Tryptophan phosphorescence signals characteristic changes in protein dynamics at physiological temperatures

Tölgyesi, F.; Ullrich, Beáta; Fidy, Judit

doi:10.1016/s0167-4838(99)00200-9

Cited by 8 publications

(6 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The amplitudes of the Arrhenius plots at pH 8.5 vary for wildtype and thermostable pNB E, but the activation energies are similar. As has been seen for other proteins (24,25), the long lifetime Arrhenius plots (Figure 4A) fit to two exponentials with activation energies of 2700 ((400) cm -1 and 4600 ((300) cm -1 . For shorter phosphorescence lifetimes, this second component is difficult to resolve, and the Arrhenius plots (Figure 4B) can be fit with a single activation energy of 3000 ((200) cm -1 , which is consistent with the lower activation energy of the long lifetime component.…”

Section: Stability Of Evolved Pnbsupporting

confidence: 79%

“…The activation energies derived from the Arrhenius plots demonstrate that the triplet state decay processes are similar for wild-type and variant pNB Es. Indeed, the pNB E phosphorescence lifetime Arrhenius plots resemble those observed for other proteins (24,25), indicating a common deactivation mechanism. The nonlinearity of the Arrhenius plot, particularly evident for the long phosphorescence lifetime (Figure 4A), has also been observed with other proteins (24,25) and has been attributed to activation of large-scale motions at higher temperatures (25).…”

Section: Thermostabilities and Activities Of Evolved Pnb Esterasessupporting

confidence: 66%

See 1 more Smart Citation

Tryptophan Phosphorescence Study of Enzyme Flexibility and Unfolding in Laboratory-Evolved Thermostable Esterases

et al. 2000

View full text Add to dashboard Cite

Directed evolution of p-nitrobenzyl esterase (pNB E) has yielded eight generations of increasingly thermostable variants. The most stable esterase, 8G8, has 13 amino acid substitutions, a melting temperature 17 degrees C higher than the wild-type enzyme, and increased hydrolytic activity toward p-nitrophenyl acetate (pNPA), the substrate used for evolution, at all temperatures. Room-temperature activities of the evolved thermostable variants range from 3.5 times greater to 4.0 times less than wild type. The relationships between enzyme stability, catalytic activity, and flexibility for the esterases were investigated using tryptophan phosphorescence. We observed no correlation between catalytic activity and enzyme flexibility in the vicinity of the tryptophan (Trp) residues. Increases in stability, however, are often accompanied by decreases in flexibility, as measured by Trp phosphorescence. Phosphorescence data also suggest that the N- and C-terminal regions of pNB E unfold independently. The N-terminal region appears more thermolabile, yet most of the thermostabilizing mutations are located in the C-terminal region. Mutational studies show that the effects of the N-terminal mutations depend on one or more mutations in the C-terminal region. Thus, the pNB E mutants are stabilized by long-range, cooperative interactions between distant parts of the enzyme.

show abstract

Section: Stability Of Evolved Pnbsupporting

confidence: 79%

Section: Thermostabilities and Activities Of Evolved Pnb Esterasessupporting

confidence: 66%

Tryptophan Phosphorescence Study of Enzyme Flexibility and Unfolding in Laboratory-Evolved Thermostable Esterases

et al. 2000

View full text Add to dashboard Cite

show abstract

“…The chaperone-like activity was less pronounced below 30 mC, and it was found enhanced above this temperature [18,19]. We demonstrated that this temperature dependence correlates well with the activation of large-scale motions in the protein above 30 mC [20]. It has also been demonstrated that chaotropic agents in small quantities enhance the protective ability of α-crystallin [21], whereas it is essentially eliminated by Mg# + at concentrations well below the physiological level or by reducing the pH value below 6 [22].…”

Section: Introductionmentioning

confidence: 56%

“…increasing thermal fluctuations and flexibility of the molecule supplying the necessary accessibility to hydrophobic regions. We investigated previously the flexibility of the molecule at various temperatures by tryptophan phosphorescence [20]. Our results indicated an increase in flexibility above 30 mC that coincides with the enhancement of the chaperone activity at the given temperature.…”

Section: Discussionmentioning

confidence: 79%

Chaperone-like activity of alpha-crystallin is enhanced by high-pressure treatment

Böde

Tölgyesi

Smeller

et al. 2003

Biochemical Journal

Self Cite

View full text Add to dashboard Cite

alpha-Crystallin, an oligomeric protein in vertebrate eye lens, is a member of the small heat-shock protein family. Several papers pointed out that its chaperone-like activity could be enhanced by increasing the temperature. We demonstrate in the present study that structural perturbations by high hydrostatic pressures up to 300 MPa also enhance this activity. In contrast with temperature-induced changes, the pressure-induced enhancement is reversible. After pressure release, the extra activity is lost with a relaxation time of 2.0+/-0.5 h. Structural alterations contributing to the higher activity were studied with IR and fluorescence spectroscopy, and light-scattering measurements. The results suggest that while the secondary structure barely changes under pressure, the interactions between the subunits weaken, the oligomers dissociate, the area of accessible hydrophobic surfaces significantly increases and the environment of tryptophan residues becomes slightly more polar. It seems that structural flexibility and the total surface area of the oligomers are the key factors in the chaperone capacity, and that the increase in the chaperone activity does not require the increase in the oligomer size as was assumed previously [Burgio, Kim, Dow and Koretz (2000) Biochem. Biophys. Res. Commun. 268, 426-432]. After pressure release, the structure of subunits are reorganized relatively quickly, whereas the oligomer size reaches its original value slowly with a relaxation time of 33+/-4 h. In our interpretation, both the fast and slow structural rearrangements have an impact on the functional relaxation.

show abstract

“…Double activation energy (E a ) with two Arrhenius equations fit. 62 3. Kinetic data analyzed with a polynomial function may yield Arrhenius behavior.…”

Section: Non-arrhenius Data Analysismentioning

confidence: 99%

Evaluation of a Non-Arrhenius Model for Therapeutic Monoclonal Antibody Aggregation

Kayser

Chennamsetty

Voynov

et al. 2011

Journal of Pharmaceutical Sciences

104

View full text Add to dashboard Cite

Tryptophan phosphorescence signals characteristic changes in protein dynamics at physiological temperatures

Cited by 8 publications

References 29 publications

Tryptophan Phosphorescence Study of Enzyme Flexibility and Unfolding in Laboratory-Evolved Thermostable Esterases

Tryptophan Phosphorescence Study of Enzyme Flexibility and Unfolding in Laboratory-Evolved Thermostable Esterases

Chaperone-like activity of alpha-crystallin is enhanced by high-pressure treatment

Evaluation of a Non-Arrhenius Model for Therapeutic Monoclonal Antibody Aggregation

Contact Info

Product

Resources

About