Oligomeric states of a thermophile

The Malvern Zetasizer Nano S was used to measure the size, molecular weight and second virial coefficient of pyrrolidone carboxyl peptidase PCP at pH 2.5. 3.0 and 7.5. The results clearly indicate the form of the molecular structure changes with pH. At low pH monomer and at higher pH tetramer, supported by crystallography. Estimated MW from empirical model to gain molecular mass from size attains good match with expectation.

Prof. Nakagawa, Dr. Ogasahara, Inst. for Protein Research, Osaka University, Japan

Introduction

This application note presents the characterization of a protein isolated from hyperthermophiles, which are often described as ultrastable due to their extremely high denaturation points. We investigate pyrrolidone carboxyl peptidase (PCP) from Pyrococcus furiosus to study the assembly behavior at different pH solution conditions.

PCP has been crystallized at acidic conditions as a tetramer, with a molecular weight of the complex of about 92 kDa. The crystallographic structure is shown in fig. 1.

Figure 1: Molecular structure of PCP at pH 4.6 (tetramer) [pdb:1IOI]
mrk841 fig1

Experimental

Wild-type PCP was expressed in Escherichia coli and purified with centrifugation, various additional filtration and chromatography steps. The final product showed a single band on SDS/ PAGE.[1]

Samples were dialyzed in different pH buffers (pH 7.5, 3.0, and 2.5) and then measured in the Zetasizer Nano without further additional filtration.

Results

All samples appear clear. Dynamic light scattering (DLS) measurements were performed in automatic mode, and all samples prepared at a series of concentrations (between 3.0 and 0.1 mg/mL) in order to determine the molecular weight by static light scattering as well.

Hydrodynamic size

This protein was crystallized as a tetramer, assuming a roughly globular molecule we would expect a size near 8.2nm diameter for the tetrameric form. We use dynamic light scattering (DLS) to analyze the fluctuations in the scattered light. These lead to the diffusion coefficient and applying knowledge of the viscosity of the diffusing medium (aqueous buffer in this case) we can obtain a hydrodynamic size of the scattering object. This definition of this size is the size of an equivalent sphere that diffuses with the same diffusion coefficient as the protein molecules. We look at the intensity size distribution shown in figure 2. At low pH the size is smallest, at neutral pH we observe the largest mean value for the distribution. The intermediate pH is nestled between the two peaks. DLS results are summarized in Table 1.

Figure 2: Size Distribution of PCP at pH 2.5 (blue), pH 3.0 (green) and pH 7.5 (red)
mrk841 fig2
Table 1: Peak information summary for PCP under different pH conditions
Buffer conditionMean size of Peak (d.nm)
Estimated MW
Width (%Pd)
pH 2.54.3 nm
20 kDa
15%
pH 3.06.7 nm
57 kDa
24%
pH 7.58.9 nm
109 kDa
19%

We notice that the blue and red peak are narrow, with a polydispersity of less than 20%. The green peak shows a broader distribution of 24%. As a rule of thumb, when proteins exhibit a peak width above 20% up to 30% this can be caused by the presence of other (oligomeric) species in solution. When we look at the green sample (pH 3.0) with the Protein Wizard from Malvern we find that the result obtained would be consistent with 74% of dimer (estimated to be 41kDa) and 26% by mass of tetramer.

The results from DLS of the other two samples at 4.3nm and 8.9nm are consistent with mainly monomer (pH 2.5) and tetramer forms (pH 7.5).

Molecular weight

In static light scattering (SLS) the absolute MW is determined by detecting the scattering signal from known concentrations of the sample. This measurement provides the mass average molecular weight of all species present in solution. The scattered intensity signal is adjusted for the signal from the buffer alone (~30 kcps) and scaled with the scattering standard toluene (307 kcps).

The refractive index increment dn/dc of many proteins is approximately 0.185 mL/g. We use this average number for the PCP samples.

The scattering intensities from the different concentrations are used in the Debye Plot facility of the Zetasizer Nano software to calculate a mass average molecular weight. We show the overlay of the plots for all three conditions in Figure 3, the numerical results for the mass and the second Virial coefficient are shown in table 2.

Figure 3: Debye plot, overlay of intensity and Kc/R for the three different samples
mrk841 fig3
Table 2: Static light scattering results from PCP, molecular weight and second Virial coefficient A2 are dependent on pH
Buffer conditionMolecular WeightA2   [mL mol/g2 ]
pH 2.531 kDa1.1 10-3
pH 3.068 kDa5.5 10-4
pH 7.594 kDa8.4 10-5

The results are in reasonable agreement and support the working hypothesis that primarily monomer exists at the most acidic buffer condition, whereas dimer and tetramer prefer slightly higher pH values.

Summary

A thermophilic protein pyrrolidone carboxyl peptidase (PCP) was characterized by dynamic and static light scattering. Results support the existence of different oligomeric forms as a function of pH of the buffer. The protein is primarily monomeric at pH2.5, dimeric at pH3 and prefers tetrameric quarternary structure at neutral pH.

 [1] K. Ogasahara, N.N. Khechinashvili, M. Nakamura, T. Yoshimoto and K. Yutani Eur. J. Biochem.268 (2001), 3233-3242

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