Achieving Secondary Structural Resolution in Kinetic Measurements of Protein Folding: A Case Study of the Folding Mechanism of Trp‐cage

Culik, Robert M.; Serrano, Arnaldo L.; Bunagan, Michelle R.; Gai, Feng

doi:10.1002/ange.201104085

Cited by 38 publications

(110 citation statements)

References 67 publications

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“…In the case of TC5b, this mutation has been reported to have a stabilizing effect (a positive Δ T m by CD) and provides a significantly greater acceleration of the folding rate. 7 This published account indicates a Φ F in excess of 1.2. The same Δ T m for a [P12W] mutation has also been observed for TC16b and a circularly permuted Trp-cage sequence and ΔΔ G U values on the order of +2 to +3 kJ/mol were derived 14 for this mutation.…”

Section: Resultsmentioning

confidence: 95%

“…Culik et al 12 reported that the fold-stabilizing G10 a mutation (Δ T m = +21 °C) increased the folding rate of TC5b by only a factor of 2. Two additional mutations, R16K and P19A, were examined in the case of TC10b, 7 and neither was reported to alter the folding rate appreciably. The P19A mutation is a particularly destabilizing mutation, Δ T m = −44 °C, as quoted by Culik et al 7 As a result, Feng Gai and co-workers have proposed that hydrophobic staple (Y3/P19) and salt-bridge (D9/R16) formation occur on the downhill side of the folding transition and only stabilize the final state; essentially, that helix formation is rate determining and sets the stage for downhill folding events thereafter.…”

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confidence: 99%

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Folding Dynamics and Pathways of the Trp-Cage Miniproteins

et al. 2014

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Using alternate measures of fold stability for a wide variety of Trp-cage mutants has raised the possibility that prior dynamics T-jump measures may not be reporting on complete cage formation for some species. NMR relaxation studies using probes that only achieve large chemical shift difference from unfolded values on complete cage formation indicate slower folding in some but not all cases. Fourteen species have been examined, with cage formation time constants (1/kF) ranging from 0.9–7.5 μs at 300 K. The present study does not change the status of the Trp-cage as a fast folding, essentially two-state system, although it does alter the stage at which this description applies. A diversity of prestructuring events, depending on the specific analogue examined, may appear in the folding scenario, but in all cases, formation of the N-terminal helix is complete either at or before the cage-formation transition state. In contrast, the fold-stabilizing H-bonding interactions of the buried Ser14 side chain and the Arg/Asp salt bridge are post-transition state features on the folding pathway. The study has also found instances in which a [P12W] mutation is fold destabilizing but still serves to accelerate the folding process.

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Section: Resultsmentioning

confidence: 95%

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confidence: 99%

Folding Dynamics and Pathways of the Trp-Cage Miniproteins

et al. 2014

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“…Indeed, the formation of such a low-temperature folding intermediate has been reported previously. 31,72 ■ CONCLUSION…”

Section: ■ Resultsmentioning

confidence: 97%

Folding Dynamics of the Trp-Cage Miniprotein: Evidence for a Native-Like Intermediate from Combined Time-Resolved Vibrational Spectroscopy and Molecular Dynamics Simulations

et al. 2013

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Trp-cage is a synthetic 20-residue miniprotein which folds rapidly and spontaneously to a well-defined globular structure more typical of larger proteins. Due to its small size and fast folding, it is an ideal model system for experimental and theoretical investigations of protein folding mechanisms. However, Trp-cage's exact folding mechanism is still a matter of debate. Here we investigate Trp-cage's relaxation dynamics in the amide I' spectral region (1530-1700 cm(-1)) using time-resolved infrared spectroscopy. Residue-specific information was obtained by incorporating an isotopic label ((13)C═(18)O) into the amide carbonyl group of residue Gly11, thereby spectrally isolating an individual 310-helical residue. The folding-unfolding equilibrium is perturbed using a nanosecond temperature-jump (T-jump), and the subsequent re-equilibration is probed by observing the time-dependent vibrational response in the amide I' region. We observe bimodal relaxation kinetics with time constants of 100 ± 10 and 770 ± 40 ns at 322 K, suggesting that the folding involves an intermediate state, the character of which can be determined from the time- and frequency-resolved data. We find that the relaxation dynamics close to the melting temperature involve fast fluctuations in the polyproline II region, whereas the slower process can be attributed to conformational rearrangements due to the global (un)folding transition of the protein. Combined analysis of our T-jump data and molecular dynamics simulations indicates that the formation of a well-defined α-helix precedes the rapid formation of the hydrophobic cage structure, implying a native-like folding intermediate, that mainly differs from the folded conformation in the orientation of the C-terminal polyproline II helix relative to the N-terminal part of the backbone. We find that the main free-energy barrier is positioned between the folding intermediate and the unfolded state ensemble, and that it involves the formation of the α-helix, the 310-helix, and the Asp9-Arg16 salt bridge. Our results suggest that at low temperature (T ≪ Tm) a folding path via formation of α-helical contacts followed by hydrophobic clustering becomes more important.

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“…The freeenergy plots were projected onto various local and global collective variables that provide information about the folding of the different regions of the protein as well as the overall protein. Despite several experimental 53 24,25,35,62,63,65,66,68,69 there is still no agreement on the nature of this intermediate.…”

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confidence: 90%

Achieving Rigorous Accelerated Conformational Sampling in Explicit Solvent

Doshi

Hamelberg

2014

J. Phys. Chem. Lett.

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Molecular dynamics simulations can provide valuable atomistic insights into biomolecular function. However, the accuracy of molecular simulations on general-purpose computers depends on the time scale of the events of interest. Advanced simulation methods, such as accelerated molecular dynamics, have shown tremendous promise in sampling the conformational dynamics of biomolecules, where standard molecular dynamics simulations are nonergodic. Here we present a sampling method based on accelerated molecular dynamics in which rotatable dihedral angles and nonbonded interactions are boosted separately. This method (RaMD-db) is a different implementation of the dual-boost accelerated molecular dynamics, introduced earlier. The advantage is that this method speeds up sampling of the conformational space of biomolecules in explicit solvent, as the degrees of freedom most relevant for conformational transitions are accelerated. We tested RaMD-db on one of the most difficult sampling problems - protein folding. Starting from fully extended polypeptide chains, two fast folding α-helical proteins (Trpcage and the double mutant of C-terminal fragment of Villin headpiece) and a designed β-hairpin (Chignolin) were completely folded to their native structures in very short simulation time. Multiple folding/unfolding transitions could be observed in a single trajectory. Our results show that RaMD-db is a promisingly fast and efficient sampling method for conformational transitions in explicit solvent. RaMD-db thus opens new avenues for understanding biomolecular self-assembly and functional dynamics occurring on long time and length scales.

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Achieving Secondary Structural Resolution in Kinetic Measurements of Protein Folding: A Case Study of the Folding Mechanism of Trp‐cage

Cited by 38 publications

References 67 publications

Folding Dynamics and Pathways of the Trp-Cage Miniproteins

Folding Dynamics and Pathways of the Trp-Cage Miniproteins

Folding Dynamics of the Trp-Cage Miniprotein: Evidence for a Native-Like Intermediate from Combined Time-Resolved Vibrational Spectroscopy and Molecular Dynamics Simulations

Achieving Rigorous Accelerated Conformational Sampling in Explicit Solvent

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