| 1. |
- Kaldmae, Margit, et al.
(author)
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A strategy for the identification of protein architectures directly from ion mobility mass spectrometry data reveals stabilizing subunit interactions in light harvesting complexes
- 2019
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In: Protein Science. - : WILEY. - 0961-8368 .- 1469-896X. ; 28:6, s. 1024-1030
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Journal article (peer-reviewed)abstract
- Biotechnological applications of protein complexes require detailed information about their structure and composition, which can be challenging to obtain for proteins from natural sources. Prominent examples are the ring-shaped phycoerythrin (PE) and phycocyanin (PC) complexes isolated from the light-harvesting antennae of red algae and cyanobacteria. Despite their widespread use as fluorescent probes in biotechnology and medicine, the structures and interactions of their noncrystallizable central subunits are largely unknown. Here, we employ ion mobility mass spectrometry to reveal varying stabilities of the PC and PE complexes and identify their closest architectural homologues among all protein assemblies in the Protein Data Bank (PDB). Our results suggest that the central subunits of PC and PE complexes, although absent from the crystal structures, may be crucial for their stability, and thus of unexpected importance for their biotechnological applications.
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| 2. |
- Kaldmae, Margit, et al.
(author)
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Gas-Phase Collisions with Trimethylamine-N-Oxide Enable Activation-Controlled Protein Ion Charge Reduction
- 2019
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In: Journal of the American Society for Mass Spectrometry. - : American Chemical Society (ACS). - 1044-0305 .- 1879-1123. ; 30:8, s. 1385-1388
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Journal article (peer-reviewed)abstract
- Modulating protein ion charge is a useful tool for the study of protein folding and interactions by electrospray ionization mass spectrometry. Here, we investigate activation-dependent charge reduction of protein ions with the chemical chaperone trimethylamine-N-oxide (TMAO). Based on experiments carried out on proteins ranging from 4.5 to 35kDa, we find that when combined with collisional activation, TMAO removes approximately 60% of the charges acquired under native conditions. Ion mobility measurements furthermore show that TMAO-mediated charge reduction produces the same end charge state and arrival time distributions for native-like and denatured protein ions. Our results suggest that gas-phase collisions between the protein ions and TMAO result in proton transfer, in line with previous findings for dimethyl- and trimethylamine. By adjusting the energy of the collisions experienced by the ions, it is possible to control the degree of charge reduction, making TMAO a highly dynamic charge reducer that opens new avenues for manipulating protein charge states in ESI-MS and for investigating the relationship between protein charge and conformation.
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| 3. |
- Poska, Helen, et al.
(author)
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Recombinant Bri3 BRICHOS domain is a molecular chaperone with effect against amyloid formation and non-fibrillar protein aggregation
- 2020
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In: Scientific Reports. - : Springer Nature. - 2045-2322. ; 10:1
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Journal article (peer-reviewed)abstract
- Molecular chaperones assist proteins in achieving a functional structure and prevent them from misfolding into aggregates, including disease-associated deposits. The BRICHOS domain from familial dementia associated protein Bri2 (or ITM2B) probably chaperones its specific proprotein region with high beta-sheet propensity during biosynthesis. Recently, Bri2 BRICHOS activity was found to extend to other amyloidogenic, fibril forming peptides, in particular, Alzheimer's disease associated amyloid-beta peptide, as well as to amorphous aggregate forming proteins. However, the biological functions of the central nervous system specific homologue Bri3 BRICHOS are still to be elucidated. Here we give a detailed characterisation of the recombinant human (rh) Bri3 BRICHOS domain and compare its structural and functional properties with rh Bri2 BRICHOS. The results show that rh Bri3 BRICHOS forms more and larger oligomers, somewhat more efficiently prevents non-fibrillar protein aggregation, and less efficiently reduces A beta 42 fibril formation compared to rh Bri2 BRICHOS. This suggests that Bri2 and Bri3 BRICHOS have overlapping molecular mechanisms and that their apparently different tissue expression and processing may result in different physiological functions.
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| 4. |
- Saluri, Mihkel, et al.
(author)
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A “grappling hook” interaction connects self-assembly and chaperone activity of Nucleophosmin 1
- 2023
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In: pnas nexus. - : Oxford University Press (OUP). - 2752-6542. ; 2:2
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Journal article (peer-reviewed)abstract
- How the self-assembly of partially disordered proteins generates functional compartments in the cytoplasm and particularly in the nucleus is poorly understood. Nucleophosmin 1 (NPM1) is an abundant nucleolar protein that forms large oligomers and undergoes liquid-liquid phase separation by binding RNA or ribosomal proteins. It provides the scaffold for ribosome assembly but also prevents protein aggregation as part of the cellular stress response. Here, we use aggregation assays and native mass spectrometry (MS) to examine the relationship between the self-assembly and chaperone activity of NPM1. We find that oligomerization of full-length NPM1 modulates its ability to retard amyloid formation in vitro. Machine learning-based structure prediction and cryo-electron microscopy reveal fuzzy interactions between the acidic disordered region and the C-terminal nucleotide-binding domain, which cross-link NPM1 pentamers into partially disordered oligomers. The addition of basic peptides results in a tighter association within the oligomers, reducing their capacity to prevent amyloid formation. Together, our findings show that NPM1 uses a grappling hook mechanism to form a network-like structure that traps aggregation-prone proteins. Nucleolar proteins and RNAs simultaneously modulate the association strength and chaperone activity, suggesting a mechanism by which nucleolar composition regulates the chaperone activity of NPM1.
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