| 1. |
- Abramsson, Mia L, et al.
(författare)
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Charge engineering reveals the roles of ionizable side chains in electrospray ionization mass spectrometry
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The role of ionizable side chains in the electrospray ionization mass spectrometry of intact proteins remains hotly debated but has not been conclusively addressed because multiple chargeable sites are present in virtually all proteins. Using engineered soluble proteins, we show that ionizable side chains are completely dispensable for charging under native conditions, but if present, they are preferential protonation sites. The absence of ionizable side chains results in identical charge state distributions under native-like and denaturing conditions, whilst co-existing conformers can be distinguished using ion mobility separation. An excess of ionizable side chains, on the other hand, effectively modulates protein ion stability. We conclude that the sum of charges is governed solely by Coulombic terms, while their locations affect the stability of the protein in the gas phase.
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| 2. |
- Abramsson, Mia L., et al.
(författare)
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Charge Engineering Reveals the Roles of Ionizable Side Chains in Electrospray Ionization Mass Spectrometry
- 2021
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Ingår i: JACS Au. - : American Chemical Society (ACS). - 2691-3704. ; 1:12, s. 2385-2393
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Tidskriftsartikel (refereegranskat)abstract
- In solution, the charge of a protein is intricately linked to its stability, but electrospray ionization distorts this connection, potentially limiting the ability of native mass spectrometry to inform about protein structure and dynamics. How the behavior of intact proteins in the gas phase depends on the presence and distribution of ionizable surface residues has been difficult to answer because multiple chargeable sites are present in virtually all proteins. Turning to protein engineering, we show that ionizable side chains are completely dispensable for charging under native conditions, but if present, they are preferential protonation sites. The absence of ionizable side chains results in identical charge state distributions under native-like and denaturing conditions, while coexisting conformers can be distinguished using ion mobility separation. An excess of ionizable side chains, on the other hand, effectively modulates protein ion stability. In fact, moving a single ionizable group can dramatically alter the gas-phase conformation of a protein ion. We conclude that although the sum of the charges is governed solely by Coulombic terms, their locations affect the stability of the protein in the gas phase.
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| 3. |
- Hochberg, Georg K. A., et al.
(författare)
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A hydrophobic ratchet entrenches molecular complexes
- 2020
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 588:7838, s. 503-508
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Tidskriftsartikel (refereegranskat)abstract
- Most proteins assemble into multisubunit complexes1. The persistence of these complexes across evolutionary time is usually explained as the result of natural selection for functional properties that depend on multimerization, such as intersubunit allostery or the capacity to do mechanical work. In many complexes, however, multimerization does not enable any known function. An alternative explanation is that multimers could become entrenched if substitutions accumulate that are neutral in multimers but deleterious in monomers; purifying selection would then prevent reversion to the unassembled form, even if assembly per se does not enhance biological function. Here we show that a hydrophobic mutational ratchet systematically entrenches molecular complexes. By applying ancestral protein reconstruction and biochemical assays to the evolution of steroid hormone receptors, we show that an ancient hydrophobic interface, conserved for hundreds of millions of years, is entrenched because exposure of this interface to solvent reduces protein stability and causes aggregation, even though the interface makes no detectable contribution to function. Using structural bioinformatics, we show that a universal mutational propensity drives sites that are buried in multimeric interfaces to accumulate hydrophobic substitutions to levels that are not tolerated in monomers. In a database of hundreds of families of multimers, most show signatures of long-term hydrophobic entrenchment. It is therefore likely that many protein complexes persist because a simple ratchet-like mechanism entrenches them across evolutionary time, even when they are functionally gratuitous.
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| 4. |
- Hochberg, Georg K A, et al.
(författare)
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Structural principles that enable oligomeric small heat-shock protein paralogs to evolve distinct functions.
- 2018
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 359:6378, s. 930-935
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Tidskriftsartikel (refereegranskat)abstract
- Oligomeric proteins assemble with exceptional selectivity, even in the presence of closely related proteins, to perform their cellular roles. We show that most proteins related by gene duplication of an oligomeric ancestor have evolved to avoid hetero-oligomerization and that this correlates with their acquisition of distinct functions. We report how coassembly is avoided by two oligomeric small heat-shock protein paralogs. A hierarchy of assembly, involving intermediates that are populated only fleetingly at equilibrium, ensures selective oligomerization. Conformational flexibility at noninterfacial regions in the monomers prevents coassembly, allowing interfaces to remain largely conserved. Homomeric oligomers must overcome the entropic benefit of coassembly and, accordingly, homomeric paralogs comprise fewer subunits than homomers that have no paralogs.
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