Kinetics and thermodynamics of the unfolding and refolding of the three-stranded alpha-helical coiled coil, Lpp-56.
Temperature-induced reversible unfolding and refolding of the three-stranded alpha-helical coiled coil, Lpp-56, were studied by kinetic and thermodynamic methods, using CD spectroscopy, dynamic light scattering, and scanning calorimetry. It was found that both unfolding and refolding reactions of this protein in neutral solution in the presence of 100 mM NaCl are characterized by unusually slow kinetics, which permits detailed investigation of the mechanism of these reactions. Kinetic analyses show that the unfolding of this coiled coil represents a single-stage first-order reaction, while the refolding represents a single-stage third-order reaction. The activation enthalpy and entropy for unfolding do not depend noticeably on temperature and are both significantly greater than those for the folding reaction, which show a significant dependence on temperature. The activation heat capacity change for the unfolding reaction is close to zero, while it is quite significant for the folding reaction. The correlation between the activation and structural parameters obtained for the Lpp-56 coiled coil suggests that interhelical van der Waals interactions are disrupted in the transition state, which is nevertheless still compact, and water has not yet penetrated into the interface; the transition from the transient state to the unfolded state results in hydration of exposed apolar groups of the interface and the disruption of helices. The low propensity for the Lpp-56 strands to fold and associate is caused by the high number of charged groups at neutral pH. On one hand, these charges give rise to considerable repulsive forces destabilizing the helical conformation of the strands. On the other hand, they align the folded helices in parallel and in register so that the apolar sides face each other, and the oppositely charged groups may form salt links, which are important for the formation of the trimeric coiled coil. A decrease in pH, which eliminates the salt links, dramatically decreases the stability of Lpp-56; its structure becomes less rigid and unfolds much faster.
Dragan
Anatoly I
AI
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218, USA.
Potekhin
Sergey A
SA
Sivolob
Andrei
A
Lu
Min
M
Privalov
Peter L
PL
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification; D002021:Buffers; D002152:Calorimetry, Differential Scanning; D003198:Computer Simulation; D019277:Entropy; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D008074:Lipoproteins / Q000737:chemistry* / Q000302:isolation & purification; D013058:Mass Spectrometry; D008956:Models, Chemical; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012542:Scattering, Radiation; D012996:Solutions / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013053:Spectrophotometry; D013696:Temperature; D013816:Thermodynamics
D001425:Bacterial Outer Membrane Proteins; D002021:Buffers; D029968:Escherichia coli Proteins; D008074:Lipoproteins; C099932:Lpp protein, E coli; D012996:Solutions