Fatty acid interactions with native and mutant fatty acid binding proteins
Fatty acid interactions with native and mutant fatty acid binding proteins
复制标题
脂肪酸与天然和突变脂肪酸结合蛋白的相互作用
DOI：
--
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发表时间：
1999
影响因子：
4.3
通讯作者：
A. Kleinfeld
中科院分区：
文献类型：
--
作者：
G. V. Richieri;R. Ogata;A. Kleinfeld
对长链脂肪酸（FA）与野生型（WT）脂肪酸结合蛋白（FABP）和工程FABP突变体的相互作用进行了监测，以确定平衡结合常数以及结合和解离的速率常数。这些测量是用荧光探针ADIFAB和ADIFAB2完成的，它们可以测定游离脂肪酸（FFA）与蛋白质和膜反应的浓度。这些研究结果表明，对于来自脂肪细胞、心脏、肠道和肝脏的WT蛋白，Kd值在nM范围内，亲和度随着FA水溶性的增加而降低。对心脏和肝脏的结合亲和力一般大于对脂肪细胞和肠道的结合亲和力。此外，速率常数的测量表明，所有FA和fabp在37°c下的结合平衡在几秒钟内就能实现。这些结果与血清（未结合）FFA水平一起表明，FABPs具有缓冲作用，有助于维持细胞内FFA浓度，从而使血清和细胞之间的FFA通量沿浓度梯度下降。结合温度依赖性的测量表明，自由能主要是焓，反应的焓来自于结合腔内FA-FABP的相互作用。通过测定与肠FABP工程化点突变体结合的热力学，研究了这些相互作用的性质。这些测量表明，结合亲和不能准确地报告蛋白质- fa相互作用的变化，因为结合熵和焓的变化倾向于补偿。例如，丙氨酸取代精氨酸106产生的结合亲和力增加30倍，因为由于FA羧酸酯和Arg106之间有利相互作用的消除而导致的焓损失被熵的增加所补偿。因此，要了解氨基酸替代对FA-FABP相互作用的影响，除了亲和力外，还需要测量焓和熵。
The interactions of long chain fatty acids (FA) with wild type (WT) fatty acid binding proteins (FABP) and engineered FABP mutants have been monitored to determine the equilibrium binding constants as well as the rate constants for binding and dissociation. These measurements have been done using the fluorescent probes, ADIFAB and ADIFAB2, that allow the determination of the free fatty acid (FFA) concentration in the reaction of FA with proteins and membranes. The results of these studies indicate that for WT proteins from adipocyte, heart, intestine, and liver, Kd values are in the nM range and affinities decrease with increasing aqueous solubility of the FA. Binding affinities for heart and liver are generally greater than those for adipocyte and intestine. Moreover, measurements of the rate constants indicate that binding equilibrium at 37øC is achieved within seconds for all FA and FABPs. These results, together with the level of serum (unbound) FFA, suggests a buffering action of FABPs that helps to maintain the intracellular concentration of FFA so that the flux of FFA between serum and cells occurs down a concentration gradient. Measurements of the temperature dependence of binding reveal that the free energy is predominately enthalpic and that the enthalpy of the reaction results from FA-FABP interactions within the binding cavity. The nature of these interactions were investigated by determining the thermodynamics of binding to engineered point mutants of the intestinal FABP. These measurements showed that binding affinities did not report accurately the changes in protein-FA interactions because changes in the binding entropy and enthalpy tend to compensate. For example, an alanine substitution for arginine 106 yields a 30 fold increase in binding affinity, because the loss in enthalpy due to the elimination of the favorable interaction between the FA carboxylate and Arg106, is more than compensated for by an increase in entropy. Thus understanding the effects of amino acid replacements on FA-FABP interactions requires measurements of enthalpy and entropy, in addition to affinity.
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DOI：
10.1016/s0021-9258(18)35872-1
发表时间：
1992-11
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
J. Sacchettini;G. Scapin;D. Gopaul;J. Gordon
通讯作者：
J. Sacchettini;G. Scapin;D. Gopaul;J. Gordon
影响因子：
2.9
作者：
Cistola, DP;Kim, K;Frieden, C
通讯作者：
Frieden, C
使用荧光探针 ADIFAB 测量脂肪酸与来自脂肪细胞、肠、心脏和肝脏的脂肪酸结合蛋白的结合平衡常数。
DOI：
--
发表时间：
1994
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Richieri,GV;Ogata,RT;Kleinfeld,AM
通讯作者：
Kleinfeld,AM
大肠杆菌衍生的大鼠肠脂肪酸结合蛋白，在 1.5 A 分辨率下结合肉豆蔻酸盐，以及在 1.74 A 分辨率下结合油酸盐的 I-FABPArg106-->Gln。
DOI：
--
发表时间：
1993
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Eads,J;Sacchettini,JC;Kromminga,A;Gordon,JI
通讯作者：
Gordon,JI
影响因子：
--
作者：
L. Banaszak;N. Winter;Zhaohui Xu;D. Bernlohr;S. Cowan;Alwyn Jones
通讯作者：
L. Banaszak;N. Winter;Zhaohui Xu;D. Bernlohr;S. Cowan;Alwyn Jones