Mascotti D P, Lohman T M
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
Biochemistry. 1993 Oct 12;32(40):10568-79. doi: 10.1021/bi00091a006.
We have examined the thermodynamics of binding of a series of oligolysines (net charge z = +2 to +10) containing one, two, or three tryptophans to several single-stranded (ss) homo-polynucleotides [poly(A), poly(C), poly(I), poly(dU), poly(dT)] and duplex (ds) DNA in order to investigate the effects of peptide charge, tryptophan content, and polynucleotide base and sugar type. Equilibrium association constants, Kobs, were measured as a function of monovalent salt concentration (KCH3CO2) and temperature by monitoring the quenching of the peptide tryptophan fluorescence upon interaction with the polynucleotides, from which the dependence of delta G(o)obs, delta H(o)obs, and delta S(o)obs on [KCH3CO2] was obtained. As observed previously with poly(U) [Mascotti, D.P., & Lohman, T.M. (1992) Biochemistry 31, 8932], the dependence of delta G(o)obs on [K+] for peptide binding to each polynucleotide is entirely entropic in origin (i.e., delta H(o)obs is independent of [K+]), consistent with the conclusion that Kobs increases with decreasing salt concentration due to the favorable increase in entropy resulting from the displacement of bound cations (K+) from the nucleic acid upon formation of the complex. For each ss polynucleotide, we find that significantly less than one potassium ion is released thermodynamically per net positive peptide charge, as determined from the value of delta log Kobs/delta log [K+]. Interestingly, (-delta log Kobs/delta log [K+])/z decreases with increasing peptide charge for poly(A), poly(C), and poly(dT), contrary to the behavior observed with poly(U) and ds-DNA, which may reflect a significant release of bound water upon formation of peptide complexes with these ss homo-polynucleotides or an increased binding of K+ to the ss polynucleotide with increasing [K+]. Alternatively, there may be conformational differences between the bound states of oligolysines of low charge, relative to oligolysines of higher charge. However, in all cases, peptides with z < +4 display different thermodynamics of binding than peptides with z > +4. The presence of tryptophan (Trp) within these peptides does not influence the salt dependence of Kobs for binding to poly(A), poly(C), or poly(dT). However, the Trp content of the peptide does contribute significantly to the thermodynamics of these interactions: Trp interactions result in a favorable contribution to delta H(o)obs, but an unfavorable contribution to delta S(o)obs, with little effect on delta G(o)obs due to entropy-enthalpy compensations. Oligolysines containing Trp also display a small, but significant, dependence of Kobs on base composition, with Kobs decreasing in the order poly(I) >> poly(dT) approximately poly(U) approximately poly(A) >> poly(C).
我们研究了一系列含有一、二或三个色氨酸的寡聚赖氨酸（净电荷z = +2至+10）与几种单链（ss）同聚核苷酸[聚（A）、聚（C）、聚（I）、聚（dU）、聚（dT）]和双链（ds）DNA结合的热力学，以研究肽电荷、色氨酸含量以及多核苷酸碱基和糖类型的影响。通过监测肽色氨酸荧光在与多核苷酸相互作用时的猝灭，测量了平衡缔合常数Kobs作为单价盐浓度（KCH3CO2）和温度的函数，由此得到了ΔG(o)obs、ΔH(o)obs和ΔS(o)obs对[KCH3CO2]的依赖性。如先前对聚（U）的观察[Mascotti, D.P., & Lohman, T.M. (1992) Biochemistry 31, 8932]，肽与每种多核苷酸结合时ΔG(o)obs对[K+]的依赖性完全源于熵（即ΔH(o)obs与[K+]无关），这与以下结论一致：由于复合物形成时结合的阳离子（K+）从核酸上被取代导致熵的有利增加，Kobs随盐浓度降低而增加。对于每种单链多核苷酸，我们发现根据Δlog Kobs/Δlog [K+]的值，每单位净正肽电荷热力学释放的钾离子显著少于一个。有趣的是，对于聚（A）、聚（C）和聚（dT），（-Δlog Kobs/Δlog [K+]）/z随肽电荷增加而降低，这与聚（U）和双链DNA的行为相反，这可能反映了与这些单链同聚核苷酸形成肽复合物时结合水的显著释放，或者随着[K+]增加K+与单链多核苷酸结合增加。或者，低电荷寡聚赖氨酸的结合状态与高电荷寡聚赖氨酸之间可能存在构象差异。然而，在所有情况下，z < +4的肽与z > +4的肽显示出不同的结合热力学。这些肽中色氨酸（Trp）的存在不影响与聚（A）、聚（C）或聚（dT）结合时Kobs的盐依赖性。然而，肽的色氨酸含量确实对这些相互作用的热力学有显著贡献：色氨酸相互作用对ΔH(o)obs有有利贡献，但对ΔS(o)obs有不利贡献，由于熵 - 焓补偿对ΔG(o)obs影响很小。含有色氨酸的寡聚赖氨酸也显示出Kobs对碱基组成有小但显著的依赖性，Kobs按聚（I）>>聚（dT）≈聚（U）≈聚（A）>>聚（C）的顺序降低。
