Luo Minkui, Fadeev Evgeny A, Groves John T
Department of Chemistry, Princeton University, Princeton, NJ 08540, USA.
J Am Chem Soc. 2005 Feb 16;127(6):1726-36. doi: 10.1021/ja044230f.
Acinetobacter haemolyticus is an antibiotic resistant, pathogenic bacterium responsible for an increasing number of hospital infections. Acinetoferrin (Af), the amphiphilic siderophore isolated from this organism, contains two unusual trans-2-octenoyl hydrocarbon chains reminiscent of a phospholipid structural motif. Here, we have investigated the membrane affinity of Af and its iron complex, Fe-Af, using small and large unilamellar phospholipid vesicles (SUV and LUV) as model membranes. Af shows a high membrane affinity with a partition coefficient, K(x)= 6.8 x 10(5). Membrane partitioning and trans-membrane flip-flop of Fe-Af have also been studied via fluorescence quenching of specifically labeled vesicle leaflets and (1)H NMR line-broadening techniques. Fe-Af is found to rapidly redistribute between lipid and aqueous phases with dissociation/partitioning rates of k(off) = 29 s(-1) and k(on) = 2.4 x 10(4) M(-1) s(-1), respectively. Upon binding iron, the membrane affinity of Af is reduced 30-fold to K'(x) = 2.2 x 10(4) for Fe-Af. In addition, trans-membrane flip-flop of Fe-Af occurs with a rate constant, k(p) = 1.2 x 10(-3) s(-1), with egg-PC LUV and a half-life time around 10 min with DMPC SUV. These properties are due to the phospholipid-like conformation of Af and the more extended conformation of Fe-Af that is enforced by iron binding. Remarkable similarities and differences between Af and another amphiphilic siderophore, marinobactin E, are discussed. The potential biological implications of Af and Fe-Af are also addressed. Our approaches using inner- and outer-leaflet-labeled fluorescent vesicles and (1)H NMR line-broadening techniques to discern Af-mediated membrane partitioning and trans-membrane diffusion are amenable to similar studies for other paramagnetic amphiphiles.
溶血不动杆菌是一种具有抗生素抗性的致病细菌，导致医院感染的数量不断增加。从该生物体中分离出的两亲性铁载体阿西铁蛋白（Af）含有两条不寻常的反式-2-辛烯酰烃链，让人联想到磷脂结构基序。在这里，我们使用小单层和大单层磷脂囊泡（SUV和LUV）作为模型膜，研究了Af及其铁络合物Fe-Af的膜亲和力。Af显示出高膜亲和力，分配系数K(x)=6.8×10⁵。还通过特异性标记的囊泡小叶的荧光猝灭和¹H NMR线宽展技术研究了Fe-Af的膜分配和跨膜翻转。发现Fe-Af在脂质相和水相之间快速重新分布，解离/分配速率分别为k(off)=29 s⁻¹和k(on)=2.4×10⁴ M⁻¹ s⁻¹。结合铁后，Af的膜亲和力降低30倍，Fe-Af的K'(x)=2.2×10⁴。此外，Fe-Af的跨膜翻转速率常数k(p)=1.2×10⁻³ s⁻¹，对于鸡蛋PC LUV而言，半衰期约为10分钟，对于DMPC SUV而言，半衰期约为10分钟。这些特性归因于Af的磷脂样构象以及铁结合所强制形成的Fe-Af的更伸展构象。讨论了Af与另一种两亲性铁载体海洋杆菌素E之间的显著异同。还探讨了Af和Fe-Af的潜在生物学意义。我们使用内叶和外叶标记的荧光囊泡以及¹H NMR线宽展技术来识别Af介导的膜分配和跨膜扩散的方法适用于对其他顺磁性两亲物进行类似研究。
