Abstract：
Hsp16.3 plays a vital role in the slow growth of Mycobacterium tuberculosis via its chaperone function. Many secretory proteins, including Hsp16.3 undergo acetylation in vivo. Seven lysine (K) residues (K64, K78, K85, K114, K119, K132 and K136) in Hsp16.3 are acetylated inside pathogen. However, how lysine acetylation affects its structure, chaperone function and pathogen\'s growth is still elusive. We examined these aspects by executing in vitro chemical acetylation (acetic anhydride modification) and by utilizing a lysine acetylation mimic mutant (Hsp16.3-K64Q/K78Q/K85Q/K114Q/K119Q/K132Q/K136Q). Far- and near-UV CD measurements revealed that the chemically acetylated proteins(s) and acetylation mimic mutant has altered secondary and tertiary structure than unacetylated/wild-type protein. The chemical modification and acetylation mimic mutation also disrupted the oligomeric assembly, increased surface hydrophobicity and reduced stability of Hsp16.3, as revealed by GF-HPLC, 4,4\'-dianilino-1,1\'-binaphthyl-5,5\'-disulfonic acid binding and urea denaturation experiments, respectively. These structural changes collectively led to an enhancement in chaperone function (aggregation and thermal inactivation prevention ability) of Hsp16.3. Moreover, when the H37Rv strain expressed the acetylation mimic mutant protein, its growth was slower in comparison to the strain expressing the wild-type/unacetylated Hsp16.3. Altogether, these findings indicated that lysine acetylation improves the chaperone function of Hsp16.3 which may influence pathogen\'s growth in host environment.
摘要：
Hsp16.3通过其伴侣功能在结核分枝杆菌的缓慢生长中起着至关重要的作用。许多分泌蛋白，包括Hsp16.3在体内经历乙酰化。Hsp16.3中的七个赖氨酸（K）残基（K64，K78，K85，K114，K119，K132和K136）在病原体内部被乙酰化。然而,赖氨酸乙酰化如何影响其结构，伴侣功能和病原体的生长仍然难以捉摸。我们通过进行体外化学乙酰化（乙酸酐修饰）和利用赖氨酸乙酰化模拟突变体（K64Q/K78Q/K85Q/K114Q/K119Q/K132Q/K136Q）检查了这些方面。Far和近UVCD测量揭示化学乙酰化的蛋白质和乙酰化模拟突变体比未乙酰化的/野生型蛋白质具有改变的二级和三级结构。化学修饰和乙酰化模拟突变也破坏了寡聚装配，如GF-HPLC所示，Hsp16.3的表面疏水性增加，稳定性降低，4,4'-二苯胺基-1,1'-联萘-5,5'-二磺酸结合和尿素变性实验，分别。这些结构变化共同导致Hsp16.3的伴侣功能（聚集和热失活预防能力）增强。此外，当H37Rv菌株表达乙酰化模拟突变蛋白时，与表达野生型/未乙酰化Hsp16.3的菌株相比，其生长较慢。总之,这些发现表明赖氨酸乙酰化改善了Hsp16.3的伴侣功能，这可能会影响病原体在宿主环境中的生长。
