β2m is a protein subunit of the class I major histocompatibility complex which is normally soluble. In the joints of patients undergoing long-term dialysis as a consequence of kidney disease, β2m forms insoluble amyloid fibrils, which are large filamentous protein aggregates [1,2]. Both in vivo and in vitro evidence suggests that Cu(II) plays a role in this process [3], but almost nothing is known about the early stages of the protein's oligomerization. Insight into the early stages of this process, should provide the stimulus for the rational design of therapeutics against this dialysis-related amyloidosis. In this application note, the value of dynamic light scattering (DLS) to study the early stages of amyloid formation by β-2-microglobulin (b2m) is examined.
To form oligomers and eventually fibrils of β2m, a 100μM solution of protein was incubated at pH 7.4 with 200μM of CuSO4. The solution was buffered at pH 7.4 using 25mM MOPS and also contained 200mM potassium acetate and 500mM urea. Control experiments were done in an identical manner, except CuSO4 was not added to the solution and 1mM EDTA was added to complex any trace metals. Light scattering measurements were conducted using a Malvern Zetasizer Nano ZS instrument. Aliquots (45µL) of the incubation mixture or the control sample were analyzed everyday for 3 weeks. Each measurement was averaged over five runs of 30 seconds. At least three replicate measurements were performed for each sample.
Light scattering measurements indicate that prior to fibril formation, β2m forms dimers, tetramers, and hexamers in the presence of Cu(II), with no odd-ordered oligomeric intermediates observed. This suggests that the oligomers form via the addition of dimeric units. Two other observations provide important insight into the early stages of β2m fibril formation: (1) the hexamer is first measured in solution at about the same time (day 4) that the first amyloid fibrils are observed; and (2) no octamers, decamers, or larger even-ordered oligomers are measured in solution. The only other measured particles have diameters that exceed 100 nm, and these particles have very broad size distributions (see Figure 1).
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Based on the light scattering data and complementary data from size-exclusion chromatography and mass spectrometry, the hexameric oligomer is hypothesized to be the nucleus necessary for subsequent formation of insoluble amyloid fibrils. The prevailing general mechanism for amyloid fibril formation is that the process proceeds through an intermediate nucleus from which subsequent growth of the amyloid fibrils rapidly occurs [4].
Such a nucleus is consistent with the lag-phase kinetics observed with all amyloid forming systems and is consistent with the observation that fibril formation can be accelerated by "seeding" the reaction. In our experiments, the coincident observation of the hexamer and amyloid fibrils in the incubated sample indicates that the hexamer could be this nucleus. Furthermore, the absence of octamers, decamers, and other larger even-ordered oligomers suggest that once the hexamer is formed, fibril formation is rapid enough that no other intermediates are measurable.
The Zetasizer Nano system from Malvern Instruments is the first commercial instrument to include the hardware and software for combined dynamic, static, and electrophoretic light scattering measurements. The wide range of sample properties available for measurement with the Zetasizer Nano system include, particle size, molecular weight, and zeta potential.
The Zetasizer Nano system was specifically designed to meet the low concentration and sample volume requirements typically associated with pharmaceutical and biomolecular applications, along with the high concentration requirements for colloidal applications. Satisfying this unique mix of requirements was accomplished via the integration of a backscatter optical system and the design of a novel cell chamber. As a consequence of these features, the Zetasizer Nano specifications for sample size and concentration exceed those for any other commercially available dynamic light scattering instrument, with a size range of 0.6nm to 6µm, and a concentration range of 0.1mg/mL lysozyme to circa 40% w/v.
Complementary to the patented and award winning hardware design, is the DTS software, providing instrument control and data analysis for the Zetasizer Nano System. The DTS software utilizes self analyzing algorithms to insure that the optical setup is optimized for each set of experimental conditions, and includes a unique SOP and Expert Mode protocols to minimize the new user learning curve.
[1] Floege, J.; Ehlerding, G. "Beta-2-microglobulin associated amyloidosis," (1996) Nephron.72, 9-26.
[2] Floege, J.; Ketteler, M. "Beta(2)-microglobulin-derived amyloidosis: An update," (2001) Kidney Int. 59, S164-S171.
[3] Morgan, C. J.; Gelfand, M.; Atreya, C.; Miranker, A. D. "Kidney dialysis-associated amyloidosis: A molecular role for Cu(II) in fiber formation," (2001) J. Mol. Biol. 309, 339-345.
[4] Rochet, J. C.; Lansbury Jr., P. T. "Amyloid fibrillogenesis: Themes and variations," (2000) Curr. Opin. Struct. Biol. 10, 60-68.
