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Applications of analytical ultracentrifugation to protein size-and-shape distribution and structure-and-function analyses - PubMed

Applications of analytical ultracentrifugation to protein size-and-shape distribution and structure-and-function analyses

Chi-Yuan Chou et al. Methods. 2011 May.

Abstract

The rebirth of modern analytical ultracentrifugation (AUC) began in 1990s. Since then many advanced AUC detectors have been developed that provide a vast range of versatile choices when characterizing the physical and chemical features of macromolecules. In addition, there have been remarkable advances in software that allow the analysis of AUC data using more sophisticated models, including quaternary structures, conformational changes, and biomolecular interactions. Here we report the application of AUC to protein size-and-shape distribution analysis and structure-and-function analysis in the presence of ligands or lipids. Using band-sedimentation velocity, quaternary structural changes and an enzyme's catalytic activity can be observed simultaneously. This provides direct insights into the correlation between quaternary structure and catalytic activity of the enzyme. On the other hand, also in this study, we have applied size-and-shape distribution analysis to a lipid-binding protein in either an aqueous or lipid environment. The sedimentation velocity data for the protein with or without lipid were evaluated using the c(s,f(r)) two-dimensional distribution model, which provides a precise and quantitative means of analyzing the protein's conformational changes.

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Figures

Fig. 1
Fig. 1

Band-forming active enzyme centrifugation of Mpro in the presence of 200 μM substrate. (A) A typical trace of absorbance at 250 nm of the enzyme during the experiment. The symbols are experimental data and the lines are the results fitted to the Lamm equation using the SEDFIT program. (B) The residual bitmap of the raw data and the best-fit results. (C) Continuous c(s) distribution from the best fit analysis. The species at S = 2.3 corresponds to the dimeric Mpro. The protein amount used is 15 μl (1 mg/ml) in PBS (pH 7.6). The substrate at 200 μM was dissolved in D2O to give a higher density, which sharpens the protein band when the centrifugation begins. Total volume is 330 μl.

Fig. 2
Fig. 2

Monitoring of enzymatic activity of Mpro during band-forming ultracentrifugation. The same cell in Fig. 1 was followed using the enzymatic reaction under identical conditions as described above. Panel (A) shows the absorbance at 405 nm trace for the released product (pNA) after the first hour of the experiment. The time interval of each spectrum from black to pink color is 10 min. Panel (B) showed the product at different time with different substrate concentrations (close circle: 5 μM; open circle: 25 μM; close triangle, 50 μM; open triangle, 100 μM; close square, 200 μM; open square, 400 μM; close diamond, 500 μM; and open diamond, 600 μM). The lines indicated the best-fit results for initial velocity calculation. Panel (C) showed the plot of initial velocities versus substrate concentrations. The line represented best-fit results according to the Michaelis–Menten equation. The inset plot shows the same data fitted to the Hill equation. The kinetic parameters derived are shown in Table 1.

Fig. 3
Fig. 3

Effects of substrate concentration on the quaternary structure of Mpro measured by band-forming ultracentrifugation. Different curves represented the continuous c(s) distribution of Mpro at substrate concentration of 0 (filled circle), 5 (open circle), 25 (filled triangle), 50 (open triangle), 100 (filled square), and 200 (open square) μM. The labels M and D showed the position of the monomer and dimer species, respectively. To clearly display the other results, that for 200 μM (Fig. 2C showed the full scale) is only partially shown. According to our previous studies, the result at 50 μM substrate, which showed a broad peak between monomer and dimer, suggested that Mpro is a rapid self-association protein.

Fig. 4
Fig. 4

Representative SV raw data subset of apoE3-(72–166) protein. The experiments were run at a rotor speed of 42,000 rpm at 20 °C. Traces were calculated at time intervals of 480 s. For clarity, only every sixth scan is shown. The A–C indicated the results of apoE3-(72–166) protein in PBS (pH 7.3) (aqueous), or 5 mM, and 50 mM DHPC (lipid) solutions, respectively.

Fig. 5
Fig. 5

Continuous c(s,fr) 2d distribution analysis of apoE3-(72–166) protein in aqueous or lipid environments. (A–C) apoE3-(72–166) in PBS, 5 mM, and 50 mM DHPC, respectively. The protein concentration was 0.5 mg/ml. The x, y, and z axes show the sedimentation coefficients (S), local concentration c(s), and anhydrous frictional ratio (fr), respectively. The colors define the local concentration of the species (red to blue: high to low). Contour plots are shown at the bottom. Insets, a grayscale of the residual bit map showing the data fitting quality. Calculation based on the SEDFIT program.

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