Photo-induced cross-linking of unmodified proteins (PICUP) allows characterization of oligomer size distribution in metastable protein mixtures. We demonstrate application of PICUP to three representative amyloidogenic peptides the 40- and 42-residue forms of amyloid β-protein, and calcitonin, and a control peptide growth-hormone releasing factor.
1. Peptide preparation
2. Solubilizing the HFIP-treated peptides and photo cross-linking
3. SDS-PAGE and sliver-staining of cross-linked peptide products
Part 4: Representative Results (Figure 1)
Figure 1: Silver-stained polyacrylamide gels showing uncross-linked (−) and photo-cross-linked (+) GRF, calcitonin, Aβ40, and Aβ42
SDS-PAGE and silver-staining analyses of PICUP-generated Aβ40 oligomers show approximately similar band intensities of monomer through tetramer, followed by a sharp decrease in the abundance of higher oligomers7. Uncross-linked Aβ40 migrates with an Mr consistent with that of monomer7. Aβ42 shows a distinct oligomer size distribution. Aβ42 oligomers comprise 2–3 groups8. The first group, monomer through trimer, displays decreasing intensity with increasing oligomer order. In the second group, a Gaussian-like distribution is observed between tetramer and heptamer, with a maximum at pentamer and hexamer. The third group, which is not shown here, contains oligomers of Mr ~30,000–60,000 Da8. These higher Mr oligomers typically are observed using SEC-isolated LMW Aβ42 but not peptide prepared by filtration or HFIP treatment6. Uncross-linked Aβ42 produces predominantly two bands, a monomer band and a broad trimer/tetramer band, which is an artefact induced by SDS5,9. Calcitonin yields an oligomer size distribution that strongly diverges from a monotonous exponential decrease in the region monomer through tetramer, suggesting pre-existence of dimers, trimers, and tetramers7. The control polypeptide, GRF, produces a monotonic oligomer distribution ranging from dimer through hexamer such that the apparent relative amounts of oligomers decrease exponentially with increasing molecular mass. In other experiments, higher oligomers also could be visualized7. The exact number and relative intensities of oligomer bands may vary somewhat among experiments depending on the actual protein concentration, the amount of protein loaded on the gel, and the time used for the development step in the silver-staining protocol.
PICUP was developed originally to study stable protein complexes2. The method was applied later to quantitative study of metastable amyloid protein assemblies, including Aβ10, prion and disease-associated PrPSc 11, and α-synuclein12. The most important factors that must be considered when designing a PICUP experiment are the reagent stoichiometry, irradiation time, and sample preparation procedure. The former two issues may require empirical optimization, whereas the latter issue largely affects interpretation of the experimental data. For amyloidogenic proteins in particular, determination of size distributions of metastable oligomers requires using aggregate-free starting preparations. The background, mechanism, instrumentation, protocol, optimization, scope, modifications, applications, and limitations of PICUP were covered in previous publications1,2,13. PICUP can be used to generate stable, soluble protein oligomers that following fractionation and purification, could be used for structural studies, cytotoxicity assays, oligomerization-inhibition studies, and as targets for development of molecular-recognition tools.
This work was supported by grants AG027818 and AG030709 from NIH/NIA, 2005/2E from the Larry L. Hillblom Foundation, IIRG-07-58334 from the Alzheimer Association, and 07-65798 from California Department of Public Health.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Dolan-Jenner 200-W incandescent lamp | Other | Dolan-Jenner Industries | Model 170-D | The heat generated by the lamp does not affect samples for short incubation periods. |
35-mm SLR camera body | Tool | Pentax | SP500 model | In our settings, a bellows is attached to the body of the camera to provide a convenient chamber for irradiation of the sample placed 10 cm away from the light source. |
Clear, thin walled PCR tubes | Other | Eppendorf | 951010006 supplied by Fisher L22-003-24 | |
Glass vials (1.8 mL) | Other | Kimble Chromatography | 60940A 2, supplied by Fisher 03-340-60 | |
GRF | Reagent | Bachem | H-3695 | |
HFIP | Reagent | TCI America | H0424 | Use in a fume hood. |
Aβ40 and Aβ42 | Reagent | UCLA Biopolymers Laboratory | ||
Calcitonin | Reagent | American Peptide | 22-1-10 | |
Tris(2,2-bipridyl)dichlororuthenium(II) hexahydrate | Reagent | Sigma | 224758-1G | Vortex until the solution is clear. Cover the RuBpy tube with foil to protect the reagent from ambient light. RuBpy is prepared freshly each time and should be used within 48 h. |
Ammonium persulfate | Reagent | Sigma | A-7460 | Vortex until the solution is clear. APS is prepared freshly each time and should be used within 48 h. |
β-mercaptoethanol | Reagent | Sigma | M7154-25 ML | Can be used when SDS-PAGE analysis is performed. |
Novex Tricine SDS Sample Buffer (2x) | Reagent | Invitrogen | LC1676 | |
XCell SureLock Mini-Cell | Tool | Invitrogen | EI0001 | |
Novex Tricine Gels (10-20%) | Other | Invitrogen | EC6625B0X | |
Novex Tricine SDS Running Buffer (10x ) | Invitrogen | LC1675 | ||
Silver Express Staining Kit | Invitrogen | LC6100 |