Time Resolved Collapse of a Folding Protein Observed with Small Angle X-Ray Scattering

Pollack, Lois; Täte, Mark W.; Finnefrock, Adam C.; Kalidas, Chitkala; Trotter, S.; Darnton, N.; Lurio, L. B.; Austin, Robert H.; Batt, Carl A.; Grüner, Sol M.; Mochrie, S. G. J.

doi:10.1103/physrevlett.86.4962

Cited by 158 publications

(144 citation statements)

References 29 publications

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“…This residual structure has only 2-7% native like persistence involving the turn between ␤-strands and between the two short helices. Native-like spatial positioning, orientation of chain segments, and compact denatured states have been observed in other systems (25)(26)(27). The present paper suggests that the acid unfolded state of IFABP is compact and the hydrodynamic radius of IFABP in the unfolded state is only 35% larger than that of the native state.…”

Section: Discussionmentioning

confidence: 53%

Measurement of microsecond dynamic motion in the intestinal fatty acid binding protein by using fluorescence correlation spectroscopy

Chattopadhyay

Saffarian

Elson

et al. 2002

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

117

149

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protein folding ͉ conformational dynamics ͉ diffusion coefficient ͉ unfolded state T he intestinal fatty acid binding protein (IFABP) belongs to a family of proteins that bind a variety of ligands (fatty acids, bile salts, and retinoids) into a large cavity located in the interior of the protein. The structure has been determined by both x-ray (1, 2) and NMR methods (3, 4). The x-ray structure shown in Fig. 1 is believed to represent the closed form of the apo protein, whereas the NMR structure suggests the open form (4). The structure of IFABP consists of 2 ␤-sheets, each containing 5 ␤-strands, and a small helical region. This protein has been an excellent model system for folding studies of ␤-sheet proteins because it is small (15 kDa), monomeric, and does not contain any proline or cysteine residues. Equilibrium unfolding transitions of IFABP monitored by steady state fluorescence or CD measurements can be fit by typical two-state model. However, two recent kinetic studies (5, 6) indicated the presence of intermediates, the first one forming within 200 sec. A second intermediate, formed with a rate constant of Ϸ2,000 sec Ϫ1 , contains the majority of the secondary structure (6). Previous 19 F NMR studies as a function of urea concentration had indicated the presence of an intermediate state in addition to the folded and unfolded forms (7). Hodsdon and Frieden (8), monitoring urea denaturation by NMR, found several unidentified resonances in heteronuclear sequential quantum correlation (HSQC) spectra at low urea concentrations, which disappeared at higher urea concentrations, again indicating the presence of intermediates. In the present study, we have explored the possibility of using fluorescence correlation spectroscopy (FCS) as a tool to measure the conformational dynamics and diffusional properties of apo IFABP in its folded, unfolded, and intermediate states.FCS is emerging as an important technique in chemistry, biophysics, and biochemistry for its applications in measuring diffusional properties and chemical kinetics at low molar concentrations (9, 10). The technique involves measuring fluorescence fluctuations resulting from the changes in the number of fluorophores due to diffusion or chemical reaction under conditions of thermodynamic equilibrium in a small observation volume.There are generally two kinds of applications of FCS in protein biophysics. First, the diffusion time and hence the diffusion coefficient of a protein can be measured very accurately. Second, by suitably optimizing the measurement conditions, FCS can be used to study protein dynamics or conformational events in the microsecond time scale (11).In earlier studies, we mutated specific residues of IFABP to cysteine and examined the properties of the mutated proteins (12) as well as proteins for which the fluorescent probe fluorescein was covalently attached to the cysteine residue (13). These studies showed that the probe attached to cysteine at position 60 (V60C) was located within the interior cavity and that the fluorescence p...

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Section: Discussionmentioning

confidence: 53%

Measurement of microsecond dynamic motion in the intestinal fatty acid binding protein by using fluorescence correlation spectroscopy

Chattopadhyay

Saffarian

Elson

et al. 2002

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

117

149

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“…The resulting distributions could then be used to study the dynamic nature of the solvation shells, for instance, via time-resolved scattering techniques. [51][52][53] It could also be used to test the accuracy of theoretical predictions, facilitating improvements in how those theories treat water molecules, ions, and cosolvents in general. Comparing theory vs. experiment for individual interaction terms (as in Figs.…”

Section: Discussionmentioning

confidence: 99%

Extracting water and ion distributions from solution x-ray scattering experiments

Nguyên

Pabit

Pollack

et al. 2016

The Journal of Chemical Physics

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Small-angle X-ray scattering measurements can provide valuable information about the solvent environment around biomolecules, but it can be difficult to extract solvent-specific information from observed intensity profiles. Intensities are proportional to the square of scattering amplitudes, which are complex quantities. Amplitudes in the forward direction are real, and the contribution from a solute of known structure (and from the waters it excludes) can be estimated from theory; hence, the amplitude arising from the solvent environment can be computed by difference. We have found that this "square root subtraction scheme" can be extended to non-zero q values, out to 0.1 Å −1 for the systems considered here, since the phases arising from the solute and from the water environment are nearly identical in this angle range. This allows us to extract aspects of the water and ion distributions (beyond their total numbers), by combining experimental data for the complete system with calculations for the solutes. We use this approach to test molecular dynamics and integral-equation (3D-RISM (three-dimensional reference interaction site model)) models for solvent structure around myoglobin, lysozyme, and a 25 base-pair duplex DNA. Comparisons can be made both in Fourier space and in terms of the distribution of interatomic distances in real space. Generally, computed solvent distributions arising from the MD simulations fit experimental data better than those from 3D-RISM, even though the total small-angle X-ray scattering patterns are very similar; this illustrates the potential power of this sort of analysis to guide the development of computational models. Published by AIP Publishing. [http://dx

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“…Data were collected (1 min per time point) by using pink beam (27,28), with defining slits set to 10 m in the vertical dimension and 40 m along the direction of flow. These conditions give a beam flux of Ϸ3 ϫ 10 10 photons per s; with a flow rate of 86 mm͞s along the outlet channel, these slits give an exposure time of 400 s. In addition to providing high temporal resolution, the short exposure time of each RNA molecule to the x-ray beam minimizes the potential for radiation damage to the RNA.…”

Section: Saxs Measurementsmentioning

confidence: 99%

Rapid compaction during RNA folding

Russell

Millett

Täte

et al. 2002

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

217

207

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We have used small angle x-ray scattering and computer simulations with a coarse-grained model to provide a time-resolved picture of the global folding process of the Tetrahymena group I RNA over a time window of more than five orders of magnitude. A substantial phase of compaction is observed on the low millisecond timescale, and the overall compaction and global shape changes are largely complete within one second, earlier than any known tertiary contacts are formed. This finding indicates that the RNA forms a nonspecifically collapsed intermediate and then searches for its tertiary contacts within a highly restricted subset of conformational space. The collapsed intermediate early in folding of this RNA is grossly akin to molten globule intermediates in protein folding.

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Time Resolved Collapse of a Folding Protein Observed with Small Angle X-Ray Scattering

Cited by 158 publications

References 29 publications

Measurement of microsecond dynamic motion in the intestinal fatty acid binding protein by using fluorescence correlation spectroscopy

Measurement of microsecond dynamic motion in the intestinal fatty acid binding protein by using fluorescence correlation spectroscopy

Extracting water and ion distributions from solution x-ray scattering experiments

Rapid compaction during RNA folding

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