Effect of Heavy Water on Protein Flexibility

Cioni, Patrizia; Strambini, Giovanni B.

doi:10.1016/s0006-3495(02)75666-x

Cited by 172 publications

(191 citation statements)

References 34 publications

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“…To answer this, we assigned and measured 15 N and 13 C α chemical shifts 18 as well as 3 J HNHA scalar couplings in the E-mutant and compared them to those of wt tau. Both proteins showed only small deviations from random coil values of 13 C α chemical shifts, in agreement with their disordered nature ( Figure S15). Direct comparison of the experimental 13 C α chemical shifts for wt and E-mutant tau demonstrated that local conformational changes induced by pseudophosphorylation are small and restricted to the vicinity of the mutation.…”

supporting

confidence: 60%

“…Rigid secondary structure is not induced, in agreement with 3 J HNHA scalar couplings ( Figure S15) and previous work on short tau peptides. 19 Despite the fact that the PHF1 epitope near the C-terminus contains only two phosphorylation sites (396 and 404), 13 C α chemical shift changes were more pronounced and affected a larger set of residues than did glutamic acid mutations in the proline-rich regions, suggesting that the PHF1 epitope is more prone to conformational changes.…”

mentioning

confidence: 99%

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Structural Impact of Proline-Directed Pseudophosphorylation at AT8, AT100, and PHF1 Epitopes on 441-Residue Tau

et al. 2011

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ABSTRACT:The intrinsically disordered protein tau becomes excessively phosphorylated and aggregates into neurofibrillary tangles in Alzheimer's disease. To obtain insight into the structural consequences of phosphorylation, we characterized a mutant protein of tau in which epitopes recognized by Alzheimer diagnostic antibodies were mimicked by mutation to glutamic acid [AT8 (S199E, S202E, T205E), AT100 (T212E and S214E), and PHF1 (S396E and S404E)]. A large number of distance restraints obtained from NMR paramagnetic relaxation enhancement in combination with ensemble conformer calculations demonstrate that pseudophosphorylation causes an opening of the transient folding of tau. Together with previous studies on the Parkinson-related protein α-synuclein, our data indicate that networks of transient long-range interactions are common properties of intrinsically disordered proteins and that their modulation is important for aggregation.A ggregation of the microtubule-associated protein tau into neurofibrillary tangles is the pathological hallmark of a variety of dementias. 1,2 For reasons not yet known, tau becomes excessively phosphorylated in Alzheimer's brains and as a result no longer binds properly to microtubules. The unbound tau is free to undergo abnormal aggregation. In vivo, hyperphosphorylation of tau precedes tangle formation. 3 At least 30 phosphorylation sites in tau filaments have been identified. 4 Phosphorylation at serine/proline and threonine/proline motifs in the flanking regions of the repeat domain of tau has only a moderate influence on tauÀmicrotubule interactions but is upregulated in Alzheimer's disease. 5 Tau is a prototypical intrinsically disordered protein that does not assume a rigid tertiary or secondary structure but populates an ensemble of interconverting structures in solution. 6,7 Because of the inherent flexibility of tau, NMR spectroscopy is the only method that allows a description of its conformations and dynamics with high resolution. 8 We have recently shown that it is possible to obtain the complete backbone resonance assignment of the longest isoform of human tau and demonstrated that 441-residue tau has a distinct domain character with an intricate network of long-range interactions. 7 Here we investigate changes in the local and global structure of 441-residue tau related to phosphorylation in the epitopes recognized by the Alzheimer diagnostic antibodies AT8 (S199E, S202E, T205E), AT100 (T212E and S214E), and PHF1 (S396E and S404E). To avoid the ambiguities of heterogeneous phosphorylation, we cloned "pseudophosphorylation" mutants of tau in which serine and threonine residues were converted into glutamic acid. The same mutant protein was previously shown to aggregate slightly faster in comparison with wild-type (wt) tau. 9 In addition, a different six-site pseudophosphorylation mutant (S199E, S202E, T205E, T231E, S396E, and S404E) had a decreased rate of elongation and a pronounced lag time of aggregation. 10 We determined cross-validated ensembles of wild-type (wt)...

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supporting

confidence: 60%

mentioning

confidence: 99%

Structural Impact of Proline-Directed Pseudophosphorylation at AT8, AT100, and PHF1 Epitopes on 441-Residue Tau

et al. 2011

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“…Generally for smaller proteins, assuming the EX2 mechanism (see "Results"), the highest ⌬G HX value estimated by H/D exchange corresponds to the overall ⌬G U value calculated from the unfolding transition curve obtained with denaturants or with heat (39 -42). On the other hand, there are some possible contributions to the increase in the ⌬G HX value, such as the existence of superprotected residues (43), the effects of solvent isotopes (by Ϯ0 -2 kcal/mol), and proline isomerization (44,45).…”

Section: Discussionmentioning

confidence: 99%

Cores and pH-dependent Dynamics of Ferredoxin-NADP+ Reductase Revealed by Hydrogen/Deuterium Exchange

Lee

Tamura

Hoshino

et al. 2007

Journal of Biological Chemistry

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NMR-detected hydrogen/deuterium (H/D) exchange of amide protons is a powerful way for investigating the residuebased conformational stability and dynamics of proteins in solution. Maize ferredoxin-NADP ؉ reductase (FNR) is a relatively large protein with 314 amino acid residues, consisting of flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADP ؉ )-binding domains. To address the structural stability and dynamics of FNR, H/D exchange of amide protons was performed using heteronuclear NMR at pD r values 8.0 and 6.0, physiologically relevant conditions mimicking inside of chloroplasts. At both pD r values, the exchange rate varied widely depending on the residues. The profiles of protected residues revealed that the highly protected regions matched well with the hydrophobic cores suggested from the crystal structure, and that the NADP ؉ -binding domain can be divided into two subdomains. The global stability of FNR obtained by H/D exchange with NMR was higher than that by chemical denaturation, indicating that H/D exchange is especially useful for analyzing the residue-based conformational stability of large proteins, for which global unfolding is mostly irreversible. Interestingly, more dynamic conformation of the C-terminal subdomain of the NADP ؉ -binding domain at pD r 8.0, the daytime pH in chloroplasts, than at pD r 6.0 is likely to be involved in the increased binding of NADP ؉ for elevating the activity of FNR. In light of photosynthesis, the present study provides the first structure-based relationship of dynamics with function for the FNR-type family in solution.Protein conformations as shown by x-ray crystallography or nuclear magnetic resonance spectroscopy are virtually dynamic entities over various time ranges in solution (1, 2). Clarifying such conformational motions at the level of the atom or residue is essential for understanding the structural stabilities of proteins and the relationships between protein structures and functions (3-5). The hydrogen/deuterium (H/D) 4 exchange of amide protons in backbones has become an important way to address the motions of a protein from small or relatively large scale motions involved in biological functions such as binding of a substrate or releasing a product to much more large scale motions like a global unfolding process (4, 6). Among several approaches, the H/D exchange combined with heteronuclear NMR spectroscopy is the most convenient and powerful way because it can provide residue-specific information for most residues (7). For many globular proteins, this approach has identified protected cores, which are often composed of secondary structures buried inside the molecules and the cooperative interactions with partner molecules (4, 8), leading to allostery (9). Detailed analyses of exchanges in the native state of cytochrome c in the presence of various concentrations of denaturants suggested a pathway to unfolding, in which groups of secondary structural elements (i.e. foldons) are unfolded sequentially through distin...

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“…(1) Some proteins have been shown to be more stable in 2 H 2 O than in H 2 O, [36][37][38] and the CD data (on which the Ising fits were based) were performed in H 2 O, whereas the HX experiments were performed in 2 H 2 O, but for both CTPR2 and CTPR3 we saw no appreciable difference in protein stability in H 2 O versus 2 H 2 O (Fig. 4a, inset).…”

Section: Native State Hydrogen Exchange (Nhx)mentioning

confidence: 95%

Mapping the Energy Landscape of Repeat Proteins using NMR-detected Hydrogen Exchange

Cortajarena¹,

Mochrie²,

Regan³

2008

Journal of Molecular Biology

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Repeat proteins contain tandem arrays of a simple structural motif. In contrast to globular proteins, repeat proteins are stabilized only by interactions between residues that are relatively close together in the sequence, with no "long-range" interactions. Our work focuses on the tetratricopeptide repeat (TPR), a 34 amino acid helix-turn-helix motif found in tandem arrays in many natural proteins. Earlier, we reported the design and characterization of a series of consensus TPR (CTPR) proteins, which are built as arrays of multiple tandem copies of a 34 amino acid consensus sequence. Here, we present the results of extensive hydrogen exchange (HX) studies of the folding-unfolding behavior of two CTPR proteins (CTPR2 and CTPR3). We used HX to detect and characterize partially folded species that are populated at low frequency in the nominally folded state. We show that for both proteins the equilibrium folding-unfolding transition is nontwo-state, but sequential, with the outermost helices showing a significantly higher probability than inner helices of being unfolded. We show that the experimentally observed unfolding behavior is consistent with the predictions of a simple Ising model, in which individual helices are treated as "spin-equivalents". The results that we present have general implications for our understanding of the thermodynamic properties of repeat proteins.

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Effect of Heavy Water on Protein Flexibility

Cited by 172 publications

References 34 publications

Structural Impact of Proline-Directed Pseudophosphorylation at AT8, AT100, and PHF1 Epitopes on 441-Residue Tau

Structural Impact of Proline-Directed Pseudophosphorylation at AT8, AT100, and PHF1 Epitopes on 441-Residue Tau

Cores and pH-dependent Dynamics of Ferredoxin-NADP+ Reductase Revealed by Hydrogen/Deuterium Exchange

Mapping the Energy Landscape of Repeat Proteins using NMR-detected Hydrogen Exchange

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