Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides

Summary

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.

Abstract

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, Parkinson's, and Huntington's diseases, hereditary amyotrophic lateral sclerosis, and type 2 diabetes. Owing to the metastable nature of these protein assemblies, it is difficult to assess their oligomer size distribution quantitatively using classical methods, such as electrophoresis, chromatography, fluorescence, or dynamic light scattering. Oligomers of amyloidogenic proteins exist as metastable mixtures, in which the oligomers dissociate into monomers and associate into larger assemblies simultaneously. PICUP stabilizes oligomer populations by covalent cross-linking and when combined with fractionation methods, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or size-exclusion chromatography (SEC), PICUP provides snapshots of the oligomer size distributions that existed before cross-linking. Hence, PICUP enables visualization and quantitative analysis of metastable protein populations and can be used to monitor assembly and decipher relationships between sequence modifications and oligomerization¹. Mechanistically, PICUP involves photo-oxidation of Ru²⁺ in a tris(bipyridyl)Ru(II) complex (RuBpy) to Ru³⁺ by irradiation with visible light in the presence of an electron acceptor. Ru³⁺ is a strong one-electron oxidizer capable of abstracting an electron from a neighboring protein molecule, generating a protein radical^1,2. Radicals are unstable, highly-reactive species and therefore disappear rapidly through a variety of intra- and intermolecular reactions. A radical may utilize the high energy of an unpaired electron to react with another protein monomer forming a dimeric radical, which subsequently loses a hydrogen atom and forms a stable, covalently-linked dimer. The dimer may then react further through a similar mechanism with monomers or other dimers to form higher-order oligomers. Advantages of PICUP relative to other photo- or chemical cross-linking methods^3,4 include short (≤1 s) exposure to non-destructive visible light, no need for pre facto modification of the native sequence, and zero-length covalent cross-linking. In addition, PICUP enables cross-linking of proteins within wide pH and temperature ranges, including physiologic parameters. Here, we demonstrate application of PICUP to cross-linking of three amyloidogenic proteins the 40- and 42-residue amyloid β-protein variants (Aβ40 and Aβ42), and calcitonin, and a control protein, growth-hormone releasing factor (GRF).

Protocol

1. Peptide preparation

1. Weigh out ~100–200 μg of lyophilized peptide using a microbalance and transfer into labeled, silicon-coated, low-adsorbent microfuge tubes. Here, we use the human sequences of Aβ40, Aβ42, calcitonin, and GRF.
2. Here, we use peptides pre-treated with 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) to obtain homogeneous, aggregate-free preparations. This step is necessary because pre-formed aggregates induce rapid aggregation of amyloidogenic proteins, which result in poor reproducibility among experiments⁵. Other methods such as filtration and SEC also can be used to obtain aggregate-free preparations for PICUP⁶.<....

Discussion

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 PrP^{Sc 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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dolan-Jenner 200-W incandescent lamp	Other	Dolan-Jenner Industries	Model 170-D	The heat generated by the lamp d–s 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 Chase	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-Aldrich	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-Aldrich	A-7460	Vortex until the solution is clear. APS is prepared freshly each time and should be used within 48 h.
β-mercapt–thanol	Reagent	Sigma-Aldrich	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