Measuring Unfolding of Proteins in the Presence of Denaturant Using Fluorescence Correlation Spectroscopy

Chattopadhyay, Krishnananda; Saffarian, Saveez; Elson, Elliot L.; Frieden, Carl

doi:10.1529/biophysj.104.053199

Cited by 101 publications

(134 citation statements)

References 32 publications

(39 reference statements)

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“…A similar dependence with pH had already been shown above where rotational correlation times obtained pointed to smaller rotating entities at low pH. It is known that the 3-D structure of the protein influences its mobility and thus the diffusion coefficient making it possible to follow unfolding processes [60]. Moreover, depending on the native structure of the protein, unfolding may lead to elongated structures (usually causing an increase in measured R H ) or to more globular forms (usually with smaller R H ).…”

Section: Resultssupporting

confidence: 81%

Translational and Rotational Motions of Albumin Sensed by a Non-Covalent Associated Porphyrin Under Physiological and Acidic Conditions: A Fluorescence Correlation Spectroscopy and Time Resolved Anisotropy Study

Andrade¹,

Costa²,

Borst

et al. 2008

J Fluoresc

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The interaction between a free-base, anionic watersoluble porphyrin, TSPP, and the drug carrier protein, bovine serum albumin (BSA) has been studied by time-resolved fluorescence anisotropy (TRFA) and fluorescence correlation spectroscopy (FCS) at two different pH-values. Both rotational correlation times and translational diffusion times of the fluorescent species indicate that TSPP binding to albumin induces very little conformational changes in the protein under physiological conditions. By contrast, at low pH, a biexponential decay is obtained where a short rotational correlation time (7 int =1.2 ns) is obtained, which is likely associated to wobbling movement of the porphyrin in the protein binding site. These physical changes are corroborated by circular dichroism (CD) data which show a 37% loss in the protein helicity upon acidification of the medium. In the presence of excess porphyrin formation of porphyrin Jaggregates is induced, which can be detected by timeresolved fluorescence with short characteristic times. This is also reflected in FCS data by an increase in molecular brightness together with a decrease in the number of fluorescent molecules passing through the detection volume of the sample.

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Section: Resultssupporting

confidence: 81%

Translational and Rotational Motions of Albumin Sensed by a Non-Covalent Associated Porphyrin Under Physiological and Acidic Conditions: A Fluorescence Correlation Spectroscopy and Time Resolved Anisotropy Study

Andrade¹,

Costa²,

Borst

et al. 2008

J Fluoresc

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“…4). Determination of the Stokes radius from the measured diffusion time, D , at pH 2, indicates that the protein is compact (22 Å), whereas in the presence of 2-3 M Gdm⅐HCl, it is close to a random coil (46 Å) (9). The far UV CD data also show that the addition of salt (100 mM KCl) at pH 2 induces more structure with the spectrum resembling that of the native state.…”

Section: Resultsmentioning

confidence: 96%

“…The procedure for FCS experiments using two-photon excitation has been described (8,9), and the same experimental procedure has been used here. Data analyses were performed with ORIGIN 7.0 (OriginLab, Northampton, MA).…”

Section: Methodsmentioning

confidence: 99%

The kinetics of conformational fluctuations in an unfolded protein measured by fluorescence methods

Chattopadhyay

Elson²,

Frieden³

2005

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

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135

143

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The simplest dynamic model for an unfolded protein is a statistical coil that continually undergoes substantial conformational fluctuations. A growing number of studies indicate that the unfolded protein is not a simple random coil but rather forms transient structures. We have directly measured the rate of conformational fluctuations of unfolded intestinal fatty acid binding protein (131 aa, 15 kDa) by using fluorescence self-quenching in combination with fluorescence correlation spectroscopy. The conformational fluctuations in this state have an apparent relaxation time, R, of 1.6 sec in 3 M guanidine-HCl at pH 7 and 20°C. The value of R increases with increasing solution viscosity, suggesting a diffusive process. In the molten globule state at pH 2, R is 2.5 sec, increasing further with the formation of salt-induced secondary structure. These measurements, which should be widely applicable to other systems, can provide important information about the still incompletely understood conformational properties of unfolded proteins and the mechanism of protein folding. Little is known, however, about the dynamics between different conformers in the unfolded state. Probing these conformational transitions is not trivial for two reasons. First, the dynamics are expected to be rapid (sec) and inaccessible to NMR and other commonly used experimental techniques. Second, the difference in spectroscopic signatures between two conformational states is small or even absent, thus making it difficult to find a suitable probe. In this study, we have used fluorescence self-quenching of tetramethyl rhodamine (TMR) as analyzed by fluorescence correlation spectroscopy (FCS) to study the dynamics of the unfolded state under different solution conditions.To perform these experiments we have incorporated a fluorescent probe in two positions of the intestinal fatty acid binding protein (IFABP), 48 residues apart, and have directly measured both the rate of diffusion of the whole molecule and the rate of internal dynamics of the chain in the unfolded state. IFABP consists of two ␤-sheets enclosing a large cavity into which the fatty acid binds (3). The protein (131 residues, 15 kDa) does not contain either cysteine or proline residues, but numerous mutations of residues to cysteine, to which fluorescence probes have been covalently attached (4) have been made with few deleterious effects. Thus, this protein provides an excellent model system for these studies. The persistence of structure in the unfolded state of IFABP was observed by Hodsdon and Frieden (5). In the present study two polar residues, in the two different ␤-sheets, have been mutated to cysteine (D59C͞E107C) and then modified with TMR-5-maleimide. Two other mutants were generated, each with only one residue replaced by cysteine and modified by TMR (D59C-TMR and E107C-TMR). Relative to the labeled single mutants, the doubly labeled mutant is significantly quenched in the presence of 2 M guanidine-HCl (Gdm⅐HCl). We conclude that this quenching (fluorescence self-quenching) ...

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“…1b) whose rate is faster than its molecular diffusion (32, 38 -40). Determination of molecular diffusion ( D ) using a suitable correlation function (Equation 1 for example) yields r H (31,32) (Fig. 1a), which provides information about the conformation of the folded, unfolded, or intermediate states of a protein.…”

Section: Resultsmentioning

confidence: 99%

“…Typically, 50 -100 nM labeled protein concentration was used. To correct for the refractive index and viscosity of urea and arginine solutions, necessary correction measures were taken using microscope correction collar and height as described previously (32). Additionally, the experiments were carried out with free TMR at each solution condition to normalize the protein data.…”

Section: Methodsmentioning

confidence: 99%

Role of Protein Stabilizers on the Conformation of the Unfolded State of Cytochrome c and Its Early Folding Kinetics

Haldar

Mitra

Chattopadhyay

2010

Journal of Biological Chemistry

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An insight into the conformation and dynamics of unfolded and early intermediate states of a protein is essential to understand the mechanism of its aggregation and to design potent inhibitor molecules. Fluorescence correlation spectroscopy has been used to study the effects of several model protein stabilizers on the conformation of the unfolded state and early folding dynamics of tetramethyl rhodamine-labeled cytochrome c from Saccharomyces cerevisiae at single molecular resolution. Special attention has been given to arginine, which is a widely used stabilizer for improving refolding yield of different proteins. The value of the hydrodynamic radius (r H ) obtained by analyzing the intensity fluctuations of the diffusing molecules has been found to increase in a two-state manner as the protein is unfolded by urea. The results further show that the presence of arginine and other protein stabilizers favors a relatively structured conformation of the unfolded states (r H of 29 Å ) over an extended one (r H of 40 Å ), which forms in their absence. Also, the time constant of a kinetic component ( R ) of about 30 s has been observed by analyzing the correlation functions, which represents formation of a collapsed state. This time constant varies with urea concentration representing an inverted Chevron plot that shows a roll-over behavior in the absence of arginine. To the best of our knowledge, this is one of the first applications of fluorescence correlation spectroscopy to study direct folding kinetics of a protein.

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Measuring Unfolding of Proteins in the Presence of Denaturant Using Fluorescence Correlation Spectroscopy

Cited by 101 publications

References 32 publications

Translational and Rotational Motions of Albumin Sensed by a Non-Covalent Associated Porphyrin Under Physiological and Acidic Conditions: A Fluorescence Correlation Spectroscopy and Time Resolved Anisotropy Study

Translational and Rotational Motions of Albumin Sensed by a Non-Covalent Associated Porphyrin Under Physiological and Acidic Conditions: A Fluorescence Correlation Spectroscopy and Time Resolved Anisotropy Study

The kinetics of conformational fluctuations in an unfolded protein measured by fluorescence methods

Role of Protein Stabilizers on the Conformation of the Unfolded State of Cytochrome c and Its Early Folding Kinetics

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