Energetics of arginine-4 substitution mutants in the N-terminal cooperativity domain of T4 gene 32 protein.
Energetics of arginine-4 substitution mutants in the N-terminal cooperativity domain of T4 gene 32 protein.
复制标题
T4 基因 32 蛋白 N 末端协同结构域精氨酸 4 取代突变体的能量学。
DOI：
10.1021/bi00092a038
复制
发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Giedroc,DP
中科院分区：
文献类型：
--
作者：
Villemain,JL;Giedroc,DP
来自噬菌体T4的基因32蛋白（gp 32）是一种序列非特异性单链（ss）核酸结合蛋白，其与ss核酸高度协同结合。已知N-末端“B”或碱性结构域（残基1-21）是gp 32高度协同结合所必需的（其中Kapp= w^ 500），因为它的去除导致蛋白质与ss核酸非协同结合（w= 1）。在本文中，我们探测的合作结合的gp 32的分子细节的一组四个单一的氨基酸取代突变体的Arg 4：赖氨酸4（R4 K gp 32），Gin 4（R4 Q gp 32），Thr 4（R4 T gp 32），和Gly 4（R4 G gp 32）的理化特性。对poly（A）的结合亲和力的定性排序为野生型> R4 K> R4 Q> R4 T> R4 G> gp 32-B（gp 32缺少前21个氨基酸）。所有gp 32的闭塞部位大小为napp= 7.5±0.5。在低晶格饱和度（i>< 0.011）的条件下，使用在10 mMTris-HCl，pH 8.1，20 ℃和NaCl浓度下收集的多次反向荧光滴定，估计野生型、R4 K、R4 Q和R4 T gp 32的Δ Tm和μ的分辨率，其中对于核糖均聚物poly（A）上的每个gp 32，Km为（2-4）× 106 M-1。直接获得所有gp 32的结合参数，或通过将Km的[NaCl]依赖性保守外推至0.20 M NaCl，20 ℃，pH8.1进行比较。然后假设n的大小不随[NaCl]变化（针对R4 T gp 32显示），从而允许在0.20 M NaCl下估计Δ Tint。我们发现，R4 K gp 32以比野生型gp 32低2-3倍的总体亲和力（A> app）结合聚腺苷酸，而每个分子的w似乎不可区分（野生型gp 32，u> n = 800-1300; R4 K gp 32，u> n = 600-1200）。令人惊讶的是，R4 Q gp 32的特征在于也不容易与野生型和R4 K蛋白区分开（α> 800-4400），而Kapp降低约10倍。该突变体还显示与poly（A）结合的[NaCl]依赖性显著降低。R4 T gp 32的结合比Q突变体弱约10倍。它表现出一个us范围从300到700和一个大大减少的[NaCl]的依赖性（d log Kmt/d log [NaCl]=-1.4从0.10到0.20 M NaCl），表明显着的扰动在Kmt和ui条款。R4 G gp 32与比R4 T gp 32降低约20倍的Kapp结合，其中在0.20 M NaCl下的大部分这种差异显然存在于η项中（来自多次滴定的α><<5-35）。最后，gp 32-B似乎以比R4 G gp 32低=>> 10倍的亲和力结合，这与R4 G gp 32中残余协同性的发现一致。突变gp 32对poly（A）的平衡亲和力的总体趋势与其相对螺旋去稳定化活性平行，如用部分双链交替DNA共聚物poly [d（AT）]测量的。我们的结论是，Arg 4的正电荷是至关重要的合作结合和螺旋不稳定活性的gp 32的方式，这是依赖于取代的性质的维护。此外，本发明还这些取代似乎对gp 32对单链核酸的[NaCl]依赖性和总体亲和力产生了比从N-末端结构域中单个侧链的丢失所预期的更有害的影响。噬菌体T4基因32蛋白（gp 32）* 是原型序列非特异性单链（ss）在DNA复制、重组和修复中起结构作用的DNA结合蛋白[综述见Karpel（1990）]。它是螺旋不稳定蛋白的范例，这种蛋白以“无限”类型的协同性与ssDNA片段跨膜结合。
Revised Manuscript Received July 6, 1993® abstract: Gene 32 protein (gp32) from bacteriophage T4 is a sequence-nonspecific single-strand (ss) nucleic acid binding protein which binds highly cooperatively to ss nucleic acids. The N-terminal “B” or basic domain (residues 1-21) is known to be required for highly cooperative binding by gp32 (where Kapp= w^ 500), since its removal results in a protein which binds ss nucleic acids noncooperatively (w= 1). In this paper, we probe the molecular details of cooperative binding by gp32 by physicochemical characterization of a set of four single amino acid substitution mutants of Arg4: Lys4 (R4K gp32), Gin4 (R4Q gp32), Thr4 (R4T gp32), and Gly4 (R4G gp32). The qualitative ranking of binding affinities to poly (A) is wild-type> R4K> R4Q> R4T> R4G> gp32-B (gp32 lacking the first 21 amino acids). The occluded site sizeis napp= 7.5±0.5 for all gp32s. Resolution of ATi „t and u for wild-type, R4K, R4Q, and R4T gp32s was estimated under conditions of low lattice saturation (i>< 0.011) using multiple reverse fluorescence titrations collected at 10 mM Tris-HCl, pH 8.1, 20 C, and a NaCl concentration where Km was (2-4) x 106 M'1 for each gp32 on the ribohomopolymer poly (A). Binding parameters for all gp32s were obtained directly or compared by conservative extrapolation of the [NaCl] dependence of Km to 0.20 M NaCl, 20 C, pH 8.1. The magnitude of «was then assumed not to vary with [NaCl](shown for R4T gp32), allowing estimation of ATi „t at 0.20 M NaCl. We find that R4K gp32 binds to poly (A) with an overall affinity (A^ app) which is 2-3-fold lower than wild-type gp32, while w for each molecule seems indistinguishable (wild-type gp32, ui 800-1300; R4K gp32, u>«= 600-1200). Surprisingly, R4Q gp32 is characterized by an us also not readily distinguishable from the wild-type and R4K proteins (o>« 800-4400), while Kapp is reduced about 10-fold. This mutant also shows a significantly reduced [NaCl] dependence of the binding to poly (A). R4T gp32 binds about 10-fold weaker than the Q mutant. It exhibits an us ranging from 300 to 700 and a substantially reduced [NaCl] dependence (d log Kmt/d log [NaCl]=-1.4 from 0.10 to 0.20 M NaCl), indicative of significant perturbations in both Kmt and ui terms. R4G gp32 binds with a Kapp about 20-fold reduced from R4T gp32, with most of this difference at 0.20 M NaCl apparently residing in the «term (o>« 5-35 from multiple titrations). Finally, gp32-Bappears to bind with a=» 10-fold lower affinity than R4G gp32, consistent with the finding of residual cooperativity in R4G gp32. The overall trend in equilibrium affinities of mutant gp32s for poly (A) parallels their relative helix-destabilizing activities as measured with the partially double-stranded alternating DNA copolymer poly [d (AT)]. We conclude that the positive charge of Arg4 is critical for maintenance of cooperative binding and helix-destabilizing activity of gp32 in a manner which is dependent upon the nature of the substitution. Further, these substitutions appear to give rise to a more deleterious effect on the [NaCl] dependence and overall affinity of gp32 for single-stranded nucleic acids than might have been anticipated from the loss of a single side chainin the N-terminal domain.Bacteriophage T4 gene 32 protein (gp32)* is the prototype sequence-nonspecific single-strand (ss) DNA binding protein which plays a structural role in DNA replication, recombination, and repair [for a review, see Karpel (1990)]. It is the paradigm for helix-destabilizing proteins which bind with an “unlimited” type of cooperativityto ssDNA segments tran-