title: Molecular Dissection of the Homotrimeric Sliding Clamp of T4 Phage: Two Domains of a Subunit Display Asymmetric Characteristics.
abstract: Sliding clamp proteins are circular dimers or trimers that encircle DNA and serve as processivity factors during DNA replication. Their presence in all the three domains of life and in bacteriophages clearly indicates their high level of significance. T4 gp45, besides functioning as the DNA polymerase processivity factor, also moonlights as the late promoter transcription determinant. Here we report a detailed biophysical analysis of gp45. The chemical denaturation of gp45 probed by circular dichroism spectroscopy, tryptophan fluorescence anisotropy, and blue-native polyacrylamide gel electrophoresis suggests that the protein follows a three-state denaturation profile and displays an intermediate molten globule-like state. The three-state transition was found to be the result of the sequential unfolding of the two domains, the N-terminal domain (NTD) and the C-terminal domain (CTD), of gp45. The experiments involving Trp fluorescence quenching by acrylamide demonstrate that the CTD undergoes substantial changes in conformation during formation of the intermediate state. Further biophysical dissection of the individual domain reveals contrasting properties of the two domains. The NTD unfolds at low urea concentrations and is also susceptible to protease cleavage, whereas the CTD resists urea-mediated denaturation and is not amenable to protease digestion even at higher urea concentrations. These experiments allow us to conclude that the two domains of gp45 differ in their dynamics. While the CTD shows stability and rigidity, we find that the NTD is unstable and flexible. We believe that the asymmetric characteristics of the two domains and the interface they form hold significance in gp45 structure and function.
authors.affiliation: Microbiology and Molecular Biology Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) , Bhopal 462023, India.
authors.forename: Manika Indrajit
authors.initials: MI
authors.lastname: Singh
authors.affiliation: Microbiology and Molecular Biology Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) , Bhopal 462023, India.
authors.forename: Vikas
authors.initials: V
authors.lastname: Jain
mesh_terms: D017122:Bacteriophage T4 / Q000378:metabolism*; D011489:Protein Denaturation; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry; D012996:Solutions; D015534:Trans-Activators / Q000737:chemistry*; D014764:Viral Proteins / Q000737:chemistry*
chemical_list: D021122:Protein Subunits; D012996:Solutions; D015534:Trans-Activators; D014764:Viral Proteins; C074555:gene 45 protein, Enterobacteria phage T4