scholarly journals Binding of a burst-phase intermediate formed in the folding of denatured D-glyceraldehyde-3-phosphate dehydrogenase by chaperonin 60 and 8-anilino-1-naphthalenesulphonic acid

1998 ◽  
Vol 331 (2) ◽  
pp. 505-511 ◽  
Author(s):  
Xiao-Ling LI ◽  
Xiang-Dong LEI ◽  
Hui CAI ◽  
Jian LI ◽  
Sheng-Li YANG ◽  
...  
Keyword(s):  
Guanidine Hydrochloride ◽  
Time Lag ◽  
Folding Intermediate ◽  
Native Conformation ◽  
Unfolded State ◽  
Partially Folded Intermediate ◽  
Chaperonin 10 ◽  
Chaperonin 60 ◽  
Burst Phase ◽  
Partially Unfolded

Upon dilution, d-glyceraldehyde-3-phosphate dehydrogenase (GADPH) that has been fully inactivated, but only partially unfolded, in dilute guanidine hydrochloride (GuHCl) recovers activity completely. The fully unfolded enzyme, however, is re-activated only to a limited extent after dilution, and refolds rapidly in a burst phase to a partially folded intermediate characterized by increases in both the emission intensity of intrinsic fluorescence and binding to 8-anilino-1-naphthalenesulphonic acid (ANS). This intermediate aggregates with a time lag of a few minutes, and the aggregation can be suppressed completely by chaperonin 60 (GroEL). Stoichiometric analysis of the suppression of GAPDH re-activation by GroEL suggests that the tetradecameric GroEL binds to a dimeric GAPDH folding intermediate. This intermediate can be re-activated by ATP or ATP/chaperonin 10 (GroES) to an extent considerably greater than that obtained on spontaneous re-activation of the fully denatured enzyme upon dilution. Probing with a fluorescent derivative of NAD+ shows that this folding intermediate does not have a native conformation at the active site. The similar profiles of the effects of GroEL and ANS on the re-activation of GAPDH denatured by different concentrations of GuHCl suggest that GroEL and ANS recognize and bind to the same folding intermediate, which is similar to the relatively stable, partially unfolded, state of the enzyme denatured in 0.5–1.0 M GuHCl. However, the complexes of the intermediate with GroEL or ANS appear to be different, in that GroEL, but not ANS, suppresses aggregation and assists folding in the presence of ATP.

scholarly journals Spectroscopic Studies on Unfolding Processes of Apo-Neuroglobin Induced by Guanidine Hydrochloride and Urea

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Cui Zhang ◽  
Chaohui Gao ◽  
Jianshuai Mu ◽  
Zhanglei Qiu ◽  
Lianzhi Li
Keyword(s):  
Structural Transition ◽  
Guanidine Hydrochloride ◽  
Fluorescence Emission ◽  
Emission Spectra ◽  
Spectroscopic Studies ◽  
Native State ◽  
Fluorescence Emission Spectra ◽  
Unfolded State ◽  
Partially Unfolded ◽  
Far Ultraviolet

Neuroglobin (Ngb), a recently discovered globin, is predominantly expressed in the brain, retina, and other nerve tissues of vertebrates. The unfolding processes of apo-neuroglobin (apoNgb) induced by guanidine hydrochloride (GdnHCl) and urea were investigated by spectroscopic methods. In the unfolding processes, apoNgb's tertiary structural transition was monitored by the changes of intrinsic fluorescence emission spectra, and its secondary structural transition was measured by the changes of far-ultraviolet circular dichroism (CD) spectra. In addition, 8-anilino-1-naphthalenesulfonic acid (ANS), a hydrophobic cluster binding dye, was also used to monitor the unfolding process of apoNgb and to explore its intermediates. Results showed that GdnHCl-induced unfolding of apoNgb was via a three-state pathway, that is, Native state(N)→ Intermediate state(I)→ Unfolded state(U), during which the intermediate was inferred by an increase in fluorescence intensity and the change of CD value. Gibbs free energy changes are 10.2 kJ·mol−1for the first unfolding transition and 14.0 kJ·mol−1for the second transition. However, urea-induced unfolding of apoNgb only underwent a two-state transition: Native state(N)→ Partially unfolded state(P). The result showed that GdnHCl can efficiently affect the conformational states of apoNgb compared with those of urea. The work will benefit to have an understanding of the unfolding mechanism of apoNgb induced by GdnHCl and urea.

Improved Stability of a Protein Vaccine Through Elimination of a Partially Unfolded State

2004 ◽  
Author(s):  
Colleen A. McHugh ◽  
Ralph F. Tammariello ◽  
Charles B. Millard ◽  
John H. Carra
Keyword(s):  
Protein Vaccine ◽  
Unfolded State ◽  
Improved Stability ◽  
Partially Unfolded

scholarly journals Immunochemical localization of a region of chaperonin-60 important for productive interaction with chaperonin-10.

1992 ◽  
Vol 267 (36) ◽  
pp. 25632-25635
Author(s):  
D.L. Burns ◽  
M Kessel ◽  
J.L. Arciniega ◽  
A Karpas ◽  
J Gould-Kostka
Keyword(s):  
Chaperonin 10 ◽  
Chaperonin 60

Cold Shock Domain of the Human Y-Box Protein YB-1. Backbone Dynamics and Equilibrium between the Native State and a Partially Unfolded State†

Biochemistry ◽  
2004 ◽  
Vol 43 (31) ◽  
pp. 10237-10246 ◽  
Author(s):  
Cathelijne P. A. M. Kloks ◽  
Marco Tessari ◽  
Geerten W. Vuister ◽  
Cornelis W. Hilbers
Keyword(s):  
Cold Shock ◽  
Backbone Dynamics ◽  
Native State ◽  
Unfolded State ◽  
Cold Shock Domain ◽  
Partially Unfolded

scholarly journals Thermal inactivation and chaperonin-mediated renaturation of mitochondrial aspartate aminotransferase

1998 ◽  
Vol 334 (1) ◽  
pp. 219-224 ◽  
Author(s):  
James M. LAWTON ◽  
Shawn DOONAN
Keyword(s):  
Escherichia Coli ◽  
Aspartate Aminotransferase ◽  
Thermal Inactivation ◽  
Active Form ◽  
Active Conformation ◽  
E Coli ◽  
Catalytically Active ◽  
Mitochondrial Aspartate Aminotransferase ◽  
Chaperonin 10 ◽  
Chaperonin 60

Mitochondrial aspartate aminotransferase is inactivated irreversibly on heating. The inactivated protein aggregates, but aggregation is prevented by the presence of the chaperonin 60 from Escherichia coli (GroEL). The chaperonin increases the rate of thermal inactivation in the temperature range 55–65 °C but not at lower temperatures. It has previously been shown [Twomey and Doonan (1997) Biochim. Biophys. Acta 1342, 37–44] that the enzyme switches to a modified, but catalytically active, conformation at approx. 55–60 °C and the present results show that this conformation is recognized by and binds to GroEL. The thermally inactivated protein can be released from GroEL in an active form by the addition of chaperonin 10 from E. coli (GroES)/ATP, showing that inactivation is not the result of irreversible chemical changes. These results suggest that the irreversibility of thermal inactivation is due to the formation of an altered conformation with a high kinetic barrier to refolding rather than to any covalent changes. In the absence of chaperonin the unfolded molecules aggregate but this is a consequence, rather than the cause, of irreversible inactivation.

scholarly journals The mechanism of folding of globular proteins. Suitability of a penicillinase from Staphylococcus Aureus as a model for refolding studies

1976 ◽  
Vol 155 (2) ◽  
pp. 325-330 ◽  
Author(s):  
B Robson ◽  
R H. Pain
Keyword(s):  
Staphylococcus Aureus ◽  
Enzymic Activity ◽  
Optical Rotatory Dispersion ◽  
Native Conformation ◽  
Guanidinium Chloride ◽  
Tyrosine Residues ◽  
Low Concentrations ◽  
Unfolded State ◽  
Solvent Molecules ◽  
Homogeneous Preparation

1. A homogeneous preparation of penicillinase (penicillin amido-β-lactamhydrolase, EC 3.5.2.6) was isolated and purified from cultures of Staphylococcus aureus by a simple two-stage procedure. 2. The native protein contains 20-30% helix as determined by optical-rotatory-dispersion and circular-dichroism measurements. Some 54(+/-5)% of the 13 tyrosine residues are exposed to solvent molecules of diameter 0.44 and 0.94 nm. 3. Conditions that allow full recovery of enzymic activity and native conformation from the fully unfolded state in 4M-guanidinium chloride were defined. 4. Refolding of the protein was shown to be inhibited by intermolecular interaction, by small changes in ionization and by low concentrations (0.025 M) of phenol.

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