May 1, 2011 (Vol. 31, No. 9)

Concentration-Gradient Dynamic Light Scattering Finds Uses in High-Throughput Setting

Protein-protein interactions are intensively studied, not only to gain understanding of cellular function, but also for pharmaceutical development. In vitro, such complexes may be difficult to separate and analyze in pure form, as they equilibrate rapidly with their component monomers and partially formed complexes.

Accordingly, methods that evaluate reversible interactions at true equilibrium are indispensable. Since any modification of the protein, either by immobilization or by labeling, can influence the interaction, free-solution, label-free methods are optimal. However, methods meeting these criteria—sedimentation equilibrium analytical ultracentrifugation, isothermal titration calorimetry, and more recently, concentration-gradient static light scattering—all require a relatively large quantity of sample when used in their standard configurations and are not ideally suited for high-throughput measurement.

In contrast, there is a dynamic light scattering (DLS) method, concentration-gradient DLS, which enables accurate and quantitative characterization of protein-protein interactions in a high-throughput manner, using only picomoles of sample. DLS, also known as quasielastic light scattering or photon correlation spectroscopy, processes the time-dependent fluctuations in scattered light to yield the hydrodynamic radius, rh, of particles in solution.

This method gives the ability to detect and characterize multiple binding stoichiometries, small-molecule inhibition of protein-protein interactions, and changes of interactions with temperature or solution conditions. Recent innovations in DLS instrumentation allow measurements from microtiter plates, enabling high-throughput measurements using only a few µL of sample per condition. These measurement volumes reduce protein requirements by several orders of magnitude over the methods listed above, and coupled with high-throughput measurements make DLS characterization of protein-protein interactions practical.

Methods and Results

If multiple species are present in solution and have an rh within a factor of ~3 of each other, then the rh measured by DLS is an average over those species, called ravg. If two proteins A and B associate to form a complex AB, then the presence of the complex increases ravg, and the increase in ravg may be used to calculate the degree of association. The method of continuous variation is a standard method whereby a parameter proportional to complex formation is measured as the molar concentration of [A] with respect to [B] is altered, e.g., from 0–100%, with the total molar concentration [A] + [B] fixed. The concentration-gradient DLS method demonstrated here is the method of continuous variation with ravg derived from DLS as the measurable parameter. It determines Kd, which gives the molar concentration of a complex given the molar concentrations of the individual species as:

[AB] = [A][B]/Kd

Concentration-gradient DLS was used to characterize the binding of the model system α-chymotrypsin and soybean trypsin inhibitor (STI), using both 10 µL/well and 1 µL/well sample volumes. Agreement of the 10 µL and 1 µL results indicated that reduction in the sample volume had minimal impact on data quality. Sample stock solutions were 500 µg/mL concentration, and the concentration gradient was produced by volumetrically combining different ratios of stock solutions.

Lower concentrations may be used, particularly for larger proteins which scatter more light such as antibodies, which may have stock concentrations of 20 µg/mL. Measurements were made with randomized placement of five replicate wells, and the error bars shown are the standard deviation of results from the five wells.

A maximum of ravg at 0.5 mole fraction is consistent with 1:1 binding. For this system, ravg reached a maximum value at ~0.75 mole fraction, indicating that STI possesses more than one binding site, as has been previously reported. The data was fit assuming STI possesses two binding sites of equal affinity along with an “incompetent fraction”, which had also been previously reported.

Measurements were made with the temperature sequence 25-20-15-10-5-25-30-35-25, and Kd determined at each temperature, with the repeat measurements at 25°C used to ensure that time and elevated temperature did not alter the system. For one series of measurements, the chymotrypsin was pretreated with the small-molecule serine protease inhibitor AEBSF. The AEBSF-treated chymotrypsin was found to no longer bind with STI, demonstrating the detection of small-molecule inhibition of binding.

The 25°C and AEBSF measurements are shown in Figure 1A, and the measurements as a function of temperature are shown in Figure 1B. Thermodynamic information was obtained using a van’t Hoff plot (Figure 1C), with results for the change in enthalpy ΔH°=12±4 kcal/mol, and the change in entropy ΔS°=70±14 cal/(mol K). The Table summerizes all measured kd values.

Figure 1. Characterization of a 2:1 α-chymotrypsin:soybean trypsin inhibitor interaction at neutral pH as a function of temperature: Data is presented as ravg vs. mole fraction of 0 to 1, with the mole fraction being the total molar concentration of chymotrypsin divided by the molar concentrations of both chymotrypsin and soybean trypsin inhibitor, yielding a range of entirely soybean trypsin inhibitor (0), to entirely chymotrypsin (1). Symbols represent measured data; fits are shown as solid lines. (A) Control measurements and small-molecule inhibition study (AEBSF). (B) Association as a function of temperature: Profile expected for no association is shown by dotted line. In A and B, error bars show S.D. (C) van’t Hoff plot: Ka values from fits in B shown in black. Error bars represent change in Ka value required to increase reduced χ2 by 1. Minimization of χ2 was used to fit the data, yielding a slope of -ΔH°/R and an intercept of ΔS°/R.

Although not optimized for reduced sample use, these nondestructive experiments used only 465 picomoles of protein (Twenty 1 µL solutions at 500 µg/mL, minimum molar mass of 21.5 kDa). As the intensity of scattered light is proportional to the product of molar mass squared and molar concentration, similar studies with antibodies (150 kDa) should require only 2.9 femtomoles (twenty 1 µL solutions at 22 ug/mL).

Although the experiments conducted involved protein-protein interactions, the method is applicable to a much broader class of reactants. Given the lack of a protein-specific labeling or immobilization procedure, extension of the technique to other biomolecules such as tRNA is straightforward. Additionally, in conjunction with other techniques, e.g., small-molecule screening or site directed mutagenesis, concentration-gradient DLS may enable the identification of residues involved in protein molecular recognition. In this manner, concentration-gradient DLS may be used not only to characterize biomolecular interactions, but also to explain them.

The Table summarizes all measured Kd values.

Michael I. Larkin ([email protected]) is a principal scientist, and Amy D. Hanlon is a former employee in the department of research and development at Wyatt Technology.

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