pH and Urea Dependence of Amide Hydrogen−Deuterium Exchange Rates in the β-Trefoil Protein Hisactophilin

Houliston, R. Scott; Liu, Chengsong; Singh, Laila M.R.; Meiering, Elizabeth M.

doi:10.1021/bi0115838

Cited by 25 publications

(28 citation statements)

References 56 publications

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“…S6A). This protection is consistent with myristoylation increasing the global protein stability, as has also been observed in chemical denaturation experiments (6,27). Furthermore, the largest increases in amide protection arising from the myristoyl group from pH 5.9 to 8.1 are observed for amides close to the N-and C-termini (Fig.…”

Section: Resultssupporting

confidence: 86%

Nonnative interactions regulate folding and switching of myristoylated protein

Shental-Bechor

Smith

MacKenzie

et al. 2012

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

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We present an integrated experimental and computational study of the molecular mechanisms by which myristoylation affects protein folding and function, which has been little characterized to date. Myristoylation, the covalent linkage of a hydrophobic C14 fatty acyl chain to the N-terminal glycine in a protein, is a common modification that plays a critical role in vital regulated cellular processes by undergoing reversible energetic and conformational switching. Coarse-grained folding simulations for the model pH-dependent actin-and membrane-binding protein hisactophilin reveal that nonnative hydrophobic interactions of the myristoyl with the protein as well as nonnative electrostatic interactions have a pronounced effect on folding rates and thermodynamic stability. Folding measurements for hydrophobic residue mutations of hisactophilin and atomistic simulations indicate that the nonnative interactions of the myristoyl group in the folding transition state are nonspecific and robust, and so smooth the energy landscape for folding. In contrast, myristoyl interactions in the native state are highly specific and tuned for sensitive control of switching functionality. Simulations and amide hydrogen exchange measurements provide evidence for increases as well as decreases in stability localized on one side of the myristoyl binding pocket in the protein, implicating strain and altered dynamics in switching. The effects of folding and function arising from myristoylation are profoundly different from the effects of other post-translational modifications.coarse-grained simulation | funnel landscape | β-trefoil P rotein folding is governed by various physicochemical forces that bias the native state, which for many proteins is also the functional state, over the many alternative nonnative states. The network of native interactions has been found in many cases to be sufficient to capture the folding mechanism and kinetics of proteins (1, 2). The discrimination between native and nonnative interactions is the foundation of the principle of minimal frustration (3) and explains the power of native topology-based models in studying folding biophysics (4). The dominant role of native interactions is manifested by the funnel-shaped energy landscape for folding that suggests folding is robust and an efficient process. The information stored in the native topology may, however, be tuned by various factors such as confining the protein in a small space, crowding agents, or conjugating the protein to other biomolecules [e.g., oligosaccharides (5) or fatty acyl chains such as myristoyl (6)]. In addition to manipulating folding characteristics by modifications or environmental conditions, nonnative interactions, which are by definition in conflict with the native state, may decorate the folding funnel (7, 8) by increasing energetic frustration (9) between interactions and therefore landscape roughness. The degree of roughness, which affects the trapping of the protein in nonnative states, depends on the particular sequence of the pr...

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Section: Resultssupporting

confidence: 86%

Nonnative interactions regulate folding and switching of myristoylated protein

Shental-Bechor

Smith

MacKenzie

et al. 2012

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

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“…Refolding kinetics of hisactophilin monitored by quenched-flow H/D exchange data showed distinct events in the refolding pathway of the protein (54). Interestingly, native state H/D exchange data showed that the ␤-trefoil architecture of hisactophilin behaves as a single cooperative unit, and no partially unfolded forms are detected (66). In the case of hFGF-1, the urea-induced equilibrium unfolding pathway (as shown in the present study), monitored by steady state fluorescence and CD, follows a two-state (native 7 unfolded) mechanism.…”

Section: Discrepancies Between Free Energy Change Of H/d Exchange (⌬Gsupporting

confidence: 61%

Identification of Rare Partially Unfolded States in Equilibrium with the Native Conformation in an All β-Barrel Protein

Chi

Kumar

Chiu

et al. 2002

Journal of Biological Chemistry

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Human acidic fibroblast growth factor 1 (hFGF-1) is an all ␤-barrel protein, and the secondary structural elements in the protein include 12 antiparallel ␤-strands arranged into a ␤-trefoil fold. In the present study, we investigate the stability of hFGF-1 by hydrogen-deuterium exchange as a function of urea concentration. Urea-induced equilibrium unfolding of hFGF-1 monitored by fluorescence and CD spectroscopy suggests that the protein unfolds by a two-state (native to denatured) mechanism. Hydrogen exchange in hFGF-1, under the experimental conditions used, occurs by the EX2 mechanism. In contrast to the equilibrium unfolding events monitored by optical probes, native state hydrogen exchange data show that the ␤-trefoil architecture of hFGF-1 does not behave as a single cooperative unit. There are at least two structurally independent units with differing stabilities in hFGF-1. ␤-Strands I, II, III, VI, VII, X, XI, and XII fit into the global unfolding isotherm. By contrast, residues in ␤-strands IV, V, VIII, and IX exchange by the subfolding isotherm and could be responsible for the occurrence of high-energy partially unfolded state(s) in hFGF-1. There appears to be a broad continuum of stabilities among the four ␤-strands (␤-strands IV, V, VIII, and IX) constituting the subglobal folding unit. The slow exchanging residues in hFGF-1 do not represent the folding nucleus of the protein.

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“…Here, the natural isotopic distribution is treated as a series of mass offsets above the monoisotopic mass. For example, incorporation of one 13 C results in a ϩ1 offset. As with back exchange, more than one combination gives rise to the same observed mass.…”

Section: Modeling Ex2 Exchangementioning

confidence: 99%

“…Such unfolding is termed cooperative or correlated exchange and is unusual for proteins under physiological conditions. As has been shown repeatedly (examples include: [6,[11][12][13][14][15] that the addition of denaturants or a change in pH (typically to a high value of 9.0 -10.5) can artificially induce EX1 kinetics. While useful for understanding stability and the folding mechanism, artificially inducing EX1 kinetics generally does not reflect physiological protein dynamics.…”

mentioning

confidence: 99%

Identification and characterization of EX1 kinetics in H/D exchange mass spectrometry by peak width analysis

Weis

Wales

Engen

et al. 2006

J. Am. Soc. Mass Spectrom.

210

269

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Proteins that undergo cooperative unfolding events display EX1 kinetic signatures in hydrogen exchange mass spectra. The hallmark bimodal isotope pattern observed for EX1 kinetics is distinct from the binomial isotope pattern for uncorrelated exchange (EX2), the normal exchange regime for folded proteins. Detection and characterization of EX1 kinetics is simple when the cooperative unit is large enough that the isotopic envelopes in the bimodal pattern are resolved in the m/z scale but become complicated in cases where the unit is small or there is a mixture of EX1 and EX2 kinetics. Here we describe a data interpretation method involving peak width analysis that makes characterization of EX1 kinetics simple and rapid. The theoretical basis for EX1 and EX2 isotopic signatures and the effects each have on peak width are described. Modeling of EX2 widening and analysis of empirical data for proteins and peptides containing purely EX2 kinetics showed that the amount of widening attributable to stochastic forward-and back exchange in a typical experiment is small and can be quantified. Proteins and peptides with both obvious and less obvious EX1 kinetics were analyzed with the peak width method. Such analyses provide the half-life for the cooperative unfolding event and the relative number of residues involved. Automated analysis of peak width was performed with custom Excel macros and the DEX software package. Peak width analysis is robust, capable of automation, and provides quick interpretation of the key information contained in

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pH and Urea Dependence of Amide Hydrogen−Deuterium Exchange Rates in the β-Trefoil Protein Hisactophilin

Cited by 25 publications

References 56 publications

Nonnative interactions regulate folding and switching of myristoylated protein

Nonnative interactions regulate folding and switching of myristoylated protein

Identification of Rare Partially Unfolded States in Equilibrium with the Native Conformation in an All β-Barrel Protein

Identification and characterization of EX1 kinetics in H/D exchange mass spectrometry by peak width analysis

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