Fast Compaction of α-Lactalbumin During Folding Studied by Stopped-flow X-ray Scattering

Arai, Munehito; Ito, Kazuki; Inobe, Tomonao; Nakao, Masaharu; Maki, Kosuke; Kamagata, Kiyoto; Kihara, Hiroshi; Amemiya, Yoshiyuki; Kuwajima, Kunihiro

doi:10.1016/s0022-2836(02)00566-1

Cited by 101 publications

(111 citation statements)

References 51 publications

Supporting

Mentioning

103

Contrasting

Unclassified

Order By: Relevance

“…Scattering patterns were recorded by a CCD-based x-ray detector, which consisted of a beryllium-windowed x-ray image intensifier (Be-XRII) (V5445P-MOD, Hamamatsu Photonics, Hamamatsu, Japan), an optical lens, a CCD image sensor, and a data acquisition system (C7300, Hamamatsu Photonics), as described previously (32,33). The experimental details and the analyses of the scattering data were as described (32). Pair distribution (P(r)) functions were calculated using the GNOM program (34).…”

Section: Methodsmentioning

confidence: 99%

Sequential Action of ATP-dependent Subunit Conformational Change and Interaction between Helical Protrusions in the Closure of the Built-in Lid of Group II Chaperonins

Kanzaki

Iizuka

Takahashi

et al. 2008

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

ATP drives the conformational change of the group II chaperonin from the open lid substrate-binding conformation to the closed lid conformation to encapsulate an unfolded protein in the central cavity. The detailed mechanism of this conformational change remains unknown. To elucidate the intra-ring cooperative action of subunits for the conformational change, we constructed Thermococcus chaperonin complexes containing mutant subunits in an ordered manner and examined their folding and conformational change abilities. Chaperonin complexes containing wild-type subunits and mutant subunits with impaired ATP-dependent conformational change ability or ATP hydrolysis activity, one by one, exhibited high protein refolding ability. The effects of the mutant subunits correlate with the number and order in the ring. In contrast, the use of a mutant lacking helical protrusion severely affected the function. Interestingly, these mutant chaperonin complexes also exhibited ATP-dependent conformational changes as demonstrated by small angle x-ray scattering, protease digestion, and changes in fluorescence of the fluorophore attached to the tip of the helical protrusion. However, their conformational change is likely to be transient. They captured denatured proteins even in the presence of ATP, whereas addition of ATP impaired the ability of the wild-type chaperonin to protect citrate synthase from thermal aggregation. These results suggest that ATP binding/ hydrolysis causes the independent conformational change of the subunit, and further conformational change for the complete closure of the lid is induced and stabilized by the interaction between helical protrusions.Chaperonins, a ubiquitous class of molecular chaperones, are double-ring assemblies of about 60-kDa subunits. Each ring has a large central cavity in which a non-native protein can undergo productive folding in an ATP-dependent manner (1, 2). Chaperonin complexes change their conformations to close their chamber and encapsulate the bound substrate, providing a protected environment for protein folding. Opening and closing of the folding chamber is controlled by a conformational cycle driven by ATP binding and hydrolysis. Chaperonins are divided in two groups as follows: group I and group II chaperonins. Group I chaperonins are found in bacteria and endosymbiotic organelles (mitochondria and chloroplasts), and subsets of bacterially related homologs in methanogens (3). On the other hand, group II chaperonins are found in archaea (known as thermosome) (4) and the eukaryotic cytosol (chaperonin-containing t-complex polypeptide-1 or TCP-1 ring complex) (1, 5). Whereas group I chaperonins are homo-oligomeric, eukaryotic and archaeal group II chaperonins are generally hetero-oligomeric (6). All chaperonins share a similar subunit architecture *

show abstract

Section: Methodsmentioning

confidence: 99%

Sequential Action of ATP-dependent Subunit Conformational Change and Interaction between Helical Protrusions in the Closure of the Built-in Lid of Group II Chaperonins

Kanzaki

Iizuka

Takahashi

et al. 2008

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…The experimental details and the analysis of the scattering data were essentially the same as described earlier (14). The sample solution in 1-mm path length mica-windowed cell was exposed to a monochromatic x-ray beam (1.5 Å).…”

Section: Methodsmentioning

confidence: 99%

“…The temperature of the sample solution was maintained at 25°C using a circulating water bath. Scattering patterns were recorded by a CCD-based x-ray detector, which consisted of a beryllium-windowed x-ray image intensifier (Be-XRII) (Hamamatsu, V5445P-MOD), an optical lens, a CCD image sensor, and a data acquisition system (Hamamatsu C7300) (14,15). The camera length was set to be 1485 or 2375 mm depending on the scattering-angle region to be observed.…”

Section: Methodsmentioning

confidence: 99%

Oligomeric Hsp33 with Enhanced Chaperone Activity

Akhtar

Srinivas

Raman

et al. 2004

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Hsp33, an Escherichia coli cytosolic chaperone, is inactive under normal conditions but becomes active upon oxidative stress. It was previously shown to dimerize upon activation in a concentration-and temperature-dependent manner. This dimer was thought to bind to aggregation-prone target proteins, preventing their aggregation. In the present study, we report small angle x-ray scattering (SAXS), steady state and time-resolved fluorescence, gel filtration, and glutaraldehyde cross-linking analysis of full-length Hsp33. Our circular dichroism and fluorescence results show that there are significant structural changes in oxidized Hsp33 at different temperatures. SAXS, gel filtration, and glutaraldehyde cross-linking results indicate, in addition to the dimers, the presence of oligomeric species. Oxidation in the presence of physiological salt concentration leads to significant increases in the oligomer population. Our results further show that under conditions that mimic the crowded milieu of the cytosol, oxidized Hsp33 exists predominantly as an oligomeric species. Interestingly, chaperone activity studies show that the oligomeric species is much more efficient compared with the dimers in preventing aggregation of target proteins. Taken together, these results indicate that in the cell, Hsp33 undergoes conformational and quaternary structural changes leading to the formation of oligomeric species in response to oxidative stress. Oligomeric Hsp33 thus might be physiologically relevant under oxidative stress.Oxidative stress generated by the production of reactive oxygen species is an inevitable consequence of aerobic life. In addition, the extracellular environment can also cause oxidative stress to the cells (1, 2). Heat shock is shown to increase oxidative stress (3). Oxidative stress, inter alia, results in misfolding and aggregation of several cellular proteins. In order to combat oxidative stress, cells produce proteins that detoxify reactive oxygen species and repair harmful changes. The Escherichia coli cytoplasm, like that of most other organisms, is largely reducing in nature although under oxidative stress, the redox status of the cell changes significantly. Several cellular proteins are regulated by redox-dependent post-translational modifications (4).Until recently, no molecular chaperone was known that could be turned on or off by a post-translational event acting as a switch. Jakob et al. (5) showed that a member of the highly conserved cytoplasmic prokaryotic molecular chaperones, Hsp33, is regulated by the redox state of the medium. Under reducing conditions, Hsp33 is inactive. Hsp33 has four absolutely conserved cysteine residues: Cys-232, -234, -265, and -268. In the reduced state, they are coordinated to a zinc atom (6). Oxidizing conditions lead to the release of zinc and formation of two intramolecular disulfide bonds, Cys-232 with Cys-234 and Cys-265 with Cys-268 (7). Upon such oxidative activation, Hsp33 binds to aggregation-prone states of proteins, preventing their aggregation and acting as...

show abstract

“…I(0) decreases to the native level in the first phase, which suggests a decrease in the amount of hydrated waters in the transition from I 1 to I 2 (12,(35)(36)(37). I 2 possesses partial tertiary contacts demonstrated by the specific binding site for ANS and the increased Trp-4 fluorescence.…”

Section: L-shaped Conformational Landscape Of Smnmentioning

confidence: 98%

Specific collapse followed by slow hydrogen-bond formation of β-sheet in the folding of single-chain monellin

Kimura

Uzawa

Ishimori

et al. 2005

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

124

View full text Add to dashboard Cite

Characterization of the conformational landscapes for proteins with different secondary structures is important in elucidating the mechanism of protein folding. The folding trajectory of singlechain monellin composed of a five-stranded ␤-sheet and a helix was investigated by using a pH-jump from the alkaline unfolded to native state. The kinetic changes in the secondary structures and in the overall size and shape were measured by circular dichroism spectroscopy and small-angle x-ray scattering, respectively. The formation of the tertiary structure was monitored by intrinsic and extrinsic fluorescence. A significant collapse was observed within 300 s after the pH-jump, leading to the intermediate with a small amount of secondary and tertiary structures but with an overall oblate shape. Subsequently, the stepwise formation of secondary and tertiary structures was detected. The current observation was consistent with the theoretical prediction that a more significant collapse precedes the formation of secondary structures in the folding of ␤-sheet proteins than that of helical proteins [Shea, J. E., Onuchic, J. N. & Brooks, C. L., III (2002) Proc. Natl. Acad. Sci. USA 99, 16064 -16068]. Furthermore, it was implied that the initial collapse was promoted by the formation of some specific structural elements, such as tight turns, to form the oblate shape.energy landscape ͉ protein folding ͉ submillisecond dynamics ͉ x-ray scattering C haracterization of the folding dynamics for proteins and their free energy landscapes offers a key to understanding protein folding phenomena (1, 2). The coarse-grained approximation of intraprotein interactions (a ''funneled'' landscape) has been proposed (3, 4) and has succeeded in explaining the structures of proteins in the folding transition state (5-8). However, the molecular basis of the funneled landscape, which is realized by a complex interplay of various intraprotein interactions and polypeptide dynamics, is largely unknown. For example, although the hydrophobic interaction and hydrogen bond (H-bond) are the major stabilizing interactions for the specific folded conformations of proteins, their contribution to the energy landscape, including the unfolded conformations, is controversial (9). Furthermore, the relationship between protein folds and the characteristics of landscapes has not been well investigated. Therefore, the observation of the entire folding process of proteins with various folds is required to characterize the free energy landscapes.We previously observed the folding dynamics of ␣-helical proteins, cytochrome c (cyt c) and apomyoglobin (apoMb), based on helical content and compactness, which mainly reflect the formation of the H-bonds and hydrophobic interactions, respectively (10-12). The characterized landscapes of these proteins demonstrated the cooperative acquisition of the helical structure and compactness after the initial collapse and suggested that the hydrophobic environment created by the collapse facilitates the subsequent conformational search. ...

show abstract

Fast Compaction of α-Lactalbumin During Folding Studied by Stopped-flow X-ray Scattering

Cited by 101 publications

References 51 publications

Sequential Action of ATP-dependent Subunit Conformational Change and Interaction between Helical Protrusions in the Closure of the Built-in Lid of Group II Chaperonins

Sequential Action of ATP-dependent Subunit Conformational Change and Interaction between Helical Protrusions in the Closure of the Built-in Lid of Group II Chaperonins

Oligomeric Hsp33 with Enhanced Chaperone Activity

Specific collapse followed by slow hydrogen-bond formation of β-sheet in the folding of single-chain monellin

Contact Info

Product

Resources

About