figure 3. Changes in PRH structure at high temperature.
(A) PRH was incubated with ANS before emission fluorescence spectra were collected between 20°C and 75°C. (B) PRH was incubated with ANS and emission fluorescence spectra were collected between 400 nm and 600 nm at 20°C and 75°C and after heating to 75°C and then cooling to 20°C (HC). (C) Fluorescence emission spectroscopy was performed to monitor intrinsic tryptophan fluorescence between 20°C and 75°C.
Figure 4
Figure 4. Changes in PRH structure in the presence of chemical denaturant.
(A) Fluorescence emission spectroscopy was performed in the presence of increasing concentrations of GITC. (B) The intensity size distribution of PRH particles in the presence of 4 M GITC determined using DLS. (C) The ratio of the maximum intensities of the major PRH peaks obtained using DLS in the presence of increasing concentrations of GITC.
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