Dynamic Light Scattering For the Noninvasive Detection of Protein Aggregation

Protein homogeneity is an important issue in many fields of research, be it the characterization of newly isolated molecules, the identification of crystallizable samples, or the preparation of formulations for therapeutic applications. In each case, the biological material must be analyzed under conditions that do not alter either its composition or its activity.

Protein heterogeneity

Many proteins share the characteristic of heterogeneity with respect to mass, charge, or shape. The causes of protein heterogeneity are multiple. Partial post-translational modifications in vivo can be a source when the protein molecules are overproduced in another organism. Incomplete cleavage by proteolytic enzymes during extraction from the cellular medium is another one since it frequently generates a mixture of polypeptide chains that differ by length and mass. Because of the presence of charged amino acid residues, the heterogeneity in mass is frequently accompanied by a heterogeneity in charge. As a consequence, oligomeric proteins formed by several subunits can exhibit complex patterns upon separation according to hydrodynamic or electric properties.

Finally, many unmodified proteins have a propensity to aggregate when they are very pure. Aggregation involves either hydrophobic or ionic interactions or disulfide bridge formation, and the aggregates are generally highly polydisperse in size. In some instances they can be in equilibrium with the monomeric form of the protein. The study of the structural features of such protein particle populations requires noninvasive analytical methods.

Protein aggregation detection methods

Great charge differences are often detectable by ion exchange chromatography, but more subtle ones may be visualized only by gel electrophoresis or isoelectric focusing. Most often, these approaches are insufficient to confirm either the presence or absence of aggregates and they alter the sample.

Major differences in particle mass, volume, or shape can usually be monitored by fractionating the sample's content on a size exclusion chromatography column or in an ultracentrifuge. These methods have the disadvantage that nonspecific and loose aggregates can be disrupted by the shear forces resulting from solvent flow around the beads composing the column or by the pressure created in the gravity field. On the other hand, field flow fractionation provides a gentler separation of the macromolecular components. Further, spectroscopic methods are a good alternative to gain insight into the composition of a mixture.

Dynamic light scattering: A noninvasive method

Dynamic light scattering (DLS), also called quasielastic light scattering (QELS) or photon correlation spectroscopy (PCS), is the least invasive of all the analytical methods. Scientists at the Institute for Molecular and Cellular Biology (IBMC) (Strasbourg, France) have been working with the DynaPro instrument (Wyatt Technology, Santa Barbara, CA) for a decade. Sedimentation of the dust particles and large aggregates that negatively affect the light scattering measurements is the method of choice, as opposed to eliminating them by filtration because filter membranes may not be entirely inert and may adsorb charged or hydrophobic particles.

In practice, the sample (12 μL of solution at 1–2 mg/mL for a protein with a relative mass of 50,000) is transferred to a square quartz cuvette that is centrifuged for 10 min at 4500 rpm in a tabletop centrifuge. The cuvette is then placed in the temperature-controlled holder of the DynaPro and it is irradiated by the visible beam of a laser. The small fraction of the incident photons that is scattered by the particles in solution is collected at a fixed angle and the signal is amplified by an avalanche diode.

The variation of the signal over time is analyzed by a correlator and is used to generate an autocorrelation curve. The autocorrelation function is calculated from the latter by exponential regression and the translational diffusion coefficient (D) of the particles from which the variance is derived. Assuming that the particles are hard spheres with a known density, their hydrodynamic radius can be calculated and the apparent molecular mass extrapolated.

The variance on D gives the polydispersity on the radius. On average, a measurement takes approximately 12 sec, and valuable statistics are typically obtained with a set of two dozen measurements. The value of the baseline and the sum of the squares characterizing the regression are important indicators of the quality of the data. Moreover, the polydispersity is an invaluable piece of information in addition to the particle parameters. Since proteins are never dissolved in pure water, corrections must be applied to take into account the composition of the solvent. In particular, the solvent viscosity and refractive index must be known. At any moment the data can be recalculated with the help of a list of standard solvents that is provided in a separate file, and the histogram of the size distribution of the particles can be displayed.

A primary advantage of the DynaPro is that the samples are contained in cuvettes, and thus they can be recovered easily. Secondly, chemicals (i.e., detergents, salts, or reducing agents) can be added to the sample in order to test their effects on the protein aggregates. Also, biochemicals such as ligands (e.g., substrates or inhibitors) can be assayed to monitor their influence on the degree of aggregation of enzymes or receptors.

Results

Figure 1 - Crystallographic and biochemical analyses have shown that the best crystals of hen egg-white lysozyme can be grown in vitro only if the protein is free of macromolecular impurities and aggregates.

At the IBMC, focus is placed on the study of the crystallogenesis of proteins and their complexes with nucleic acids. The goal is to find solvent conditions under which these entities crystallize, and crystals that are of the best quality for structural studies by X-ray or neutron crystallography (Figure 1). A DLS analysis is routinely performed prior to setting up crystallization assays in addition to specific activity assays, as well as electrophoresis, size exclusion chromatography, and ultracentrifugation analyses. The reason for this is that monodisperse protein samples crystallize more often and produce better crystals than polydisperse ones. DLS can thus provide important information on the possibility of obtaining crystals. It can also be used to search for an adequate solvent instead of setting up a large number of useless crystallization assays.

Two striking examples illustrate the power of DLS measurements. First, it is possible to show within a few minutes that a single cycle of sample freezing is sufficient to produce protein aggregates. After freezing at –20 °C, a sample that was initially monodisperse became polydisperse. Second, it was surprising to discover that some proteins can be subjected to a tremendous aggregation (that is invisible to the naked eye) after the addition of a low concentration of a nonionic or a zwitterionic detergent (such as beta-octylglucoside, lauryldiamino oxide, and 3-[(3-cholamidopropyl)dimethylammonio]- 1-propanesulfonate [CHAPS]). This was all very unexpected because these compounds are exclusively known as solubilizing agents of protein aggregates. All these DLS measurements were performed in less than an hour and were reproducible.

Conclusion

DLS is an indispensable tool for the accurate characterization of the state of proteins or nucleoprotein complexes in solution. It is a simple, rapid, and noninvasive method for verifying the presence of aggregates in biological samples.

Dr. Lorber is Senior Scientist, Crystallogenesis Group, Dept. UPR9002, Institute for Molecular and Cellular Biology of CNRS, F-67084 Strasbourg, France; tel.: +33 3 8841 7008; fax: +33 3 8860 2218; e-mail: b.lorber@ibmc.u-strasbg.fr.

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