The complexity of the cellular medium can affect proteins’ properties, and, therefore, in-cell characterization of proteins is essential. We explored the stability and conformation of the first baculoviral IAP repeat (BIR) domain of X chromosome-linked inhibitor of apoptosis (XIAP), BIR1, as a model for a homodimer protein in human HeLa cells. We employed double electron–electron resonance (DEER) spectroscopy and labeling with redox stable and rigid Gd³⁺ spin labels at three representative protein residues, C12 (flexible region), E22C, and N28C (part of helical residues 26 to 31) in the N-terminal region. In contrast to predictions by excluded-volume crowding theory, the dimer–monomer dissociation constant K_D was markedly higher in cells than in solution and dilute cell lysate. As expected, this increase was partially recapitulated under conditions of high salt concentrations, given that conserved salt bridges at the dimer interface are critically required for association. Unexpectedly, however, also the addition of the crowding agent Ficoll destabilized the dimer while the addition of bovine serum albumin (BSA) and lysozyme, often used to represent interaction with charged macromolecules, had no effect. Our results highlight the potential of DEER for in-cell study of proteins as well as the complexities of the effects of the cellular milieu on protein structures and stability.

细胞培养基的复杂性会影响蛋白质的特性，因此，蛋白质的细胞内表征至关重要。我们探索了X染色体连锁的凋亡抑制因子（XIAP）BIR1的第一个杆状病毒IAP重复（BIR）域的稳定性和构象，作为人类HeLa细胞中同型二聚体蛋白的模型。我们采用了双电子-电子共振（DEER）光谱，并在氧化还原稳定和刚性Gd ³⁺自旋标记上的三个代表性蛋白质残基C12（柔性区域），E22C和N28C（螺旋残基26至31的一部分）进行标记。 N端区域。与排除体积拥挤理论的预测相反，二聚体-单体解离常数K _D与溶液和稀释的细胞裂解液相比，细胞中的α-α明显高于α-β。正如预期的那样，鉴于在缔合过程中关键需要二聚体界面处的保守盐桥，因此在高盐浓度条件下，这种增加部分得以概括。然而，出乎意料的是，添加拥挤剂Ficoll也使二聚体不稳定，而添加通常用来表示与带电大分子相互作用的牛血清白蛋白（BSA）和溶菌酶却没有效果。我们的研究结果突出了DEER在细胞内蛋白质研究中的潜力以及细胞环境对蛋白质结构和稳定性的影响的复杂性。