Trapping the On-pathway Folding Intermediate of Im7 at Equilibrium

Spence, Graham R.; Capaldi, Andrew P.; Radford, Sheena E.

doi:10.1016/j.jmb.2004.05.049

Cited by 42 publications

(90 citation statements)

References 49 publications

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“…Kinetic folding experiments on experimentally designed IM7 mutants have shown that the populations of the native and intermediate states can be shifted significantly with single-and double-residue mutations (14). Based on our site-specific localization of frustration, we devised two strategies to redesign the folding landscape.…”

Section: Resultsmentioning

confidence: 99%

Consequences of localized frustration for the folding mechanism of the IM7 protein

Sutto

Lätzer²,

Hegler³

et al. 2007

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

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In the laboratory, IM7 has been found to have an unusual folding mechanism in which an ''on-pathway'' intermediate with nonnative interactions is formed. We show that this intermediate is a consequence of an unusual cluster of highly frustrated interactions in the native structure. This cluster is involved in the binding of IM7 to its target, Colicin E7. Redesign of residues in this cluster to eliminate frustration is predicted by simulations to lead to faster folding without the population of an intermediate ensemble.computational design ͉ folding landscape ͉ nonnative intermediate F or proteins to fold to an organized native ensemble, they have evolved to form a highly favorable, stabilizing set of native interactions (1). Proteins, it seems, have a funneled energy landscape toward the native state (1-3). However, real protein landscapes may not be perfectly funneled but probably display some landscape ruggedness because of the possibility of forming favorable but nonnative interactions during the folding process. Energy landscape theory shows that, if the landscape were too rugged, numerous long-lived intermediate states that energetically compete with the native state would become populated (4). The rarity of the experimental finding of such intermediates having significant nonnative structure strongly argues that proteins largely conform to the ''principle of minimal frustration'' (2).When populated kinetic folding intermediates have been observed, it has generally turned out to be the case that these metastable thermodynamic states are not caused by energetic frustration but by a nonuniform compensation of the entropy and energy changes upon forming native contacts. The existence of such productive, on route, folding intermediates can be said to be topology-driven and can often be predicted from the native structure alone by using a perfectly funneled landscape (5-8).IM7 is an 86-residue-long protein that is known to fold through an on-pathway intermediate state. An intermediate populated in the folding of IM7 has been shown to possess a nonnative packing of three of the four helices around a specific hydrophobic core (9). In this article, we explore the relationship between nonnative interactions, the presence of the stable folding intermediate, and the residual frustration found in the native structure. Our analysis suggests that this seeming exception to the pattern expected for minimally frustrated proteins is, in fact, consistent with the ideas of energy landscape theory touching on the behavior of frustrated, random heteropolymers.We first demonstrate that the intermediate of IM7 is not a direct consequence of the topology of IM7 by simulating its folding with a sequence-independent energy function that yields a perfectly funneled landscape [a so-called Go Hamiltonian (10) Capaldi et al. (9), who inferred the presence of a three-helix core of helices I, II, and IV. We note that this ensemble has numerous native-like interactions but also is stabilized by some nonnative interactions.Equipped with an ...

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Section: Resultsmentioning

confidence: 99%

Consequences of localized frustration for the folding mechanism of the IM7 protein

Sutto

Lätzer²,

Hegler³

et al. 2007

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

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“…This results in the population of a hyperfluorescent partially folded species at equilibrium ( Figure 1B) that closely resembles the kinetic intermediate species in many of its properties (13). In the variant H3G6, the six residues that comprise helix III in native Im7* are substituted with (Gly) 6 , also preventing formation of the native state.…”

Section: Uvrr Indicates That the Environment Local To Trp75 Is Non-namentioning

confidence: 99%

“…Spectra were acquired in 5 min and normalized using the sulfate band at 981 cm -1 after baseline correction. The protein concentration was adjusted to 150 µM using the corresponding extinction coefficient determined at 280 nm for each protein (13). marized in Table 1.…”

Section: A Comparison Of the Solution Structures Of Im7* And Im9* By mentioning

confidence: 99%

“…Interestingly, however, the fluorescence intensity of Trp75 in these variants differs ( Figure 1B). Thus, while the fluorescence intensity of Trp75 in the folded states of L53AI54A and YY is hyperfluorescent (being greater than that of their respective unfolded states), akin to the properties of the kinetic folding intermediate of wild-type Im7* (4-6), the folded and unfolded states of the variant H3G6 ( Figure 1B) have equal intensity (13). A major unresolved question about these proteins, therefore, is the origin of their hyperfluorescence and the nature of the side chain interactions responsible for tailoring the fluorescence of Trp75 in these species.…”

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confidence: 99%

“…Using 229 nm excitation (10), the tryptophan and tyrosine Raman bands are selectively enhanced (11,12), enabling UVRR spectroscopy to provide structural and environmental information concerning the side chains of these aromatic residues. Here, we report UVRR studies of the native structures of Im7* and Im9*, together with three variants of Im7* [termed L53AI54A, H3G6, and YY (inset of Figure 1A)], that trap a partially folded state of Im7* at equilibrium by preventing the binding of helix III to the developing structure (13). These mutations selectively destabilize native Im7* such that the on-pathway folding intermediate becomes the ground state of folding in the absence of a denaturant.…”

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confidence: 99%

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Ultraviolet Resonance Raman Studies Reveal the Environment of Tryptophan and Tyrosine Residues in the Native and Partially Folded States of the E Colicin-Binding Immunity Protein Im7

et al. 2005

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Understanding the nature of partially folded proteins is a challenging task that is best accomplished when several techniques are applied in combination. Here we present ultraviolet resonance Raman (UVRR) spectroscopy studies of the E colicin-binding immunity proteins, Im7* and Im9*, together with a series of variants of Im7* that are designed to trap a partially folded state at equilibrium. We show that the environments of the tryptophan and tyrosine residues in native wild-type Im7* and Im9* are indistinguishable, in contrast with models for their structures based on X-ray and NMR methods. In addition, we show that there is a general increase in the hydrophobicity in the environment of Trp75 in all of the variants compared with wild-type Im7*. These data suggest that a significant rearrangement of the tryptophan pocket occurs in the variants, which, together with an overall decrease in solvent accessibility of Trp75 as judged by time-resolved fluorescence lifetime measurements and fluorescence quenching experiments, rationalize the unusual fluorescence properties of the variants reported previously. The data highlight the power of UVRR in analyzing the structural properties of different conformational states of the same protein and reveal new information about the structural rearrangements occurring during Im7* folding, not possible using other spectroscopic methods alone. Finally, we describe a previously unreported dependence of the tryptophan Fermi doublet on excitation wavelength in the ultraviolet region revealed by these protein spectra. We corroborated this observation using tryptophan-containing model compounds and conclude that the conventional interpretation of this UVRR feature at these wavelengths is unreliable.The biological role of the E colicin-binding immunity proteins Im2, Im7, Im8, and Im9 is to protect the host cell from the toxic endonuclease activity of a coexpressed cognate colicin, by binding to an exosite in the DNase domain, inhibiting access of the substrate DNA to the active site of the colicin by steric hindrance and electrostatic repulsion (1, 2). The small size of the immunity proteins (85-87 amino acid residues), their high level of sequence identity (approximately 60%), the single conserved tryptophan, and their lack of disulfide bonds, prosthetic groups, and cis-prolines in the native state make the immunity protein family ideal for protein folding studies. Despite the high degree of similarity between Im7 and Im9 in both sequence and structure, these proteins fold by mechanisms with different levels of kinetic complexity at neutral pH, suggesting that local sequence variations, rather than topology or global stability, are responsible for the observed folding kinetics of these proteins (3-7). Im7* 1 and Im9* (hexahistidinetagged analogues of Im7 and Im9) contain four helices ( Figure 1A), each of which displays polar side chains on the surface of the protein, while the majority of hydrophobic side chains are buried, forming a well-packed hydrophobic core. Im7* and Im...

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Chaperone–substrate interactions monitored via a robust TEM-1 β-lactamase fragment complementation assay

Bai

et al. 2017

Biotechnol Lett

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Trapping the On-pathway Folding Intermediate of Im7 at Equilibrium

Cited by 42 publications

References 49 publications

Consequences of localized frustration for the folding mechanism of the IM7 protein

Consequences of localized frustration for the folding mechanism of the IM7 protein

Ultraviolet Resonance Raman Studies Reveal the Environment of Tryptophan and Tyrosine Residues in the Native and Partially Folded States of the E Colicin-Binding Immunity Protein Im7

Chaperone–substrate interactions monitored via a robust TEM-1 β-lactamase fragment complementation assay

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