Solvation and Desolvation Dynamics in Apomyoglobin Folding Monitored by Time-resolved Infrared Spectroscopy

Nishiguchi, Shingo; Goto, Yuji; Takahashi, Satoshi

doi:10.1016/j.jmb.2007.08.003

Cited by 21 publications

(35 citation statements)

References 77 publications

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“…4), In support, Δω I1;MG is large for both 15 N and 1 H (1.0 and 0.5 ppm, respectively). Recent studies of apoMb folding and unfolding kinetics (26,27) suggest that the initial event during unfolding and the final event during refolding involve solvation and desolvation, respectively. Indeed, it has been suggested that desolvation of backbone amides may contribute to the energy barrier in the final ratelimiting step of the folding pathway (27).…”

Section: Discussionmentioning

confidence: 99%

“…Recent studies of apoMb folding and unfolding kinetics (26,27) suggest that the initial event during unfolding and the final event during refolding involve solvation and desolvation, respectively. Indeed, it has been suggested that desolvation of backbone amides may contribute to the energy barrier in the final ratelimiting step of the folding pathway (27). Although our data do not directly probe solvation effects, it is likely that the N → I1 transition, driven by the undocking of the F helix, is accompanied by substantial hydration of the empty heme-binding pocket.…”

Section: Discussionmentioning

confidence: 99%

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Measurement of protein unfolding/refolding kinetics and structural characterization of hidden intermediates by NMR relaxation dispersion

Meinhold

Wright

2011

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

112

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Detailed understanding of protein function and malfunction hinges on the ability to characterize transiently populated states and the transitions between them. Here, we use 15 N, 1 H N , and 13 CO NMR R 2 relaxation dispersion to investigate spontaneous unfolding and refolding events of native apomyoglobin. Above pH 5.0, dispersion is dominated by processes involving fluctuations of the F-helix region, which is invisible in NMR spectra. Measurements of R 2 dispersion for residues contacted by the F-helix region in the native (N W ithin the cell, unfolding and refolding of proteins occurs constantly, and spontaneous unfolding and misfolding processes play a central role in the formation of amyloid fibrils (1). In addition, fluctuations between native protein structure and partially or fully unfolded states have functional significance for binding, allosteric regulation, translocation across membranes, protein trafficking, secretion, and degradation (2). Despite the importance of spontaneous unfolding for protein function and cellular proteostasis, little is known about the transient, partially unfolded states that are formed. Detailed structural characterization of such states is difficult because of their inherently low populations and the conformational heterogeneity present when both native and partially unfolded states simultaneously exist in solution.Carr-Purcell-Meiboom-Gill (CPMG)-based R 2 relaxation dispersion experiments are unique in their ability to measure the kinetics of microsecond-millisecond exchange processes involving transient states with populations as sparse as 1%, and, in addition, allow determination of the associated NMR chemical shift changes (Δω) (3-5). In these experiments, the effective transverse relaxation rate R eff 2 is deconvoluted into the contribution from the exchange process, R ex , which varies with the pulsing frequency in the CPMG refocusing element, and the intrinsic relaxation rate R 0 2 . Here, we apply a series of amide and carbonyl R 2 dispersion experiments to investigate transient unfolding and refolding events within the native (N) state freeenergy landscape of apomyoglobin (apoMb).ApoMb is an ideal model system for studies of protein folding/ unfolding equilibria. The folded state is marginally stable at neutral pH, adopting a similar tertiary structure to the holoprotein except for disorder in the EF loop, F helix, FG loop, and N-terminal region of the G helix (residues 82-104), which appear to fluctuate between a folded native-like conformational substate and an ensemble of locally unfolded states (6, 7). ApoMb forms an equilibrium molten globule (MG) state at pH 4.1, amenable to direct investigation by NMR, which contains a significant number of native contacts and an overall compactness close to that of the N state (8-10). Kinetic refolding experiments show rapid formation of a burst-phase intermediate that resembles the equilibrium MG and contains native-like topology in the most structured regions (10-12). Folding to the N state from the MG is the overall ra...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Measurement of protein unfolding/refolding kinetics and structural characterization of hidden intermediates by NMR relaxation dispersion

Meinhold

Wright

2011

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

112

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“…In contrast, main-chain amides and hydrophilic side chains in the unfolded polypeptides possess much stronger electrostatic and hydrogen-bonding interactions with water (5). It has been suggested that the dehydration of main-chain amides, required for the burial of main chains in the native proteins, should burden proteins with a significant energetic barrier for folding (5,14). Furthermore, a partial dehydration of amides by side chains is suggested to regulate the propensity for secondary structure formation (15,16).…”

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confidence: 99%

“…These suggestions might be related to the search phase of protein folding, which occurs after the collapse in the time domain from milliseconds to seconds and involves the formation of correct secondary and tertiary structures. A recent kinetic IR study on the collapsed folding intermediates of apomyoglobin (apoMb) identified the solvated helices that persist until the final folding phase and demonstrated the difficulty of detaching water around main-chain amides in an ␣-helical protein (14). In this study, we observed the folding of a ␤-sheet protein, single-chain monellin (SMN), using kinetic IR spectroscopy to examine whether the slow dehydration of main-chain amides is still observable or not and to establish the roles of the amide hydration in protein folding.…”

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confidence: 99%

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Dehydration of main-chain amides in the final folding step of single-chain monellin revealed by time-resolved infrared spectroscopy

Kimura

Maeda

Nishiguchi

et al. 2008

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

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Kinetic IR spectroscopy was used to reveal ␤-sheet formation and water expulsion in the folding of single-chain monellin (SMN) composed of a five-stranded ␤-sheet and an ␣-helix. The timeresolved IR spectra between 100 s and 10 s were analyzed based on two consecutive intermediates, I 1 and I2, appearing within 100 s and with a time constant of Ϸ100 ms, respectively. The initial unfolded state showed broad amide I corresponded to a fluctuating conformation. In contrast, I 1 possessed a feature at 1,636 cm ؊1 for solvated helix and weak features assignable to turns, demonstrating the rapid formation of helix and turns. I 2 possessed a line for solvated helix at 1,637 cm ؊1 and major and minor lines for ␤-sheet at 1,625 and 1,680 cm ؊1 , respectively. The splitting of the major and minor lines is smaller than that of the native state, implying an incomplete formation of the ␤-sheet. Furthermore, both major and minor lines demonstrated a low-frequency shift compared to those of the native state, which was interpreted to be caused by hydration of the CAO group in the ␤-sheet. Together with the identification of solvated helix, the core domain of I 2 was interpreted as being hydrated. Finally, slow conversion of the water-penetrated core of I 2 to the dehydrated core of the native state was observed. We propose that both the expulsion of water, hydrogen-bonded to main-chain amides, and the completion of the secondary structure formation contribute to the energetic barrier of the rate-limiting step in SMN folding.protein folding dynamics ͉ ␤-sheet

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Intrinsically Disordered Proteins and Induced Folding Studied by Fourier Transform Infrared Spectroscopy

Natalello

Doglia

2010

Instrumental Analysis of Intrinsically Disordered Proteins

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Solvation and Desolvation Dynamics in Apomyoglobin Folding Monitored by Time-resolved Infrared Spectroscopy

Cited by 21 publications

References 77 publications

Measurement of protein unfolding/refolding kinetics and structural characterization of hidden intermediates by NMR relaxation dispersion

Measurement of protein unfolding/refolding kinetics and structural characterization of hidden intermediates by NMR relaxation dispersion

Dehydration of main-chain amides in the final folding step of single-chain monellin revealed by time-resolved infrared spectroscopy

Intrinsically Disordered Proteins and Induced Folding Studied by Fourier Transform Infrared Spectroscopy

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