Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023:2551:189-201.
doi: 10.1007/978-1-0716-2597-2_13.

Cross-Linking Mass Spectrometry Analysis of Metastable Compact Structures in Intrinsically Disordered Proteins

Affiliations

Cross-Linking Mass Spectrometry Analysis of Metastable Compact Structures in Intrinsically Disordered Proteins

Dailu Chen et al. Methods Mol Biol. 2023.

Abstract

Protein assembly into beta-sheet-rich amyloids is a common phenomenon in neurodegenerative diseases including Alzheimer's (AD) and Parkinson's (PD). The proteins implicated in amyloid deposition are often intrinsically disordered proteins (IDPs) and are characterized by not folding into a defined globular conformation. The amyloidogenic properties of IDPs are determined by the presence of short sequence elements, referred to as amyloid motifs, that drive ordered aggregation (Thompson MJ, Sievers SA, Karanicolas J et al. Proc Natl Acad Sci USA 103(11):4074-8, 2006; Goldschmidt L, Teng PK, Riek R et al. Proc Natl Acad Sci USA 107(8):3487-92, 2010]. The microtubule-associated protein tau adopts amyloid assemblies in over 20 different diseases commonly referred to as tauopathies. However, native tau is aggregation-resistant despite encoding at least three amyloid motifs (Chen D, Drombosky KW, Hou Z et al. Nat Commun 10(1):2493, 2019). Recent cryogenic electron microscopy (cryo-EM) structures of tau amyloid fibrils isolated from patient brains showed the involvement of amyloid motifs in the fibril core (Fitzpatrick AWP, Falcon B, He S et al. Nature 547(7662):185-90, 2017; Falcon B, Zhang W, Murzin AG et al. Nature 561(7721):137-40, 2018; Zhang W, Tarutani A, Newell KL et al. Nature 580(7802):283-7, 2020). How does tau change from an aggregation-resistant state to an aggregation-prone state? Consistent with the fibril structures, we hypothesize that tau must change conformation to expose the amyloid motifs that allow self-association into beta-sheet-rich aggregates. This would suggest that the amyloid motifs are likely buried in natively folded tau to prevent self-assembly. We developed an approach that couples cross-linking mass spectrometry (XL-MS) with temperature denaturation to probe the loss of contacts as a proxy to measure protein unfolding with sequence resolution. Using this method, we demonstrated that disease-associated mutations in tau located near an amyloid motif disrupt the protective local structure, promote amyloid motif exposure, and thus lead to aggregation (Chen D, Drombosky KW, Hou Z et al. Nat Commun 10(1):2493, 2019). In this chapter, we describe the detailed protocol for this approach. We anticipate that our protocol can be generalized to other IDPs and will help discover critical structural elements to better understand important biological questions including protein aggregation.

Keywords: Amyloid motifs; Chemical cross-linking and mass spectrometry; IDPs; Intrinsically disordered proteins; Protein aggregation; Protein folding; Protein misfolding; Secondary structures.

PubMed Disclaimer

Similar articles

Cited by

References

    1. Thompson MJ, Sievers SA, Karanicolas J et al (2006) The 3D profile method for identifying fibril-forming segments of proteins. Proc Natl Acad Sci U S A 103(11):4074–4078 - DOI - PubMed - PMC
    1. Goldschmidt L, Teng PK, Riek R et al (2010) Identifying the amylome, proteins capable of forming amyloid-like fibrils. Proc Natl Acad Sci U S A 107(8):3487–3492 - DOI - PubMed - PMC
    1. Chen D, Drombosky KW, Hou Z et al (2019) Tau local structure shields an amyloid-forming motif and controls aggregation propensity. Nat Commun 10(1):2493 - DOI - PubMed - PMC
    1. Fitzpatrick AWP, Falcon B, He S et al (2017) Cryo-EM structures of tau filaments from Alzheimer’s disease. Nature 547(7662):185–190 - DOI - PubMed - PMC
    1. Falcon B, Zhang W, Murzin AG et al (2018) Structures of filaments from Pick’s disease reveal a novel tau protein fold. Nature 561(7721):137–140 - DOI - PubMed - PMC

Publication types

LinkOut - more resources