Conformational dynamics of DnaB helicase upon DNA and nucleotide binding: analysis by intrinsic tryptophan fluorescence quenching.
DnaB helicase of E. coli unwinds duplex DNA in the replication fork using the energy of ATP hydrolysis. We have analyzed structural and conformational changes in the DnaB protein in various nucleotides and DNA bound intermediate states by fluorescence quenching analysis of intrinsic fluorescence of native tryptophan (Trp) residues in DnaB. Fluorescence quenching analysis indicated that Trp48 in domain alpha is in a hydrophobic environment and resistant to fluorescence quenchers such as potassium iodide (KI). In domain beta, Trp294 was found to be in a partially hydrophobic environment, whereas Trp456 in domain gamma appeared to be in the least hydrophobic environment. Binding of oligonucleotides to DnaB helicase resulted in a significant attenuation of the fluorescence quenching profile, indicating a change in conformation. ATPgammaS or ATP binding appeared to lead to a conformation in which Trp residues had a higher degree of solvent exposure and fluorescence quenching. However, the most dramatic increase of Trp fluorescence quenching was observed with ADP binding with a possible conformational relaxation. Site-specific Trp --> Cys mutants of DnaB helicase demonstrated that conformational change upon ADP binding could be attributed exclusively to a conformational transition in the alpha domain leading to an increase in the solvent exposure of Trp48. However, formation of DnaB.ATPgammaS.DNA ternary complex led to a conformation with a fluorescence quenching profile similar to that observed with DnaB alone. The DnaB.ADP.DNA ternary complex produced a quenching curve similar to that of DnaB.ADP complex pointing to a change in conformation due to ATP hydrolysis. There are at least four identifiable structural/conformational states of DnaB helicase that are likely important in the helicase activity. The noncatalytic alpha domain in the N-terminus appeared to undergo the most significant conformational changes during nucleotide binding and hydrolysis. This is the first reported elucidation of the putative role of domain alpha, which is essential for DNA helicase action. We have correlated these results with partial structural models of alpha, beta, and gamma domains
Flowers
Stephen
S
Department of Molecular Biology, School of Osteopathic Medicine & Graduate School of Biomedical Sciences, University of Medicine & Dentistry of New Jersey, Stratford, New Jersey 08084, USA.
Biswas
Esther E
EE
Biswas
Subhasis B
SB
D000244:Adenosine Diphosphate / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001665:Binding Sites / Q000235:genetics; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D053488:DnaB Helicases; D004789:Enzyme Activation / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000737:chemistry; D011193:Potassium Iodide / Q000737:chemistry; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence / Q000379:methods; D017186:Titrimetry; D014364:Tryptophan / Q000235:genetics
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D009841:Oligonucleotides; D011994:Recombinant Proteins; D011193:Potassium Iodide; C022571:adenosine 5'-O-(3-thiotriphosphate); D000244:Adenosine Diphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D004265:DNA Helicases; D053488:DnaB Helicases