Thermostability of Irreversible Unfolding α-Amylases Analyzed by Unfolding Kinetics

A polyacrylamide-gel-electrophoresis method has been developed that permits the analysis of conformational changes that occur during the thermal denaturation of macromolecules. A stable transverse temperature gradient was produced in an aluminium heating jacket clamped around a vertical polyacrylamide slab gel. After temperature equilibration, gels were loaded with either a layer of protein solution (20-200 micrograms/gel) or a solution of double-stranded DNA (20 micrograms/gel) and electrophoresis begun. At the end of the run the gels were stained and the effect of temperature on mobility observed. The technique proved informative both for the irreversible unfolding of proteins (Drosophila alcohol dehydrogenase and lactic acid dehydrogenase) and for a protein that was reversibly denatured by heat (beta-lactamase). In the latter case a clear transition between the native enzyme and a slower-migrating denatured state was observed. The patterns obtained were analogous to the type produced by the transverse-urea-gradient-electrophoretic method of Creighton [(1979) J. Mol. Biol. 129, 253-264]. The method also resolved a complex mixture of double-stranded-DNA restriction-digest fragments.

Download Full-text

Reversible and irreversible unfolding of eukaryote chromosomes by force

Proceedings of the First Joint BMES/EMBS Conference. 1999 IEEE Engineering in Medicine and Biology 21st Annual Conference and the 1999 Annual Fall Meeting of the Biomedical Engineering Society (Cat. No.99CH37015) ◽

10.1109/iembs.1999.802114 ◽

2003 ◽

Author(s):

J.F. Marko ◽

M. Poirier ◽

S. Eroglu ◽

D. Chatenay

Keyword(s):

Irreversible Unfolding

Download Full-text

Folding/Unfolding Kinetics of Apomyoglobin

Molecular Biology ◽

10.1007/s11008-005-0109-6 ◽

2005 ◽

Vol 39 (6) ◽

pp. 884-891

Author(s):

E. N. Baryshnikova ◽

B. S. Melnik ◽

G. V. Semisotnov ◽

V. E. Bychkova

Keyword(s):

Unfolding Kinetics ◽

Kinetics Of

Download Full-text

Design and Properties of Ligand-Conjugated Guanine Oligonucleotides for Recovery of Mutated G-Quadruplexes

Molecules ◽

10.3390/molecules23123228 ◽

2018 ◽

Vol 23 (12) ◽

pp. 3228

Author(s):

Shuntaro Takahashi ◽

Boris Chelobanov ◽

Ki Kim ◽

Byeang Kim ◽

Dmitry Stetsenko ◽

...

Keyword(s):

Cancer Progression ◽

Polyaromatic Hydrocarbons ◽

Fine Tuning ◽

Vascular Endothelial ◽

Quadruplex Dna ◽

Guanine Quadruplex ◽

Anthracene Derivatives ◽

Novel Strategy ◽

Endothelial Growth Factor ◽

Unfolding Kinetics

The formation of a guanine quadruplex DNA structure (G4) is known to repress the expression of certain cancer-related genes. Consequently, a mutated G4 sequence can affect quadruplex formation and induce cancer progression. In this study, we developed an oligonucleotide derivative consisting of a ligand-containing guanine tract that replaces the mutated G4 guanine tract at the promoter of the vascular endothelial growth factor (VEGF) gene. A ligand moiety consisting of three types of polyaromatic hydrocarbons, pyrene, anthracene, and perylene, was attached to either the 3′ or 5′ end of the guanine tract. Each of the ligand-conjugated guanine tracts, with the exception of anthracene derivatives, combined with other intact guanine tracts to form an intermolecular G4 on the mutated VEGF promoter. This intermolecular G4, exhibiting parallel topology and high thermal stability, enabled VEGF G4 formation to be recovered from the mutated sequence. Stability of the intramolecular G4 increased with the size of the conjugated ligand. However, suppression of intermolecular G4 replication was uniquely dependent on whether the ligand was attached to the 3′ or 5′ end of the guanine tract. These results indicate that binding to either the top or bottom guanine quartet affects unfolding kinetics due to polarization in DNA polymerase processivity. Our findings provide a novel strategy for recovering G4 formation in case of damage, and fine-tuning processes such as replication and transcription.

Download Full-text

Temperature Dependence of the Folding and Unfolding Kinetics of the GCN4 Leucine Zipper via 13Cα-NMR

Biophysical Journal ◽

10.1016/s0006-3495(01)76073-0 ◽

2001 ◽

Vol 80 (2) ◽

pp. 939-951 ◽

Cited By ~ 21

Author(s):

Marilyn Emerson Holtzer ◽

G. Larry Bretthorst ◽

D. André d’Avignon ◽

Ruth Hogue Angeletti ◽

Lisa Mints ◽

...

Keyword(s):

Temperature Dependence ◽

Leucine Zipper ◽

Unfolding Kinetics ◽

Kinetics Of

Download Full-text

Thermodynamic Stability of Annexin V E17G: Equilibrium Parameters from an Irreversible Unfolding Reaction†,‡

Biochemistry ◽

10.1021/bi962163z ◽

1997 ◽

Vol 36 (7) ◽

pp. 1657-1668 ◽

Cited By ~ 61

Author(s):

Thomas Vogl ◽

Claudia Jatzke ◽

Hans-Jürgen Hinz ◽

Jörg Benz ◽

Robert Huber

Keyword(s):

Thermodynamic Stability ◽

Annexin V ◽

Irreversible Unfolding

Download Full-text

Irreversible Unfolding of Myoglobin in an Aqueous Solution by Supercritical Carbon Dioxide

Journal of Agricultural and Food Chemistry ◽

10.1021/jf000081i ◽

2000 ◽

Vol 48 (10) ◽

pp. 4535-4539 ◽

Cited By ~ 18

Author(s):

Hiroya Ishikawa ◽

Mitsuya Shimoda ◽

Akiyoshi Yonekura ◽

Keiko Mishima ◽

Kiyoshi Matsumoto ◽

...

Keyword(s):

Carbon Dioxide ◽

Aqueous Solution ◽

Supercritical Carbon Dioxide ◽

Irreversible Unfolding ◽

Supercritical Carbon

Download Full-text

The role of an evolutionarily conserved cis-proline in the thioredoxin-like domain of human class Alpha glutathione transferase A1-1

Biochemical Journal ◽

10.1042/bj20021765 ◽

2003 ◽

Vol 372 (1) ◽

pp. 241-246 ◽

Cited By ~ 24

Author(s):

Chris NATHANIEL ◽

Louise A. WALLACE ◽

Jonathan BURKE ◽

Heini W. DIRR

Keyword(s):

Glutathione Transferase ◽

Conformational Stability ◽

Wild Type ◽

Catalytic Function ◽

Evolutionarily Conserved ◽

Glutathione S Transferases ◽

Equilibrium Unfolding ◽

The Stability ◽

Unfolding Kinetics

The thioredoxin-like fold has a βαβαββα topology, and most proteins/domains with this fold have a topologically conserved cis-proline residue at the N-terminus of β-strand 3. This residue plays an important role in the catalytic function and stability of thioredoxin-like proteins, but is reported not to contribute towards the stability of glutathione S-transferases (GSTs) [Allocati, Casalone, Masulli, Caccarelli, Carletti, Parker and Di Ilio (1999) FEBS Lett. 445, 347–350]. In order to further address the role of the cis-proline in the structure, function and stability of GSTs, cis-Pro-56 in human GST (hGST) A1-1 was replaced with a glycine, and the properties of the P56G mutant were compared with those of the wild-type protein. Not only was the catalytic function of the mutant dramatically reduced, so was its conformational stability, as indicated by equilibrium unfolding and unfolding kinetics experiments with urea as denaturant. These findings are discussed in the context of other thioredoxin-like proteins.

Download Full-text