Characterizing Protein Folding Transition States Using Ψ-Analysis

Pandit, Adarsh; Krantz, Bryan A.; Dothager, Robin S.; Sosnick, Tobin R.

doi:10.1385/1-59745-189-4:83

Cited by 9 publications

(19 citation statements)

References 34 publications

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“…The dissociation constant is expected to change from K U Zn ϭ 180 nM in the unfolded state to K F Zn ϭ 1.8 nM in the folded monomer. Similar, folding-induced changes in Zn 2ϩ affinity have been reported for azurin (22) and, more generally, for mutational insertion of His pairs to probe folding transition states (23). As a control of Zn 2ϩ binding to folded SOD1 mut we measured K F Zn directly by ITC, yielding a value of Ϸ13 nM (Fig.…”

Section: Formal Relationship Between Metal-binding and Protein Stabilsupporting

confidence: 61%

Functional features cause misfolding of the ALS-provoking enzyme SOD1

Nordlund

Leinartaité

Saraboji

et al. 2009

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

105

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The structural integrity of the ubiquitous enzyme superoxide dismutase (SOD1) relies critically on the correct coordination of Cu and Zn. Loss of these cofactors not only promotes SOD1 aggregation in vitro but also seems to be a key prerequisite for pathogenic misfolding in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). We examine here the consequences of Zn 2؉ loss by selectively removing the Zn site, which has been implicated as the main modulator of SOD1 stability and disease competence. After Zn-site removal, the remaining Cu ligands can coordinate a nonnative Zn 2؉ ion with M affinity in the denatured state, and then retain this ion throughout the folding reaction. Without the restriction of a metallated Zn site, however, the Cu ligands fail to correctly coordinate the nonnative Zn 2؉ ion: Trapping of a water molecule causes H48 to change rotamer and swing outwards. The misligation is sterically incompatible with the native structure. As a consequence, SOD1 unfolds locally and interacts with neighboring molecules in the crystal lattice. The findings point to a critical role for the native Zn site in controlling SOD1 misfolding, and show that even subtle changes of the metal-loading sequence can render the wild-type protein the same structural properties as ALS-provoking mutations. This frustrated character of the SOD1 molecule seems to arise from a compromise between optimization of functional and structural features. functional evolution ͉ protein disease ͉ protein misfolding

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Section: Formal Relationship Between Metal-binding and Protein Stabilsupporting

confidence: 61%

Functional features cause misfolding of the ALS-provoking enzyme SOD1

Nordlund

Leinartaité

Saraboji

et al. 2009

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

105

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“…A related ψ value analysis method, although much less used, is more definitive. It finds the same distinct partially formed native-like structure for the entire folding population (36). These results favor the distinct pathway hypothesis.…”

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confidence: 71%

The nature of protein folding pathways

Englander

Mayne

2014

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

322

368

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How do proteins fold, and why do they fold in that way? This Perspective integrates earlier and more recent advances over the 50-y history of the protein folding problem, emphasizing unambiguously clear structural information. Experimental results show that, contrary to prior belief, proteins are multistate rather than two-state objects. They are composed of separately cooperative foldon building blocks that can be seen to repeatedly unfold and refold as units even under native conditions. Similarly, foldons are lost as units when proteins are destabilized to produce partially unfolded equilibrium molten globules. In kinetic folding, the inherently cooperative nature of foldons predisposes the thermally driven amino acid-level search to form an initial foldon and subsequent foldons in later assisted searches. The small size of foldon units, ∼20 residues, resolves the Levinthal time-scale search problem. These microscopic-level search processes can be identified with the disordered multitrack search envisioned in the "new view" model for protein folding. Emergent macroscopic foldon-foldon interactions then collectively provide the structural guidance and free energy bias for the ordered addition of foldons in a stepwise pathway that sequentially builds the native protein. These conclusions reconcile the seemingly opposed new view and defined pathway models; the two models account for different stages of the protein folding process. Additionally, these observations answer the "how" and the "why" questions. The protein folding pathway depends on the same foldon units and foldon-foldon interactions that construct the native structure.protein folding | hydrogen exchange | protein structure

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“…32,50 Secondly, many TSEs contain a high fraction of the native structure and topology (e.g., the TSE has $70% of the native state's relative contact order). [20][21][22]32,51 This high level eliminates many otherwise possible transition state structures, and further reduces the amount of heterogeneity.…”

Section: Resultsmentioning

confidence: 99%

“…[1][2][3][4][5][6] This divergence persists in part because it is very difficult to measure this quantity experimentally, especially for proteins that fold in a two-state manner. [7][8][9][10][11][12][13][14] For example, the primary methods for determining transition state structure, f [15][16][17] and w analyses, [18][19][20][21][22][23] suffer from ambiguities when the measured values are fractional, that is, not 0 or 1.…”

Section: Introductionmentioning

confidence: 99%

A “Link‐Psi” strategy using crosslinking indicates that the folding transition state of ubiquitin is not very malleable

2012

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Using a combined crosslinking-w analysis strategy, we examine whether the structural content of the transition state of ubiquitin can be altered. A synthetic dichloroacetone crosslink is first introduced across two b strands. Whether the structural content in the transition state ensemble has shifted towards the region containing the crosslink is probed by remeasuring the w value at another region (w identifies the degree to which an inserted bi-Histidine metal ion binding site is formed in the transition state). For sites around the periphery of the obligate transition state nucleus, we find that the resulting changes in w values are near or at our detection limit, thereby indicating that the structural content of the transition state has not measurably changed upon crosslinking. This work demonstrates the utility of the simultaneous application of crosslinking and w-analysis for examining potential transition state heterogeneity in globular proteins.

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Characterizing Protein Folding Transition States Using Ψ-Analysis

Cited by 9 publications

References 34 publications

Functional features cause misfolding of the ALS-provoking enzyme SOD1

Functional features cause misfolding of the ALS-provoking enzyme SOD1

The nature of protein folding pathways

A “Link‐Psi” strategy using crosslinking indicates that the folding transition state of ubiquitin is not very malleable

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