Pre-Unfolding Resonant Oscillations of Single Green Fluorescent Protein Molecules

Baldini, Giancarlo; Cannone, Fabio; Chirico, Giuseppe

doi:10.1126/science.1115001

Cited by 53 publications

(67 citation statements)

References 40 publications

(2 reference statements)

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“…This possibility is indeed suggested by the earlier observation that different regions of the polypeptide chain contract in an asynchronous manner in the initial folding reaction (9). Singlemolecule studies have indicated that such gradual diffusive motion may occur over milliseconds (52) or even longer (53). In this context, it should be noted that the time scale of folding of other proteins reported to fold or unfold in a downhill manner appears to be in a similar (5-500 s) time domain at Ϸ25°C (3,7,16,17).…”

Section: Absence Of a Significant Free Energy Barrier During The Sub-msmentioning

confidence: 50%

“…Many hundreds of such contacts may form during a folding reaction. As expected, such diffusive fluctuations are slower in a more compact conformation than they are in U (52,55). The slow (20-200 s) fluctuations are thought to represent concerted chain motions, and it might be that such slow diffusive chain motions lead to the formation of I E on an Ϸ200-s time scale.…”

Section: Absence Of a Significant Free Energy Barrier During The Sub-msmentioning

confidence: 84%

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Barrierless evolution of structure during the submillisecond refolding reaction of a small protein

Sinha

Udgaonkar

2008

Proc. Natl. Acad. Sci. U.S.A.

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To determine whether a protein folding reaction can occur in the absence of a dominant barrier is crucial for understanding its complexity. Here direct ultrafast kinetic measurements have been used to study the initial submillisecond (sub-ms) folding reaction of the small protein barstar. The cooperativity of the initial folding reaction has been explored by using two probes: fluorescence resonance energy transfer, through which the contraction of two intramolecular distances is measured, and the binding of 8-anilino-1-naphthalene sulfonic acid, through which the formation of hydrophobic clusters is monitored. A fast chain contraction is shown to precede the formation of hydrophobic clusters, indicating that the sub-ms folding reaction is not cooperative. The observed rate constant of the sub-ms folding reaction monitored by 8-anilino-1-naphthalene sulfonic acid fluorescence has been found to be the same in stabilizing conditions (low urea concentrations), in which specific structure is formed, and in marginally stabilizing conditions (higher urea concentrations), where virtually no structure is formed in the product of the sub-ms folding reaction. The observation that the folding rate is independent of the folding conditions suggests that the initial folding reaction occurs in the absence of a dominant free energy barrier. These results provide kinetic evidence that the formation of specific structure need not be slowed down by any significant free energy barrier during the course of a very fast protein folding reaction.noncooperative ͉ submillisecond protein folding

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Section: Absence Of a Significant Free Energy Barrier During The Sub-msmentioning

confidence: 50%

Section: Absence Of a Significant Free Energy Barrier During The Sub-msmentioning

confidence: 84%

Barrierless evolution of structure during the submillisecond refolding reaction of a small protein

Sinha

Udgaonkar

2008

Proc. Natl. Acad. Sci. U.S.A.

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“…For example Lu et al 10 found accumulations of diagonal correlations in the consecutive Preface xi on-times of cholesterol oxidase enzyme, Edman and Rigler found oscillations in the fluorescence of labeled horse radish peroxidase, 11 and Baldini et al in the dynamics of fluorescent proteins excited with two photons. 12 Vlad and Ross, in Chapter 8, discuss two possible theoretical models that may explain these intriguing experimental results.…”

Section: Prefacementioning

confidence: 99%

Front Matter

2008

Theory and Evaluation of Single-Molecule Signals

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“…fluorescence ͉ single enzyme S ingle-molecule experiments on proteins have revealed novel phenomena, including fluctuations in the rates of enzyme catalysis (1-3), on-off blinking behavior in proteins (4)(5)(6), and oscillations under a near-denatured condition of a green fluorescent protein (7). The on-off blinking behavior of proteins and the fluctuations in the enzyme catalysis rate in the experiments were interpreted in terms of fluctuations of the protein/enzyme between various conformational substates (2,3).…”

Section: ؊1mentioning

confidence: 99%

An interpretation of fluctuations in enzyme catalysis rate, spectral diffusion, and radiative component of lifetimes in terms of electric field fluctuations

Prakash

Marcus

2007

Proc. Natl. Acad. Sci. U.S.A.

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Time-dependent fluctuations in the catalysis rate (␦k(t)) observed in single-enzyme experiments were found in a particular study to have an autocorrelation function decaying on the same time scale as that of spectral diffusion ␦0(t). To interpret this similarity, the present analysis focuses on a factor in enzyme catalysis, the local electrostatic interaction energy (E) at the active site and its effect on the activation free energy barrier. We consider the slow fluctuations of the electrostatic interaction energy (␦E(t)) as a contributor to ␦k(t) and relate the latter to ␦0(t). The resulting relation between ␦k(t) and ␦0(t) is a dynamic analog of the solvatochromism used in interpreting solvent effects on organic reaction rates. The effect of the postulated ␦E(t) on fluctuations in the radiative component (␦␥ r ؊1(t)) of the fluorescence decay of chromophores in proteins also is examined, and a relation between ␦␥ r ؊1 (t) and ␦0(t) is obtained. Experimental tests will determine whether the correlation functions for ␦k(t), ␦0(t), and ␦␥ r ؊1 are indeed similar for any enzyme. Measurements of dielectric dispersion, (), for the enzyme discussed elsewhere will provide further insight into the correlation function for ␦E(t). They also will determine whether fluctuations in the nonradiative component ␥ nr ؊1 of the lifetime decay has a different origin, fluctuations in distance for example.fluorescence ͉ single enzyme S ingle-molecule experiments on proteins have revealed novel phenomena, including fluctuations in the rates of enzyme catalysis (1-3), on-off blinking behavior in proteins (4-6), and oscillations under a near-denatured condition of a green fluorescent protein (7). The on-off blinking behavior of proteins and the fluctuations in the enzyme catalysis rate in the experiments were interpreted in terms of fluctuations of the protein/enzyme between various conformational substates (2, 3). The rates of enzyme-catalyzed reactions themselves occur typically on the milliseconds-to-seconds time scale, and the millisecond dynamics of enzymes are believed to contribute to the ''functional dynamics'' of the enzyme (8). Single-molecule enzyme experiments on cholesterol oxidase revealed an autocorrelation function of the rate of catalysis that decayed on a time scale about the same as that for the decay of the autocorrelation function of the spectral diffusion of a chromophore in the same enzyme in the absence of the substrate (1), thus suggesting a common origin for the two fluctuations.Significant insight into the functioning of enzymes has been achieved by using computer simulations (9-11) and empirical valence bond (9) and hybrid quantum mechanical/molecular mechanics (10) methods, among others. In these studies the catalysis is affected by several factors: electrostatic effects of the enzyme on the substrate (9) and coupling of protein fluctuations with motions of the substrate-coenzyme pair in the transition state (e.g., ref. 8). Electrostatic effects have been seen to be significant in enzymatic catalysis (9,(1...

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Pre-Unfolding Resonant Oscillations of Single Green Fluorescent Protein Molecules

Cited by 53 publications

References 40 publications

Barrierless evolution of structure during the submillisecond refolding reaction of a small protein

Barrierless evolution of structure during the submillisecond refolding reaction of a small protein

Front Matter

An interpretation of fluctuations in enzyme catalysis rate, spectral diffusion, and radiative component of lifetimes in terms of electric field fluctuations

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