Improved Structural Characterizations of the drkN SH3 Domain Unfolded State Suggest a Compact Ensemble with Native-like and Non-native Structure

Marsh, Joseph A.; Neale, Chris; Jack, Fernando E.; Choy, Wing‐Yiu; Lee, Anna Y.; Crowhurst, Karin A.; Forman-Kay, Julie D.

doi:10.1016/j.jmb.2007.01.038

Cited by 111 publications

(181 citation statements)

References 58 publications

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“…Although significant progress has been made recently in the interpretation of NMR observables for disordered peptide and protein systems, [76][77][78][79] combining multiple and independent structural constraints for a system with significant disorder often leads to an inadequate description of the ensemble diversity. 77 By contrast, molecular dynamics simulations of disordered systems have the opposite challenge where the simulated ensemble is directly observable with good statistical confidence, but the accuracy is difficult to assess because of incomplete convergence and uncertainties of the underlying empirical force fields.…”

Section: Resultsmentioning

confidence: 99%

Structure and Dynamics of the Aβ_21–30 Peptide from the Interplay of NMR Experiments and Molecular Simulations

et al. 2008

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We combine molecular dynamics simulations and new high-field NMR experiments to describe the solution structure of the Aβ [21][22][23][24][25][26][27][28][29][30] peptide fragment that may be relevant for understanding structural mechanisms related to Alzheimer's disease. By using two different empirical force-field combinations, we provide predictions of the three-bond scalar coupling constants ( 3 J H N H α), chemical-shift values, 13 C relaxation parameters, and rotating-frame nuclear Overhauser effect spectroscopy (ROESY) crosspeaks that can then be compared directly to the same observables measured in the corresponding NMR experiment of Aβ [21][22][23][24][25][26][27][28][29][30] . We find robust prediction of the 13 C relaxation parameters and medium-range ROESY crosspeaks by using new generation TIP4P-Ew water and Amber ff99SB protein force fields, in which the NMR validates that the simulation yields both a structurally and dynamically correct ensemble over the entire Aβ 21-30 peptide. Analysis of the simulated ensemble shows that all medium-range ROE restraints are not satisfied simultaneously and demonstrates the structural diversity of the Aβ 21-30 conformations more completely than when determined from the experimental medium-range ROE restraints alone. We find that the structural ensemble of the Aβ 21-30 peptide involves a majority population (~60%) of unstructured conformers, lacking any secondary structure or persistent hydrogen-bonding networks. However, the remaining minority population contains a substantial percentage of conformers with a β-turn centered at Val24 and Gly25, as well as evidence of the Asp23 to Lys28 salt bridge important to the fibril structure. This study sets the stage for robust theoretical work on Aβ 1-40 and Aβ 1-42 , for which collection of detailed NMR data on the monomer will be more challenging because of aggregation and fibril formation on experimental timescales at physiological conditions. In addition, we believe that the interplay of modern molecular simulation and high-quality NMR experiments has reached a fruitful stage for characterizing structural ensembles of disordered peptides and proteins in general.

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Section: Resultsmentioning

confidence: 99%

Structure and Dynamics of the Aβ_21–30 Peptide from the Interplay of NMR Experiments and Molecular Simulations

et al. 2008

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“…However, the intrinsic disorder raises the question of whether the structure of the interaction site preexists in the IDP or is induced upon binding. A surge of recent evidence has established that both denatured proteins and IDPs can contain residual structure and even adopt compact folds in solution (10)(11)(12)(13). The residual structure of an IDP is believed to play a role in initiating the folding process, in protein-protein interactions, or in preventing aggregation (10,14,15).…”

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confidence: 99%

Intrinsically disordered γ-subunit of cGMP phosphodiesterase encodes functionally relevant transient secondary and tertiary structure

Song

Guo

Muradov

et al. 2008

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

123

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The retinal phosphodiesterase (PDE6) inhibitory ␥-subunit (PDE␥) plays a central role in vertebrate phototransduction through alternate interactions with the catalytic ␣␤-subunits of PDE6 and the ␣-subunit of transducin (␣t). Detailed structural analysis of PDE␥ has been hampered by its intrinsic disorder. We present here the NMR solution structure of PDE␥, which reveals a loose fold with transient structural features resembling those seen previously in the x-ray structure of PDE␥46-87 when bound to ␣t in the transitionstate complex. NMR mapping of the interaction between PDE␥46-87 and the chimeric PDE5/6 catalytic domain confirmed that Cterminal residues 74 -87 of PDE␥ are involved in the association and demonstrated that its W70 indole group, which is critical for subsequent binding to ␣t, is left free at this stage. These results indicate that the interaction between PDE␥ and ␣t during the phototransduction cascade involves the selection of preconfigured transient conformations.NMR spectroscopy ͉ protein recognition ͉ transient structure ͉ visual transduction P hototransduction, the primary event of vision is critically regulated by the ␥-subunit of cGMP phosphodiesterase (PDE␥). In the dark, by binding tightly to the catalytic ␣␤-subunits of cGMP PDE (PDE6) PDE␥ keeps PDE6 inactive; yet upon photoexcitation, interaction of PDE␥ with the ␣-subunit of activated transducin (␣ t ) relieves its inhibitory constraint on PDE6. For termination of phototransduction, PDE␥ interacts with both ␣ t and RGS9, a regulator of G protein signaling, to accelerate ␣ t GTPase activity (1). Beyond phototransduction, the importance of PDE␥ in photoreceptor cell viability is demonstrated by rapid retinal degeneration in the PDE␥-knockout mouse (2), and growing evidence suggests that PDE␥ also interacts with some signaling proteins in nonphototransduction pathways (3).Biochemical studies have shown that the central polycationic region (residues 24-45) and the C-terminal region of PDE␥ constitute two distinct sites of interactions with ␣ t or PDE6 (4). The crystal structure of a ternary complex representing a partial model for the GTPase-activating protein (GAP) complex, consisting of a fragment of PDE␥ (residues 46-87), ␣ t chimera (␣ t/i1 ), and the catalytic core of RGS9, revealed that the C-terminal region of PDE␥ contains three discontinuous helices (5). PDE␥ 46-87 interacts with ␣ t in a GTP-dependent manner, mainly through residues in these helices or their linkers, and the indole side chain of W70 plays the key role in this interaction. Residue V66 of PDE␥ interacts with RGS9, which further tightens the association between ␣ t and RGS9. The C-terminal region of PDE␥ also interacts with the catalytic domain of PDE6; however, studies have suggested that this interaction involves the 11 C-terminal residues of PDE␥, a region distinct from the ␣ t interaction site (6).It has been demonstrated that PDE␥ belongs to the growing family of intrinsically disordered proteins (IDPs) (7,8). Presumably, the inherent structural plasticity of...

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“…It seems unlikely that pockets of structure of the type observed in several proteins could account for these results, although in some cases they may play an important role. [85][86][87] There is little evidence from the CD spectra for the presence of a-helices in the unfolded proteins (Fig. 4), perhaps because the helices form transiently in the DSE and will generally be shorter than the helices in folded proteins.…”

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confidence: 99%

Urea denatured state ensembles contain extensive secondary structure that is increased in hydrophobic proteins

Pace

Huyghues-Despointes

et al. 2010

Protein Science

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The goal of this article is to gain a better understanding of the denatured state ensemble (DSE) of proteins through an experimental and computational study of their denaturation by urea. Proteins unfold to different extents in urea and the most hydrophobic proteins have the most compact DSE and contain almost as much secondary structure as folded proteins. Proteins that unfold to the greatest extent near pH 7 still contain substantial amounts of secondary structure. At low pH, the DSE expands due to charge-charge interactions and when the net charge per residue is high, most of the secondary structure is disrupted. The proteins in the DSE appear to contain substantial amounts of polyproline II conformation at high urea concentrations. In all cases considered, including staph nuclease, the extent of unfolding by urea can be accounted for using the data and approach developed in the laboratory of Wayne Bolen (Auton et al., Proc Natl Acad Sci 2007; 104:15317-15323).

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Improved Structural Characterizations of the drkN SH3 Domain Unfolded State Suggest a Compact Ensemble with Native-like and Non-native Structure

Cited by 111 publications

References 58 publications

Structure and Dynamics of the Aβ_21–30 Peptide from the Interplay of NMR Experiments and Molecular Simulations

Structure and Dynamics of the Aβ_21–30 Peptide from the Interplay of NMR Experiments and Molecular Simulations

Intrinsically disordered γ-subunit of cGMP phosphodiesterase encodes functionally relevant transient secondary and tertiary structure

Urea denatured state ensembles contain extensive secondary structure that is increased in hydrophobic proteins

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Improved Structural Characterizations of the drkN SH3 Domain Unfolded State Suggest a Compact Ensemble with Native-like and Non-native Structure

Cited by 111 publications

References 58 publications

Structure and Dynamics of the Aβ21–30 Peptide from the Interplay of NMR Experiments and Molecular Simulations

Structure and Dynamics of the Aβ21–30 Peptide from the Interplay of NMR Experiments and Molecular Simulations

Intrinsically disordered γ-subunit of cGMP phosphodiesterase encodes functionally relevant transient secondary and tertiary structure

Urea denatured state ensembles contain extensive secondary structure that is increased in hydrophobic proteins

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Structure and Dynamics of the Aβ_21–30 Peptide from the Interplay of NMR Experiments and Molecular Simulations

Structure and Dynamics of the Aβ_21–30 Peptide from the Interplay of NMR Experiments and Molecular Simulations