Coupling between external viscosity and the intramolecular dynamics of ribonuclease T1: a two-phase model for the quenching of protein fluorescence

Somogyi, B; Punyiczki, Mária; Hedstrom, Jack; Norman, John A. T.; Prendergast, Franklyn G.; Rosenberg, Andreas

doi:10.1016/0167-4838(94)90137-6

Cited by 12 publications

(7 citation statements)

References 30 publications

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“…Because molar, submolar acrylamide affinities are not uncommon with proteins, binding could make a significant contribution to the overall quenching rate, especially when large [Q] values are employed. Binding could thus account for the smaller gradient of lifetime Stern−Volmer plots relative to the corresponding intensity plots and for the observation of very short-lived components in the fluorescence decay in the presence of acrylamide …”

Section: Discussionmentioning

confidence: 99%

“…Until now, a ratio k q / k d ≪ 1 has often been interpreted as the hindered diffusion of Q through the globular fold and therefore as a measure of its structural flexibility. , For k q to reflect the permeability of a protein to Q, quenching of buried Trp residues must be limited by the slow migration of Q through the protein matrix. However, whereas it is generally accepted that small neutral diatomic (O 2 , CO, and NO) and to a lesser extent triatomic molecules (H 2 S, CS 2 ) can migrate through the protein matrix on the nanosecond time scale of fluorescence, the debate is open for larger solutes such as acrylamide. ,,, For molecules of this size and larger ones, it is argued that Q penetration is most unlikely as the globular fold tends to be compact with rather few and small cavities, which render it largely impermeable. This point of view is also supported by a molecular dynamics simulation that predicted a sharp dependence of protein penetration rate on molecular size.…”

Section: Introductionmentioning

confidence: 99%

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Fluorescence Quenching of Buried Trp Residues by Acrylamide Does Not Require Penetration of the Protein Fold

Strambini

Gonnelli

2009

J. Phys. Chem. B

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The accessibility of acrylamide to buried Trp residues in proteins, as attested by dynamic quenching of their fluorescence emission, is often interpreted in terms of migration of the quencher (Q) through the globular fold. The quencher penetration mechanism, however, has long been debated because, on one hand, solutes the size of acrylamide are not expected to diffuse within the protein matrix on the nanosecond time scale of fluorescence and, on the other hand, alternative reactions pathways where Q remains in the solvent cannot be ruled out. To test the Q penetration hypothesis, we compared the quenching rates of acrylamide analogs of increasing molecular size (acrylonitrile, acrylamide, and bis-acrylamide) on the buried Trp residues of RNaseT1 and parvalbumin. The results show that the largest molecule, bis-acrylamide, is also the most efficient quencher and that in general the quenching rate is not correlated to quencher size, as expected for a penetration mechanism. Whereas these results rule out significant internal Q migration in the times of fluorescence, it is also demonstrated that up to a depth of burial of 3 A, through-space interactions with acrylamide in the solvent satisfactorily account for the small rate constants reported for these proteins. More generally, this analysis emphasizes that reduced dynamic quenching of protein fluorescence by acrylamide rather than reporting on the structural rigidity of the globular fold reflects the distance of closest approach between the internal chromophore and Q in the aqueous phase.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Fluorescence Quenching of Buried Trp Residues by Acrylamide Does Not Require Penetration of the Protein Fold

Strambini

Gonnelli

2009

J. Phys. Chem. B

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“…37 Similar observations have been reported for other systems as well. 38 A distinction between internal friction and solvent friction was used in the interpretation of the conformational relaxation of myoglobin. 9 The refolding kinetics displayed a Kramers'-like viscosity dependence only at high viscosities (∼ 20 cP).…”

Section: Interpretation Of ξ As An Internal Friction Termmentioning

confidence: 99%

Diffusional Barrier in the Unfolding of a Small Protein

Pradeep

Udgaonkar

2007

Journal of Molecular Biology

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“…show, however, that the interpretation of acrylamide quenching data is not straightforward. In some cases the quenching by penetrating acrylamide molecules can play a dominant role (Somogyi et al, 1994). Furthermore, proteins are able to bind acrylamide (Punyiczki et al, 1993;Matk6 et al, 1980), and the bound acrylamide molecules can also contribute to the quenching.…”

Section: Mg2tmentioning

confidence: 99%

Spectroscopic study of conformational changes in subdomain 1 of G-actin: influence of divalent cations

Nyitrai¹,

Hild²,

Belágyi³

et al. 1997

Biophysical Journal

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Temperature dependence of the fluorescence intensity and anisotropy decay of N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine attached to Cys374 of actin monomer was investigated to characterize conformational differences between Ca- and Mg-G-actin. The fluorescence lifetime is longer in Mg-G-actin than that in Ca-G-actin in the temperature range of 5-34 degrees C. The width of the lifetime distribution is smaller by 30% in Mg-saturated actin monomer at 5 degrees C, and the difference becomes negligible above 30 degrees C. The semiangle of the cone within which the fluorophore can rotate is larger in Ca-G-actin at all temperatures. Electron paramagnetic resonance measurements on maleimide spin-labeled (on Cys374) monomer actin gave evidence that exchange of Ca2+ for Mg2+ induced a rapid decrease in the mobility of the label immediately after the addition of Mg2+. These results suggest that the C-terminal region of the monomer becomes more rigid as a result of the replacement of Ca2+ by Mg2+. The change can be related to the difference between the polymerization abilities of the two forms of G-actin.

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Coupling between external viscosity and the intramolecular dynamics of ribonuclease T1: a two-phase model for the quenching of protein fluorescence

Cited by 12 publications

References 30 publications

Fluorescence Quenching of Buried Trp Residues by Acrylamide Does Not Require Penetration of the Protein Fold

Fluorescence Quenching of Buried Trp Residues by Acrylamide Does Not Require Penetration of the Protein Fold

Diffusional Barrier in the Unfolding of a Small Protein

Spectroscopic study of conformational changes in subdomain 1 of G-actin: influence of divalent cations

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