Structural Analysis of the Rate-limiting Transition States in the Folding of Im7 and Im9: Similarities and Differences in the Folding of Homologous Proteins

Friel, Claire T.; Capaldi, Andrew P.; Radford, Sheena E.

doi:10.1016/s0022-2836(02)01249-4

Cited by 127 publications

(170 citation statements)

References 35 publications

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“…2A encodes the fractional helicity and order parameters of the I state on the structure of N, with increasing tube thickness corresponding to higher helical character. It is clear that a region extending from the C-terminal residues of helix II through helix III is not formed in the I state, with H47 situated in the intervening loop, consistent with results from other measurements (47).…”

Section: Discussionsupporting

confidence: 90%

“…Previous φ value analysis studies by Radford and coworkers have established that the nonnative interactions stabilizing the intermediate are disrupted in the final, rate-limiting TS, after which helix III and related native contacts form, locking the structure into its native conformation (47). Our work highlights the changes that occur at the level of H40 and H47 that are conserved across a range of Im proteins, showing that the imidazole side chains of these residues undergo an exposed-to-ordered transition that leads to stabilization of the resulting native state.…”

Section: Discussionmentioning

confidence: 99%

“…The methodology is used to study the folding reaction of the 87-residue four-helix bundle Im7 protein, which proceeds through the formation of an on-pathway intermediate (45,46). The kinetics and thermodynamics of folding have been studied extensively with tryptophan fluorescence, equilibrium denaturation and φ value analysis (29,(47)(48)(49) and it has been established that the stability of the intermediate relative to the native state increases with decreasing pH over a range where histidine side chains typically titrate (50). However, the inability to measure site-specific electrostatic interactions has limited an atomic-level understanding of the role(s) of the histidine residues in the folding process.…”

mentioning

confidence: 99%

“…The Im7 protein folds via an on-pathway intermediate to the native conformation, a process that has been studied in detail previously using a variety of biophysical methods (29,(45)(46)(47)(48)(49), including NMR spectroscopy (48,(68)(69)(70). CPMG relaxation dispersion and CEST measurements performed in our laboratory at 10°C, pH 6.6 can be well analyzed assuming a two-state exchange mechanism, I !…”

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confidence: 99%

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Measurement of histidine pK_avalues and tautomer populations in invisible protein states

Hansen

Kay

2014

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

120

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The histidine imidazole side chain plays a critical role in protein function and stability. Its importance for catalysis is underscored by the fact that histidines are localized to active sites in ∼50% of all enzymes. NMR spectroscopy has become an important tool for studies of histidine side chains through the measurement of sitespecific pK a s and tautomer populations. To date, such studies have been confined to observable protein ground states; however, a complete understanding of the role of histidine electrostatics in protein function and stability requires that similar investigations be extended to rare, transiently formed conformers that populate the energy landscape, yet are often "invisible" in standard NMR spectra. Here we present NMR experiments and a simple strategy for studies of such conformationally excited states based on measurement of histidine 13 C γ , 13 C δ2 chemical shifts and 1 H e -13 C e onebond scalar couplings. The methodology is first validated and then used to obtain pK a values and tautomer distributions for histidine residues of an invisible on-pathway folding intermediate of the colicin E7 immunity protein. Our results imply that the side chains of H40 and H47 are exposed in the intermediate state and undergo significant conformational rearrangements during folding to the native structure. Further, the pK a values explain the pH-dependent stability differences between native and intermediate states over the pH range 5.5-6.5 and they suggest that imidazole deprotonation is not a barrier to the folding of this protein.

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Section: Discussionsupporting

confidence: 90%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Measurement of histidine pK_avalues and tautomer populations in invisible protein states

Hansen

Kay

2014

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

120

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“…Especially the recent identification of the ubiquity of intrinsically disordered proteins (29,30) raises the possibility that the protein structures at the time of gene duplication and speciation were not so rigid as has usually been considered normal for the existing natural proteins. Coevolution of function and structure under slightly different selection pressures acting on a protein family with such disordered structures would lead to the examples of the present-day proteins with similar native structures but with partially different folding cores (7,(31)(32)(33). It should be also noted that the frequent involvement of the functionally important site in folding cores (8,21,22) can be interpreted as a natural consequence of the functional selection in which the structure around the functional site is early developed in the evolutionary history.…”

Section: Resultsmentioning

confidence: 99%

Correlation between evolutionary structural development and protein folding

Nagao

Terada

Yomo

et al. 2005

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

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Evolution should have played important roles in determining folding mechanisms and structures of proteins. In this article we discuss how the folding mechanisms had been affected by the early stage of evolution through which the uniqueness of structure had developed. Although the process of such early-time evolution has remained a mystery, a plausible scenario is that the evolution of proteins toward the ordered structures was guided by functional selection pressure as demonstrated in vitro and in silico. We examine the in silico functional selection of sequences and show that there is a significant correlation between two different processes toward the unique 3D structure, the evolutionary development of structure through sequence selection, and the folding process of the resultant sequence. This finding could be rephrased as protein folding recapitulates the emergence of topology in the molecular evolution. The correlation suggests a guideline for engineering foldable proteins.functional selection ͉ molecular evolution ͉ superfunnel ͉ folding mechanism ͉ energy landscape E rnst Haeckel's statement that ontogeny recapitulates phylogeny, or the embryonic developmental process is a repeat of the evolutionary process, has been a subject of intense intellectual argument since the 19th century (1). Although his claim has been regarded as incorrect in its literal sense, comparison of the developmental process and the evolutionary history has been productive as witnessed in the recent advancement in evolutionary developmental biology or ''evo-devo.'' The early stages of development are typically conserved among the evolutionarily related species, whereas the later stages tend to differ (2-4). Such a correlation between the developmental and evolutionary processes should be based on the bottom-up features of these structure formation processes, which are typical in natural organisms but are rare in artifacts. Both processes share the feature that the cumulative effects of small modifications in the local rules within each cell and between cells progressively brings about the intricate balance among the local rules found within the present-day organisms.Here, we should point out that such local and cumulative features of the structural organization are not limited to embryonic development but are ubiquitous in cellular and molecular biological structural formation processes. In this article, we take protein folding as an example of such a biological structure formation process. Proteins fold to unique native structures according to the evolutionarily designed interactions among amino acid residues. Again, both the folding process and the evolutionary changes of sequences are cumulative processes of local changes, and thus we may be able to expect the correlation between these processes. The intrinsic relationships between the evolutionary history and the folding mechanisms have been suggested by the similarities and differences among folding mechanisms of multiple proteins (5-9). Amino acid sequence is conserved at the ...

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Overview of Protein Folding Mechanisms: Experimental and Theoretical Approaches to Probing Energy Landscapes

Morris

Searle

2012

CP Protein Science

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We present an overview of the current experimental and theoretical approaches to studying protein folding mechanisms, set against current models of the folding energy landscape. We describe how stability and folding kinetics can be determined experimentally and how this data can be interpreted in terms of the characteristic features of various models from the simplest two-state pathway to a multi-state mechanism. We summarize the pros and cons of a range of spectroscopic methods for measuring folding rates and present a theoretical framework, coupled with protein engineering approaches, for elucidating folding mechanisms and structural features of folding transition states. A series of case studies are used to show how experimental kinetic data can be interpreted in the context of non-native interactions, populated intermediates, parallel folding pathways, and sequential transition states. We also show how computational methods now allow transient species of high energy, such as folding transition states, to be modeled on the basis of experimental Φ-value analysis derived from the effects of point mutations on folding kinetics.

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Structural Analysis of the Rate-limiting Transition States in the Folding of Im7 and Im9: Similarities and Differences in the Folding of Homologous Proteins

Cited by 127 publications

References 35 publications

Measurement of histidine pK_avalues and tautomer populations in invisible protein states

Measurement of histidine pK_avalues and tautomer populations in invisible protein states

Correlation between evolutionary structural development and protein folding

Overview of Protein Folding Mechanisms: Experimental and Theoretical Approaches to Probing Energy Landscapes

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Structural Analysis of the Rate-limiting Transition States in the Folding of Im7 and Im9: Similarities and Differences in the Folding of Homologous Proteins

Cited by 127 publications

References 35 publications

Measurement of histidine pKavalues and tautomer populations in invisible protein states

Measurement of histidine pKavalues and tautomer populations in invisible protein states

Correlation between evolutionary structural development and protein folding

Overview of Protein Folding Mechanisms: Experimental and Theoretical Approaches to Probing Energy Landscapes

Contact Info

Product

Resources

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Measurement of histidine pK_avalues and tautomer populations in invisible protein states

Measurement of histidine pK_avalues and tautomer populations in invisible protein states