Very Rapid, Ionic Strength-Dependent Association and Folding of a Heterodimeric Leucine Zipper

Wendt, H.; Leder, Lukas; Härmä, Harri; Jelesarov, Ilian; Baici, Antonio; Bosshard, Hans Rudolf

doi:10.1021/bi961672y

Cited by 93 publications

(99 citation statements)

References 59 publications

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“…Screening the charges on Pep-1F and S-protein reduces k on by a factor of 26 ( 4 but has little effect on k off ( Figure 5, Table 2), indicating that the separation of charges at the transition barrier is nativelike. A similar behavior has been observed for the folding of a leucine zipper (23) and for the binding of barstar to barnase (24). The formation of RNaseS* may be either encounter-limited or folding-limited ( Figure 7).…”

Section: Formation Of Rnases* Is Described By the Two-statesupporting

confidence: 67%

“…p, N, and pN are defined in eq 1, and I r is an intermediate. The parameters obtained from fitting the entire range of data in Figure 3 to this model are K M ) 18 ( 1 µM and k 23 ) 390 ( 80 s -1 . The value of k 23 is within the range that may be reliably measured by stopped-flow spectrophotometry given the instrumental dead time of 2 ms.…”

Section: Helicity Of Pep-1fmentioning

confidence: 99%

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Kinetic Mechanism of a Partial Folding Reaction. 1. Properties of the Reaction and Effects of Denaturants

Goldberg

Baldwin

1998

Biochemistry

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The bimolecular association rate constant (k on ) and dissociation rate constant (k off ) of the complex between fluorescein-labeled S-peptide analogues and folded S-protein are reported. This is the first kinetic study of a protein folding reaction in which most of the starting material is already folded and only a small part (one additional helix) becomes ordered; it provides a folding landscape with a small conformational entropy barrier, and one in which kinetic traps are unlikely. Refolding and unfolding are measured under identical strongly native conditions, and the reaction is found to be two-state at low reactant concentrations. The dissociation constant (K d ) of the complex and the properties of the transition state may be calculated from the rate constants without extrapolation. The folded complex is formed fast (k on ) 1.8 × 10 7 M -1 s -1 ) and is very stable (K d ) 6 pM) at 10°C, 10 mM MOPS, pH 6.7. Charge interactions stabilize the complex by 1.4 kcal mol -1 . The charge effect enters in the refolding reaction: increasing the salt concentration reduces k on dramatically and has little effect on k off . Urea and GdmCl destabilize the complex by decreasing k on and increasing k off . The slopes (m-values) of plots of ln K d vs [cosolvent] are 0.75 ( 0.04 and 2.8 ( 0.3 kcal mol -1 M -1 for urea and GdmCl, respectively. The ratio m on /(m on + m off ) is 0.54 ( 0.04 for urea and 0.57 ( 0.1 for GdmCl, where m on is the m-value for k on and m off is the m-value for k off , indicating that more than half of the sites for interaction with either cosolvent are buried in the ensemble of structures present at the transition state.The refolding kinetics of numerous small proteins obey simple exponential kinetics, indicating that intermediates are not populated and that these polypeptides cross a free-energy barrier before reaching their native states (for recent examples see refs 1 and 2). This free energy barrier may be the wall of a kinetic trap or a decrease in conformational entropy before the formation of stabilizing protein-protein interactions (3, 4). There may also be an enthalpic barrier in simple refolding reactions (5). Both the depth of kinetic traps and the height of the conformational entropy barrier to refolding are expected to decrease dramatically with decreasing chain length, but the shortest stable proteins are of the order of 60 residues. We present data for the reaction where p is a fluorescently labeled S-peptide analogue, N is the folded form of S-protein, and pN is the S-peptide analogue/S-protein (RNaseS* 1 ) complex. Our aim is to characterize the free energy barrier on a folding landscape which has a low conformational entropy barrier and is unlikely to contain deep kinetic traps. The conformational entropy lost during folding is chiefly that of the 15 residue S-peptide analogue.Protein folding reactions with exponential time courses may be well described by the transition state theory (6). The observed rate constant of such a reaction is the product K q k q , where K q ...

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Section: Formation Of Rnases* Is Described By the Two-statesupporting

confidence: 67%

Section: Helicity Of Pep-1fmentioning

confidence: 99%

Kinetic Mechanism of a Partial Folding Reaction. 1. Properties of the Reaction and Effects of Denaturants

Goldberg

Baldwin

1998

Biochemistry

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“…The disparate salt effects on k a and k d are observed on many protein-protein pairs. [33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48] The binding of U1A with U1SLII shows exactly this salt behavior 7,14 and is thus expected to be modeled by a transient complex close to the native complex.…”

Section: Introductionmentioning

confidence: 99%

Prediction of Salt and Mutational Effects on the Association Rate of U1A Protein and U1 Small Nuclear RNA Stem/Loop II

Qin

Zhou

2007

J. Phys. Chem. B

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We have developed a computational approach for predicting protein-protein association rates (Alsallaq and Zhou, Structure 2007, 15, 215). Here we expand the range of applicability of this approach to protein-RNA binding and report the first results for protein-RNA binding rates predicted from atomistic modeling. The system studied is the U1A protein and stem/loop II of the U1 small nuclear RNA. Experimentally it was observed that the binding rate is significantly reduced by increasing salt concentration while the dissociation changes little with salt concentration, and charges distant from the binding site make marginal contribution to the binding rate. These observations are rationalized. Moreover, predicted effects of salt and charge mutations are found to be in quantitative agreement with experimental results.

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“…Related peptides have been very extensively studied and have been shown to exist in a two-state equilibrium between an unfolded state and fully ␣-helical dimers (16)(17)(18)(19)(20)(21)(22). Kinetic studies suggest that the ␣-helical secondary structure and the double-helical folded structure form concomitantly (19)(20)(21)(22)(23)(24).…”

mentioning

confidence: 99%

Dynamics and folding of single two-stranded coiled-coil peptides studied by fluorescent energy transfer confocal microscopy

Talaga

Lau

Röder

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

237

221

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We report single-molecule measurements on the folding and unfolding conformational equilibrium distributions and dynamics of a disulfide crosslinked version of the two-stranded coiled coil from GCN4. The peptide has a fluorescent donor and acceptor at the N termini of its two chains and a Cys disulfide near its C terminus. Thus, folding brings the two N termini of the two chains close together, resulting in an enhancement of fluorescent resonant energy transfer. End-toend distance distributions have thus been characterized under conditions where the peptide is nearly fully folded (0 M urea), unfolded (7.4 M urea), and in dynamic exchange between folded and unfolded states (3.0 M urea). The distributions have been compared for the peptide freely diffusing in solution and deposited onto aminopropyl silanized glass. As the urea concentration is increased, the mean end-to-end distance shifts to longer distances both in free solution and on the modified surface. The widths of these distributions indicate that the molecules are undergoing millisecond conformational fluctuations. Under all three conditions, these fluctuations gave nonexponential correlations on 1-to 100-ms time scale. A component of the correlation decay that was sensitive to the concentration of urea corresponded to that measured by bulk relaxation kinetics. The trajectories provided effective intramolecular diffusion coefficients as a function of the end-to-end distances for the folded and unfolded states. Single-molecule folding studies provide information concerning the distributions of conformational states in the folded, unfolded, and dynamically interconverting states.

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Very Rapid, Ionic Strength-Dependent Association and Folding of a Heterodimeric Leucine Zipper

Cited by 93 publications

References 59 publications

Kinetic Mechanism of a Partial Folding Reaction. 1. Properties of the Reaction and Effects of Denaturants

Kinetic Mechanism of a Partial Folding Reaction. 1. Properties of the Reaction and Effects of Denaturants

Prediction of Salt and Mutational Effects on the Association Rate of U1A Protein and U1 Small Nuclear RNA Stem/Loop II

Dynamics and folding of single two-stranded coiled-coil peptides studied by fluorescent energy transfer confocal microscopy

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