Abramsson Mia L, Sahin Cagla, Hopper Jonathan T S, Branca Rui M M, Danielsson Jens, Xu Mingming, Chandler Shane A, Österlund Nicklas, Ilag Leopold L, Leppert Axel, Costeira-Paulo Joana, Lang Lisa, Teilum Kaare, Laganowsky Arthur, Benesch Justin L P, Oliveberg Mikael, Robinson Carol V, Marklund Erik G, Allison Timothy M, Winther Jakob R, Landreh Michael
Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Tomtebodavägen 23A, 171 65 Stockholm, Sweden.
Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Ole Maaløes vej 5, 2200 Copenhagen, Denmark.
JACS Au. 2021 Nov 29;1(12):2385-2393. doi: 10.1021/jacsau.1c00458. eCollection 2021 Dec 27.
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 of the charges is governed solely by Coulombic terms, their affect the stability of the protein in the gas phase.
在溶液中，蛋白质的电荷与其稳定性紧密相连，但电喷雾电离会扭曲这种联系，这可能会限制天然质谱法揭示蛋白质结构和动力学的能力。由于几乎所有蛋白质中都存在多个可电离位点，完整蛋白质在气相中的行为如何依赖于可电离表面残基的存在和分布一直难以回答。通过蛋白质工程，我们发现可电离侧链在天然条件下对于电荷化是完全不必要的，但如果存在，它们就是优先质子化位点。可电离侧链的缺失会导致在类似天然和变性条件下电荷态分布相同，而共存的构象体可以通过离子淌度分离来区分。另一方面，过量的可电离侧链会有效调节蛋白质离子的稳定性。实际上，移动单个可电离基团就能显著改变蛋白质离子的气相构象。我们得出结论，虽然电荷的[此处原文缺失相关内容]仅由库仑项决定，但其[此处原文缺失相关内容]会影响蛋白质在气相中的稳定性。
