| 1. |
- Kaldmae, Margit, et al.
(författare)
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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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Ingår i: Protein Science. - : WILEY. - 0961-8368 .- 1469-896X. ; 28:6, s. 1024-1030
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Tidskriftsartikel (refereegranskat)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. |
- Kaldmäe, Margit, et al.
(författare)
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A “spindle and thread” mechanism unblocks p53 translation by modulating N-terminal disorder
- 2022
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Ingår i: Structure. - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 30:5, s. 733-742, e1-e7
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Tidskriftsartikel (refereegranskat)abstract
- Disordered proteins pose a major challenge to structural biology. A prominent example is the tumor suppressor p53, whose low expression levels and poor conformational stability hamper the development of cancer therapeutics. All these characteristics make it a prime example of “life on the edge of solubility.” Here, we investigate whether these features can be modulated by fusing the protein to a highly soluble spider silk domain (NT∗). The chimeric protein displays highly efficient translation and is fully active in human cancer cells. Biophysical characterization reveals a compact conformation, with the disordered transactivation domain of p53 wrapped around the NT∗ domain. We conclude that interactions with NT∗ help to unblock translation of the proline-rich disordered region of p53. Expression of partially disordered cancer targets is similarly enhanced by NT∗. In summary, we demonstrate that inducing co-translational folding via a molecular “spindle and thread” mechanism unblocks protein translation in vitro.
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| 3. |
- Saluri, Mihkel, et al.
(författare)
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A “grappling hook” interaction connects self-assembly and chaperone activity of Nucleophosmin 1
- 2023
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Ingår i: pnas nexus. - : Oxford University Press (OUP). - 2752-6542. ; 2:2
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Tidskriftsartikel (refereegranskat)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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| 4. |
- Yen, Hsin-Yung, et al.
(författare)
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Electrospray ionization of native membrane proteins proceeds via a charge equilibration step
- 2022
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Ingår i: RSC Advances. - : Royal Society of Chemistry (RSC). - 2046-2069. ; 12:16, s. 9671-9680
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Tidskriftsartikel (refereegranskat)abstract
- Electrospray ionization mass spectrometry is increasingly applied to study the structures and interactions of membrane protein complexes. However, the charging mechanism is complicated by the presence of detergent micelles during ionization. Here, we show that the final charge of membrane proteins can be predicted by their molecular weight when released from the non-charge reducing saccharide detergents. Our data indicate that PEG detergents lower the charge depending on the number of detergent molecules in the surrounding micelle, whereas fos-choline detergents may additionally participate in ion–ion reactions after desolvation. The supercharging reagent sulfolane, on the other hand, has no discernible effect on the charge of detergent-free membrane proteins. Taking our observations into the context of protein-detergent interactions in the gas phase, we propose a charge equilibration model for the generation of native-like membrane protein ions. During ionization of the protein-detergent complex, the ESI charges are distributed between detergent and protein according to proton affinity of the detergent, number of detergent molecules, and surface area of the protein. Charge equilibration influenced by detergents determines the final charge state of membrane proteins. This process likely contributes to maintaining a native-like fold after detergent release and can be harnessed to stabilize particularly labile membrane protein complexes in the gas phase.
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