Folding Kinetics of a Naturally Occurring Helical Peptide: Implication of the Folding Speed Limit of Helical Proteins

Mukherjee, Smita; Chowdhury, P. K.; Bunagan, Michelle R.; Gai, Feng

doi:10.1021/jp801721p

Cited by 36 publications

(60 citation statements)

References 63 publications

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“…Our data are consistent with pIL's barrierless, down-hill folding to both the folded state and the misfolded state 2 (27,28). The speed of down-hill folding is limited by diffusion of the polypeptide chain (29) and is approximately 1 × 10 6 s −1 for proteins comparable to pIL in size (20,30,31). In contrast to the folded state and misfolded state 2, there is a small (1 AE 3 k B T) and a large (11 AE 3 k B T) energy barrier for pIL to fold into misfolded states 3 and 4, with corresponding folding rates of 2.9 × 10 5 s −1…”

Section: A Model Of Helix Staggering and Sliding In Pil Folding And Msupporting

confidence: 82%

Single-molecule observation of helix staggering, sliding, and coiled coil misfolding

Gao

Sirinakis

et al. 2012

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

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The biological functions of coiled coils generally depend on efficient folding and perfect pairing of their α-helices. Dynamic changes in the helical registry that lead to staggered helices have only been proposed for a few special systems and not found in generic coiled coils. Here, we report our observations of multiple staggered helical structures of two canonical coiled coils. The partially folded structures are formed predominantly by coiled coil misfolding and occasionally by helix sliding. Using high-resolution optical tweezers, we characterized their energies and transition kinetics at a single-molecule level. The staggered states occur less than 2% of the time and about 0.1% of the time at zero force. We conclude that dynamic changes in helical registry may be a general property of coiled coils. Our findings should have broad and unique implications in functions and dysfunctions of proteins containing coiled coils.leucine zipper | protein misfolding C oiled coils widely mediate protein-protein interactions and form rigid structures such as scaffolds, spacers, and levers (1) that are often involved in generating (2), transducing (3), or sensing forces (4) in cells. The biological functions of coiled coils critically depend upon their affinity, specificity, and dynamics of helix pairing. Although the structures and dynamics of coiled coils have been extensively studied, it is unclear whether coiled coils containing staggered helices can form through protein misfolding or helix sliding (5-8). A further understanding of these alternative structures and their mechanisms of formation is needed to better understand the functions and dysfunctions of coiled coils (3).Because helices in coiled coils have a characteristic seven amino acid repeat (abcdef gÞ n , their functional folding and assembly generally requires to pair hydrophobic residues in the a and d positions in the dimerization interface and other residues near the interface. However, due to the periodic α-helical structure, alternative structures can form in which one helix shifts its registry, often by one heptad repeat, relative to the other. Although the resultant staggered helical structures were first proposed when the high-resolution crystal structure of the coiled coil GCN4 leucine zipper domain was obtained (5), these alternative conformations have only been observed in a few coiled coils with special sequence patterns deviating from the canonical heptad repeat (3,7,8). The difficulty in finding these different conformations is due to functional conformations of most coiled coils being much more stable than staggered conformations. The differential stability can be achieved by specific pairing of polar residues or complementary packing of nonpolar residues in the dimerization interface and by forming interhelical salt bridges between residues near the interface (1, 5, 9). In the GCN4 leucine zipper, substitution of a single asparagine buried in the dimerization interface for a nonpolar residue converts the two-stranded coiled coil to a mixture ...

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Section: A Model Of Helix Staggering and Sliding In Pil Folding And Msupporting

confidence: 82%

Single-molecule observation of helix staggering, sliding, and coiled coil misfolding

Gao

Sirinakis

et al. 2012

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

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“…Although the actual event associated with this final step is unknown, information about the Ub pathway garnered from ψ-analysis ( 10 ) suggests a folding event involving either the consolidation of the existing four strand/helix TS structure defined by the sites with non-zero ψ, or the initial formation of the 3 10 helix (the next major structural element to form after the TS). Our k trans rate is slower than the rate measure by Gai et al of helix formation for naturally-occurring sequences ( 27 ), which is, in turn, an order of magnitude slower than for alanine-rich helices. Our lower transmission rate may be due to the complicated nature of the remaining folding steps (e.g.…”

Section: Discussioncontrasting

confidence: 63%

Metal Binding Kinetics of Bi-Histidine Sites Used in ψ Analysis: Evidence of High-Energy Protein Folding Intermediates

2009

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The zinc-specific fluorophore, Zinpyr-1, is used in competition assays to determine the kinetic and thermodynamic parameters of Zn2+ binding to engineered bi-Histidine sites located in Ubiquitin and the B domain of protein A (BdpA). These binding sites are used in ψ-analysis studies to investigate structure formation in the folding transition state identified by the change in folding rate upon the addition of metal ions. For Ubiquitin, the on-rate binding constant and binding affinity for a site located along an α-helix are measured to be ~107 M-1s-1 and 3 μM, respectively. For a site located across two β-strands, the metal binding affinity was too weak to measure in the dye competition assays (Kd > 55 μM). The equilibrium-determined values for the Zn2+-induced stabilization of Ubiquitin and BdpA match the values derived from changes in the global folding and unfolding rates. Therefore, metal-ion binding is in fast equilibrium during the transit over the free energy barrier. Accordingly, the folding rate must be slower than the product of the fractional population of a high energy intermediate with the metal site formed and the metal binding on-rate constant. The known folding rate of 20 s-1 at 1.5 M guanidinium chloride in 400 μM Zn2+ provides an upper bound for the stability of such intermediates, ΔGU-I < +4 kcal·mol-1. These results support a view of the apparent two-state protein folding reaction surface as a fast pre-equilibrium between the denatured state and a series of high energy species. The net folding rate is a product of the equilibrium constant of the highest energy species and a transmission rate. For Ubiquitin, we estimate the transmission rate to be ~104 s-1. Implications to the role of unfolded chain diffusion on folding rates and barrier heights are discussed.

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“…A relaxation time constant for helix folding is typically 0.2 ~ 2 μs. 47 In our experimental conditions, the TAZ2 and p53 AD2 concentrations are in the order of 10 −4 M. Here, the binding rate constant is 10 6 s −1 (= 10 10 M −1 s −1 × 10 −4 M), which corresponds to 1 μs. This value is comparable to the helix folding rate.…”

Section: Discussionmentioning

confidence: 92%

Quantitative Analysis of Multisite Protein–Ligand Interactions by NMR: Binding of Intrinsically Disordered p53 Transactivation Subdomains with the TAZ2 Domain of CBP

2012

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Determination of affinities and binding sites involved in protein-ligand interactions is essential for understanding molecular mechanisms in biological systems. Here we combine singular value decomposition, and global analysis of NMR chemical shift perturbations caused by protein-protein interactions to determine the number and location of binding sites on the protein surface and to measure the binding affinities. Using this method we show that the isolated AD1 and AD2 binding motifs, derived from the intrinsically disordered N-terminal transactivation domain of the tumor suppressor p53, both interact with the TAZ2 domain of the transcriptional coactivator CBP at two binding sites. Simulations of titration curves and lineshapes show that a primary dissociation constant as small as 1~10 nM can be accurately estimated by NMR titration methods, provided that the primary and secondary binding processes are coupled. Unexpectedly, the site of binding of AD2 on the hydrophobic surface of TAZ2 overlaps with the binding site for AD1, but AD2 binds TAZ2 more tightly. The results highlight the complexity of interactions between intrinsically disordered proteins and their targets. Furthermore, the association rate of AD2 to TAZ2 is estimated to be 1.7 × 1010 M−1 s−1, approaching the diffusion-controlled limit and indicating that intrinsic disorder plus complementary electrostatics can significantly accelerate protein binding interactions.

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Folding Kinetics of a Naturally Occurring Helical Peptide: Implication of the Folding Speed Limit of Helical Proteins

Cited by 36 publications

References 63 publications

Single-molecule observation of helix staggering, sliding, and coiled coil misfolding

Single-molecule observation of helix staggering, sliding, and coiled coil misfolding

Metal Binding Kinetics of Bi-Histidine Sites Used in ψ Analysis: Evidence of High-Energy Protein Folding Intermediates

Quantitative Analysis of Multisite Protein–Ligand Interactions by NMR: Binding of Intrinsically Disordered p53 Transactivation Subdomains with the TAZ2 Domain of CBP

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