Sun He Mirabel, Evans Kacie A, Powers Morgan, Xi Zhenyu, Lantz Carter, Laganowsky Arthur, Rye Hays, Russell David H
Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States.
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843, United States.
J Phys Chem B. 2025 Aug 28;129(34):8668-8679. doi: 10.1021/acs.jpcb.5c03261. Epub 2025 Aug 19.
Native electrospray ionization-mass spectrometry (nESI-MS) enables studies of intact proteins, protein complexes, and protein-ligand complexes. Variable temperature (vT)-nESI-MS, where the temperature of the solution contained in the ESI emitter can be varied from 2 to 100 °C, adds new capabilities for dissecting the thermodynamics for protein-ligand binding. Here, vT-nESI-MS and ion mobility spectrometry (IMS) are used to compare the effects of temperature and nESI buffers on nucleotide (ADP) binding for the GroEL single ring mutant (SR1). Temperature-dependent shifts for average charge states () and rotationally averaged collision cross sections (CCS) for both apo- and nucleotide-bound SR1 complexes (SR1-ADP, = 1-7) indicate that nESI buffers alter structure, stabilities, and dynamics. These studies report nucleotide (ADP) binding affinities () and insight into cooperativity and enthalpy-entropy compensation (EEC). Specifically, we focus on three commonly used native ESI buffers: ammonium acetate (AmAc), triethylammonium acetate (TEAA), and ethylenediammonium acetate (EDDA). In AmAc solutions, ADP binding is highly cooperative at low temperatures (2-21 °C) but is significantly diminished at higher temperatures (21-31 °C). While cooperative ADP binding is only observed at low temperatures (4 °C) for TEAA solutions, it is absent in EDDA solutions. Collectively, these findings illustrate very different influences of ammonium and alkyl ammonium ions on the SR1 conformation and dynamics as manifested by changes in (change of solvent-accessible surface area) and thermodynamics for nucleotide binding. Moreover, temperature-dependent changes in and ligand binding provide additional experimental data that support prior work on the effects of hydration on cold protein folding. These results also align with recent computational work for the effects of hydration water on protein binding sites as well as membrane protein complex-lipid binding. The observed temperature-dependent changes in , buffer-dependent nucleotide binding, EEC, and changes in heat capacity strongly suggest that ADP influences the conformational states of the SR1 complex. Note, however, that large-scale structural changes in the SR1 complex are not observed in the IMS CCS experiments. Collectively, these results suggest that ADP binding alters key structural and/or dynamic properties of SR1, changes that are not observed in the overall, macroscopic structure of the complex. We suggest that SR1-ADP binding is an archetypal example of "allostery without (measurable) conformational change".
原生电喷雾电离质谱（nESI-MS）可用于完整蛋白质、蛋白质复合物以及蛋白质-配体复合物的研究。可变温度（vT）-nESI-MS能够将电喷雾电离发射器中溶液的温度在2至100°C之间变化，为剖析蛋白质-配体结合的热力学提供了新的能力。在此，vT-nESI-MS和离子淌度谱（IMS）被用于比较温度和nESI缓冲液对GroEL单环突变体（SR1）核苷酸（ADP）结合的影响。脱辅基和核苷酸结合的SR1复合物（SR1-ADP， = 1-7）的平均电荷态（）和旋转平均碰撞截面（CCS）的温度依赖性变化表明，nESI缓冲液会改变结构、稳定性和动力学。这些研究报告了核苷酸（ADP）的结合亲和力（），并深入了解了协同性和焓-熵补偿（EEC）。具体而言，我们重点关注三种常用的原生ESI缓冲液：醋酸铵（AmAc）、三乙铵醋酸盐（TEAA）和乙二铵醋酸盐（EDDA）。在AmAc溶液中，ADP结合在低温（2-21°C）下具有高度协同性，但在较高温度（21-31°C）下会显著降低。虽然对于TEAA溶液，仅在低温（4°C）下观察到协同性ADP结合，但在EDDA溶液中则不存在。总体而言，这些发现表明铵离子和烷基铵离子对SR1构象和动力学的影响截然不同，这体现在（溶剂可及表面积的变化）和核苷酸结合的热力学变化中。此外，和配体结合的温度依赖性变化提供了额外的实验数据，支持了先前关于水合作用对冷蛋白折叠影响的研究。这些结果也与最近关于水合水对蛋白质结合位点以及膜蛋白复合物-脂质结合影响的计算工作一致。观察到的、缓冲液依赖性核苷酸结合、EEC以及热容变化的温度依赖性强烈表明，ADP会影响SR1复合物的构象状态。然而，请注意，在IMS CCS实验中未观察到SR1复合物的大规模结构变化。总体而言，这些结果表明，ADP结合会改变SR1的关键结构和/或动态特性，但这些变化在复合物的整体宏观结构中并未观察到。我们认为，SR1-ADP结合是“无（可测量）构象变化的变构”的典型例子。
