Aggarwal Mayank, Boone Christopher D, Kondeti Bhargav, Tu Chingkuang, Silverman David N, McKenna Robert
Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, 1600 SW Archer Road, PO Box 100245, Gainesville, FL 32610, USA.
Acta Crystallogr D Biol Crystallogr. 2013 May;69(Pt 5):860-5. doi: 10.1107/S0907444913002771. Epub 2013 Apr 19.
Protein X-ray crystallography has seen a progressive shift from data collection at cool/room temperature (277-298 K) to data collection at cryotemperature (100 K) because of its ease of crystal preparation and the lessening of the detrimental effects of radiation-induced crystal damage, with 20-25%(v/v) glycerol (GOL) being the preferred choice of cryoprotectant. Here, a case study of the effects of cryoprotectants on the kinetics of carbonic anhydrase II (CA II) and its inhibition by the clinically used inhibitor acetazolamide (AZM) is presented. Comparative studies of crystal structure, kinetics, inhibition and thermostability were performed on CA II and its complex with AZM in the presence of either GOL or sucrose. These results suggest that even though the cryoprotectant GOL was previously shown to be directly bound in the active site and to interact with AZM, it affects neither the thermostability of CA II nor the binding of AZM in the crystal structure or in solution. However, addition of GOL does affect the kinetics of CA II, presumably as it displaces the water proton-transfer network in the active site.
由于易于制备晶体以及减少辐射诱导的晶体损伤的有害影响，蛋白质X射线晶体学已逐渐从在低温/室温（277 - 298 K）下收集数据转向在低温（100 K）下收集数据，20 - 25%（v/v）的甘油（GOL）是首选的冷冻保护剂。在此，展示了一个关于冷冻保护剂对碳酸酐酶II（CA II）动力学及其被临床使用的抑制剂乙酰唑胺（AZM）抑制的影响的案例研究。在存在GOL或蔗糖的情况下，对CA II及其与AZM的复合物进行了晶体结构、动力学、抑制作用和热稳定性的比较研究。这些结果表明，尽管冷冻保护剂GOL先前已被证明直接结合在活性位点并与AZM相互作用，但它既不影响CA II的热稳定性，也不影响AZM在晶体结构或溶液中的结合。然而，添加GOL确实会影响CA II的动力学，推测是因为它取代了活性位点中的水质子转移网络。
