title: Stabilization of native protein fold by intein-mediated covalent cyclization.
abstract: A mutant version of the N-terminal domain of Escherichia coli DnaB helicase was used as a model system to assess the stabilization against unfolding gained by covalent cyclization. Cyclization was achieved in vivo by formation of an amide bond between the N and C termini with the help of a split mini-intein. Linear and circular proteins were constructed to be identical in amino acid sequence. Mutagenesis of Phe102 to Glu rendered the protein monomeric even at high concentration. A difference in free energy of unfolding, DeltaDeltaG, between circular and linear protein of 2.3(+/-0.5) kcal mol(-1) was measured at 10 degrees C by circular dichroism. A theoretical estimate of the difference in conformational entropy of linear and circular random chains in a three-dimensional cubic lattice model predicted DeltaDeltaG=2.3 kcal mol(-1), suggesting that stabilization by protein cyclization is driven by the reduced conformational entropy of the unfolded state. Amide-proton exchange rates measured by NMR spectroscopy and mass spectrometry showed a uniform, approximately tenfold decrease of the exchange rates of the most slowly exchanging amide protons, demonstrating that cyclization globally decreases the unfolding rate of the protein. The amide proton exchange was found to follow EX1 kinetics at near-neutral pH, in agreement with an unusually slow refolding rate of less than 4 min(-1) measured by stopped-flow circular dichroism. The linear and circular proteins differed more in their unfolding than in their folding rates. Global unfolding of the N-terminal domain of E.coli DnaB is thus promoted strongly by spatial separation of the N and C termini, whereas their proximity is much less important for folding.
authors.affiliation: Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia.
authors.forename: Neal K
authors.initials: NK
authors.lastname: Williams
authors.forename: Edvards
authors.initials: E
authors.lastname: Liepinsh
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Watt
authors.forename: Pavel
authors.initials: P
authors.lastname: Prosselkov
authors.forename: Jacqueline M
authors.initials: JM
authors.lastname: Matthews
authors.forename: Phil
authors.initials: P
authors.lastname: Attard
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Beck
authors.forename: Nicholas E
authors.initials: NE
authors.lastname: Dixon
authors.forename: Gottfried
authors.initials: G
authors.lastname: Otting
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000577:Amides / Q000737:chemistry; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003500:Cyclization; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D053488:DnaB Helicases; D019277:Entropy; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D047668:Inteins / Q000235:genetics / Q000502:physiology*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011522:Protons; D021241:Spectrometry, Mass, Electrospray Ionization; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology
chemical_list: D000577:Amides; D011522:Protons; D014508:Urea; D000251:Adenosine Triphosphatases; C506730:dnaB protein, E coli; D004265:DNA Helicases; D053488:DnaB Helicases