Proteins and related biomacromolecules are complex entities that exhibit fascinating behavior when interacting with other biomolecules. Light scattering provides a simple and effective means for characterizing the essential biophysical properties of proteins: molar mass, size, charge, interactions, conjugation and conformation.
- The DAWN® and miniDAWN® multi-angle light scattering detectors interfaces with any SEC system for determination of absolute molar mass via SEC-MALS and ASTRA® software.
- Protein sizes are determined by adding a WyattQELS™ DLS module into the DAWN or miniDAWN.
- The DAWN also combines with the Calypso® composition-gradient system for the study of biomolecular interactions via CG-MALS and CALYPSO™ software.
- Rapid assessments of purity, aggregation and oligomerization may be made through size distributions via dynamic light scattering using a microwell-plate-based DynaPro® Plate Reader or a DynaPro® NanoStar® microcuvette-based instrument and the DYNAMICS® software.
- Molecular charge of fragile biomolecules is determined by combining simultaneous electrophoretic mobility and size measurements in the Mobius™ using DYNAMICS.
Molar mass is the key to identifying proteins, their oligomers or complexes, yet all too many protein researchers rely on simplistic, invalid analysis of molecular weight by SDS-PAGE or traditional size exclusion chromatography (SEC). These techniques invoke assumptions of conformation and ideal matrix interactions that may fool scientists with ambiguous or completely incorrect results, leading to fundamentally inaccurate interpretation of their data for scientific publications.
Multi-angle light scattering coupled with SEC (SEC-MALS) provides accurate molecular weight determination of proteins, oligomers and complexes, regardless of conformation or non-ideal column interactions. That's because SEC-MALS constitutes a rigorous, first-principles analysis of molar mass that does not rely on retention time or calibration with reference molecules. The only function of the SEC column is to separate molecules by size, while MALS determines molar mass of eluting proteins independently.
Conjugated & membrane proteins
Membrane proteins solubilized with detergent are particularly difficult to analyze by traditional techniques or even by mass spectroscopy because of the surfactant micelle surrounding the protein. Denaturing SDS-PAGE dissociates native oligomers and precludes their identification, while cross-linked mass spectroscopy can create oligomers that do not exist in solution.
Likewise, heavily glycosylated proteins cannot be represented by reference standards or common models for globular proteins, and so are not amenable to analysis by traditional techniques.
These challenges are met by multi-angle light scattering coupled with SEC (SEC-MALS) which can distinguish between a protein and its associated detergent or carbohydrate by combining data from three detectors downstream of SEC separation: UV, MALS and differential refractive index (dRI). ASTRA's Conjugate Analysis algorithm calculates the molar masses of both the proteinaceous component and the conjugated or micellar component. The true oligomeric or complexated state of the protein, as well as the degree of glycosylation, are determined unambiguously.
Purification & aggregates
Scientists carrying out detailed mechanistic studies of proteins and their biological function can't afford to work with poor quality material. Light scattering offers two distinct means of assessing the quality and purity of protein samples: dynamic light scattering (DLS) and size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS).
DLS is an excellent means for obtaining rapid, qualitative estimates of aggregation and impurities in a protein solution, with minimal sample consumption – as little as 1.25 µL. No separation step is required; just pipette a few drops into a microcuvette or microwell plate, and you'll have a size distribution within seconds. Large sub-micron aggregates in particular are readily identified with DLS, as well as the presence of soluble aggregates through the polydispersity parameter. Batch (unfractionated) DLS even allows for recovery of precious samples, even if only a few microliters were used in the measurement.
While DLS measures coarse distributions of size (hydrodynamic radii), you may need to distinguish and quantify small aggregates such as dimers and trimers, or obtain accurate identification of impurities or degradants. SEC-MALS performs true separations with absolute molar mass measurements in order to obtain reliable indications of just which proteins and degradants are present in solution.
Native oligomeric state
Just what is a 'native oligomeric state'? The answer may surprise you since 'native' is a relative term. Most biological oligomers consist of proteins in a dynamic equilibrium between monomers and specific oligomers (e.g., dimers or tetramers). The ratio of oligomers to monomers depends on the concentration as well as buffer pH and ionic strength, and therefore changes with analysis conditions. True identification of a native oligomer must be performed wholly in solution since the solution conditions dictate the degree of oligomerization, and even the final oligomeric number.
Multi-angle light scattering (MALS) is one of the premier methods for identifying native oligomers and determining their stoichiometry. An initial diagnosis of oligomerization is often provided by SEC-MALS which might indicate that a protein's molar mass differs significantly from monomer sequence weight, or the mass may vary over the eluting peak, decreasing with decreasing concentration on both sides of the apex. Verification may be obtained by a few additional SEC-MALS measurements consisting of different starting concentrations.
In order to obtain a rigorous analysis of oligomeric state and the equilibrium dissociation constant Kd, composition-gradient multi-angle light scattering (CG-MALS) measures self-association as a series of stop-flow injections into a MALS detector. This procedure allows for complete equilibration as well as full analysis of oligomeric stoichiometry even if more than one oligomers are present.
Dynamic light scattering (DLS), while not as quantitative as MALS, is a rapid and productive means for estimating oligomerization properties through the concentration dependence of the average molecular size.
The binding of proteins into reversible complexes underlies much of biology as we know it, whether for purposes of signaling, formation of cellular structures, cellular division, immune response or other processes crucial to maintaining a healthy, functioning organism. Many of these protein-protein interactions are difficult to analyze by traditional means such as cellular assays, gel-shift assays, or even biophysical techniques such as surface plasmon resonance or isothermal titration calorimetry, as a result of complex phenomena such as multi-valence, co-operativity, multi-protein assembly, or combined self- and hetero-association.
One of the most versatile techniques for characterizing protein and other biomolecular interactions in solution, without labeling or immobilization, is composition-gradient multi-angle light scattering (CG-MALS). In this technique, the Calypso II automatically prepares a series of compositions or concentrations and delivers them as stop-flow injections to a DAWN or miniDAWN MALS detector. The CALYPSO software uses these MALS measurements in order to determine the weight-averaged molar mass of the solution as a function of composition, then assess the fit of the data to an association scheme in order to decide if the data support the specific model and quantify the binding constants (Kd). The software provides an essentially infinite variety of association models consisting of one or two interacting species, permitting the analysis of self-association, hetero-association, combined self- and hetero-association, multi-valent interactions and co-operative binding.
The real power of CG-MALS derives from its direct assessment of molar mass, which removes all ambiguity from the determination of oligomeric state and stoichiometry of multi-protein complexes. Method development is simple and straightforward, assisted by the simulation capabilities of the CALYPSO software.
Light scattering in the service of SAXS and SANS
Beam time is expensive and hard to come by. In order to make the most of your SAXS and SANS measurements you'll want to assure the quality of your biomolecules before they reach the chamber.
DLS is a quick and easy method to check for gross aggregation in your samples. You can use at little as 1 μL in the DynaPro NanoStar's microCuvette, and even recover it if necessary! Just 4 μL are necessary with our proprietary disposable cuvettes.
For further characterization of oligomers as well as comparison with the x-ray or neutron scattering results, SEC-MALS combining your favorite SEC apparatus with a miniDAWN MALS detector and Optilab RI detector is highly desirable. See Bazin et al., "Structure and primase-mediated activation of a bacterial dodecameric replicative helicase" Nucleic Acids Res., 2015, 1
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