scholarly journals The Conformational Stabilities of Tropomyosins

1976 ◽  
Vol 29 (6) ◽  
pp. 405 ◽  
Author(s):  
EF Woods
Keyword(s):  
Guanidine Hydrochloride ◽  
Coiled Coil ◽  
Helical Structure ◽  
Globular Proteins ◽  
Free Energies ◽  
Native State ◽  
Denatured State ◽  
Partially Unfolded ◽  
The Stability ◽  
Partially Unfolded States

The stability to denaturation by heat and guanidine hydrochloride of seven vertebrate (including skeletal, cardiac and smooth muscle) tropomyosins and three invertebrate tropomyosins was examined. The transition profiles were discontinuous and in many cases distinct plateaux were observed which indicated the presence of unique partially unfolded states at intermediate temperatures and guanidine hydrochloride concentrations. The denaturation by guanidine hydrochloride could be described in the majority of cases by a model in which the native state unfolds to a partially unfolded stable intermediate which then unfolds to the completely denatured state. On this basis it was possible to estimate the free energies of unfolding in water. It was shown that part of the IX-helical structure of tropomyosin is only marginally stable and the free energy of unfolding in water of this segment is less than values found for globular proteins, whereas another segment (or segments) has a stability comparable to that found for globular proteins. The stepwise unfolding may be explained in terms of the coiled-coil interactions in tropomyosin.

scholarly journals Structural dissection of alkaline-denatured pepsin

2004 ◽  
Vol 18 (2) ◽  
pp. 227-236 ◽  
Author(s):  
Yuji O. Kamatari ◽  
Christopher M. Dobson ◽  
Takashi Konno
Keyword(s):  
Limited Proteolysis ◽  
Helical Structure ◽  
Cd Spectroscopy ◽  
Core Region ◽  
Detailed Knowledge ◽  
Native State ◽  
Denatured State ◽  
Residual Structure ◽  
Alkaline Denaturation ◽  
Folded Core

Pepsin, a gastric aspartic proteinase, is a zymogen‒derived protein that undergoes irreversible alkaline denaturation at pH 6–7. Detailed knowledge of the structure of the alkaline‒denatured state is an important step in understanding the mechanism of the formation of the active enzyme. It has been established in a number of studies that the alkaline‒denatured state of pepsin (the IPstate) is composed of a compact C‒terminal lobe and a largely unstructured N‒terminal lobe. In the present study, we have investigated the residual structure in the IPstate in more detail, using limited proteolysis to isolate and characterize a tightly folded core region from this partially denatured pepsin. The isolated core region corresponds to the 141 C‒terminal residues of the pepsin molecule, which in the fully native state forms one of the two lobes of the structure. A comparative study using NMR and CD spectroscopy has revealed, however, that the N‒terminal lobe contributes a substantial amount of additional residual structure to the IPstate of pepsin. CD spectra indicate in addition that significant non‒native α-helical structure is present in the C‒terminal lobe of the structure when the N‒terminal lobe of pepsin is either unfolded or removed by proteolysis. This study demonstrates that the structure of pepsin in the IPstate is significantly more complex than that of a fully folded C‒terminal lobe connected to an unstructured N‒terminal lobe. The “misfolding” in this state could inhibit the proper refolding of the protein when returned to conditions that stabilize the native state.

Reversible inactivation and dissociation of glutamine synthetase of Neurospora crassa by urea

1971 ◽  
Vol 17 (4) ◽  
pp. 451-459 ◽  
Author(s):  
G. W. Ward ◽  
M. Kapoor
Keyword(s):  
Glutamine Synthetase ◽  
Protective Effect ◽  
Neurospora Crassa ◽  
The State ◽  
Native State ◽  
Reversible Inactivation ◽  
Partial Inactivation ◽  
Partially Unfolded ◽  
The Stability

The effect of urea on the stability and the state of aggregation of glutamine synthetase is reported. Substrates and modulators of this enzyme exert a protective effect against urea-induced loss of activity. On partial inactivation by urea, the enzyme appears to consist of a dimeric species and an intermediate, partially unfolded form. Subsequent to the removal of urea, the presence of substrates and effectors is necessary to bring about a reversal of inactivation and a return of the enzyme lo the native or near-native state.

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.

scholarly journals Folding pathway of the pyridoxal 5′-phosphate C-S lyase MalY from Escherichia coli

2005 ◽  
Vol 389 (3) ◽  
pp. 885-898 ◽  
Author(s):  
Mariarita Bertoldi ◽  
Barbara Cellini ◽  
Douglas V. Laurents ◽  
Carla Borri Voltattorni
Keyword(s):  
Escherichia Coli ◽  
Gel Filtration ◽  
Native State ◽  
Equilibrium Conditions ◽  
Lyase Activity ◽  
Three Stages ◽  
Soluble Aggregates ◽  
Equilibrium Transition ◽  
Partially Unfolded ◽  
The Stability

MalY from Escherichia coli is a bifunctional dimeric PLP (pyridoxal 5′-phosphate) enzyme acting as a β-cystathionase and as a repressor of the maltose system. The spectroscopic and molecular properties of the holoenzyme, in the untreated and NaBH4-treated forms, and of the apoenzyme have been elucidated. A systematic study of the urea-induced unfolding of MalY has been monitored by gel filtration, cross-linking, ANS (8-anilino-1-naphthalenesulphonic acid) binding and by visible, near- and far-UV CD, fluorescence and NMR spectroscopies under equilibrium conditions. Unfolding proceeds in at least three stages. The first transition, occurring between 0 and 1 M urea, gives rise to a partially active dimeric species that binds PLP. The second equilibrium transition involving dimer dissociation, release of PLP and loss of lyase activity leads to the formation of a monomeric equilibrium intermediate. It is a partially unfolded molecule that retains most of the native-state secondary structure, binds significant amounts of ANS (a probe for exposed hydrophobic surfaces) and tends to self-associate. The self-associated aggregates predominate at urea concentrations of 2–4 M for holoMalY. The third step represents the complete unfolding of the enzyme. These results when compared with the urea-induced unfolding profiles of apoMalY and NaBH4-reduced holoenzyme suggest that the coenzyme group attached to the active-site lysine residue increases the stability of the dimeric enzyme. Both holo- and apo-MalY could be successfully refolded into the active enzyme with an 85% yield. Further refolding studies suggest that large misfolded soluble aggregates that cannot be refolded could be responsible for the incomplete re-activation.

scholarly journals Denaturing action of urea and guanidine hydrochloride towards two thermophilic esterases

2002 ◽  
Vol 367 (3) ◽  
pp. 857-863 ◽  
Author(s):  
Pompea DEL VECCHIO ◽  
Giuseppe GRAZIANO ◽  
Vincenzo GRANATA ◽  
Guido BARONE ◽  
Luigi MANDRICH ◽  
...  
Keyword(s):  
Structural Information ◽  
Guanidine Hydrochloride ◽  
Globular Proteins ◽  
Archaeoglobus Fulgidus ◽  
Large Set ◽  
Steady State Fluorescence ◽  
Key Factor ◽  
Alicyclobacillus Acidocaldarius ◽  
The Stability ◽  
Thermal Stability Of

The stability of two thermophilic esterases, AFEST from Archaeoglobus fulgidus and EST2 from Alicyclobacillus acidocaldarius, against the denaturing action of urea and guanidine hydrochloride has been investigated by means of steady-state fluorescence and circular dichroism measurements. Experimental results indicate that the two enzymes, even though very resistant to temperature and urea, show a resistance to guanidine hydrochloride weaker than expected on the basis of data collected so far for a large set of globular proteins. Structural information available for AFEST and EST2 and ideas that emerged from studies on the molecular origin of the greater thermal stability of thermophiles allow the suggestion of a reliable rationale. The present results may be an indication that the optimization of charge—charge interactions on the protein surface is a key factor for the stability of the two esterases.

scholarly journals Perplexing cooperative folding and stability of a low-sequence complexity, polyproline 2 protein lacking a hydrophobic core

2017 ◽  
Vol 114 (9) ◽  
pp. 2241-2246 ◽  
Author(s):  
Zachary P. Gates ◽  
Michael C. Baxa ◽  
Wookyung Yu ◽  
Joshua A. Riback ◽  
Hui Li ◽  
...  
Keyword(s):  
Room Temperature ◽  
Polypeptide Chain ◽  
Helical Structure ◽  
Residue Level ◽  
Hydrophobic Core ◽  
Native State ◽  
Sequence Complexity ◽  
Unfolded State ◽  
The Stability ◽  
Stabilizing Factors

The burial of hydrophobic side chains in a protein core generally is thought to be the major ingredient for stable, cooperative folding. Here, we show that, for the snow flea antifreeze protein (sfAFP), stability and cooperativity can occur without a hydrophobic core, and without α-helices or β-sheets. sfAFP has low sequence complexity with 46% glycine and an interior filled only with backbone H-bonds between six polyproline 2 (PP2) helices. However, the protein folds in a kinetically two-state manner and is moderately stable at room temperature. We believe that a major part of the stability arises from the unusual match between residue-level PP2 dihedral angle bias in the unfolded state and PP2 helical structure in the native state. Additional stabilizing factors that compensate for the dearth of hydrophobic burial include shorter and stronger H-bonds, and increased entropy in the folded state. These results extend our understanding of the origins of cooperativity and stability in protein folding, including the balance between solvent and polypeptide chain entropies.

scholarly journals Mechanism of the small ATP-independent chaperone Spy is substrate specific

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Rishav Mitra ◽  
Varun V. Gadkari ◽  
Ben A. Meinen ◽  
Carlo P. M. van Mierlo ◽  
Brandon T. Ruotolo ◽  
...  
Keyword(s):  
Relative Affinity ◽  
Complex Protein ◽  
Partially Unfolded ◽  
Substrate Proteins ◽  
Partially Unfolded States

AbstractATP-independent chaperones are usually considered to be holdases that rapidly bind to non-native states of substrate proteins and prevent their aggregation. These chaperones are thought to release their substrate proteins prior to their folding. Spy is an ATP-independent chaperone that acts as an aggregation inhibiting holdase but does so by allowing its substrate proteins to fold while they remain continuously chaperone bound, thus acting as a foldase as well. The attributes that allow such dual chaperoning behavior are unclear. Here, we used the topologically complex protein apoflavodoxin to show that the outcome of Spy’s action is substrate specific and depends on its relative affinity for different folding states. Tighter binding of Spy to partially unfolded states of apoflavodoxin limits the possibility of folding while bound, converting Spy to a holdase chaperone. Our results highlight the central role of the substrate in determining the mechanism of chaperone action.

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
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