Marti D N, Jelesarov I, Bosshard H R
Institute of Biochemistry, University of Zürich, CH-8057 Zürich, Switzerland.
Biochemistry. 2000 Oct 24;39(42):12804-18. doi: 10.1021/bi001242e.
Residues of opposite charge often populate heptad positions g (heptad i on chain 1) and e' (heptad i + 1 on chain 2) in dimeric coiled coils and may stabilize the dimer by formation of interchain ion pairs. To investigate the contribution to stability of such electrostatic interactions we have designed a disulfide-linked heterodimeric zipper (AB zipper) consisting of the acidic chain Ac-E-VAQLEKE-VAQAEAE-NYQLEQE-VAQLEHE-CG-NH(2) and the basic chain Ac-E-VQALKKR-VQALKAR-NYAAKQK-VQALRHK-CG-NH(2) in which all e and g positions are occupied by either E or K/R to form a maximum of seven interhelical salt bridges. Temperature-induced denaturation experiments monitored by circular dichroism reveal a stable coiled coil conformation below 50 degrees C and in the pH range 1.2-8.0. Stability is highest at pH approximately 4.0 [DeltaG(U) (37 degrees C) = 5.18 +/- 0.51 kcal mol(-)(1)]. The solution structure of the AB zipper at pH 5.65 has been elucidated on the basis of homonuclear (1)H NMR data collected at 800 MHz [heavy atom rmsd's for the ensemble of 50 calculated structures are 0.47 +/- 0.13 A (backbone) and 0.95 +/- 0.16 A (all)]. Both chains of the AB zipper are almost entirely in alpha-helical conformation and form a superhelix with a left-handed twist. Overhauser connectivities reveal close contacts between g position residues (heptad i on chain 1) and residues d/f (heptad i on chain 1), residues a/d (heptad i + 1 on chain 1), and residue a' (heptad i + 1 on chain 2). Residues in position e (heptad i on chain 1) are in contact with residues a/b/d/f (heptad i on chain 1) and residue d' (heptad i on chain 2). These connectivities hint at a relatively defined alignment of the side chains across the helix interface. Partial H-bond formation between the functional groups of residues g and e'(+1) is observed in the calculated structures. NMR pH titration experiments disclose pK(a) values for Glu delta-carboxylate groups: 4.14 +/- 0.02 (E(1)), 4.82 +/- 0.07 (E(6)), 4.52 +/- 0.01 (E(8)), 4.37 +/- 0.03 (E(13)), 4.11 +/- 0.02 (E(15)), 4.41 +/- 0.07 (E(20)), 4.82 +/- 0.03 (E(22)), 4.65 +/- 0.04 (E(27)), 4.63 +/- 0.03 (E(29)), 4.22 +/- 0.02 (E(1)(')). By comparison with pK(a) of Glu in unfolded peptides ( approximately 4. 3 +/- 0.1), our pK(a) data suggest marginal or even unfavorable contribution of charged Glu to the stability of the AB zipper. The electrostatic energy gained from interhelical ion pairs is likely to be surpassed by hydrophobic energy terms upon protonation of Glu, due to increased hydrophobicity of uncharged Glu and, thus, better packing against apolar residues at the chain interface.
在二聚体卷曲螺旋中，带相反电荷的残基通常占据七肽重复序列的g位（链1上的七肽重复序列i）和e'位（链2上的七肽重复序列i + 1），并且可能通过形成链间离子对来稳定二聚体。为了研究这种静电相互作用对稳定性的贡献，我们设计了一种二硫键连接的异源二聚体拉链（AB拉链），其由酸性链Ac-E-VAQLEKE-VAQAEAE-NYQLEQE-VAQLEHE-CG-NH(2)和碱性链Ac-E-VQALKKR-VQALKAR-NYAAKQK-VQALRHK-CG-NH(2)组成，其中所有的e位和g位均被E或K/R占据，以形成最多七个螺旋间盐桥。通过圆二色性监测的温度诱导变性实验表明，在50℃以下以及pH值为1.2 - 8.0的范围内，该卷曲螺旋构象稳定。在pH约为4.0时稳定性最高[ΔG(U)(37℃)= 5.18±0.51 kcal mol⁻¹]。基于在800 MHz下收集的同核¹H NMR数据，阐明了AB拉链在pH 5.65时的溶液结构[50个计算结构的集合的重原子均方根偏差为0.47±0.13 Å（主链）和0.95±0.16 Å（全部）]。AB拉链的两条链几乎完全处于α-螺旋构象，并形成具有左手扭曲的超螺旋。Overhauser连接性揭示了g位残基（链1上的七肽重复序列i）与d/f位残基（链1上的七肽重复序列i）、a/d位残基（链1上的七肽重复序列i + 1）以及a'位残基（链2上的七肽重复序列i + 1）之间的紧密接触。e位（链1上的七肽重复序列i）的残基与a/b/d/f位（链1上的七肽重复序列i）的残基以及d'位（链2上的七肽重复序列i）的残基接触。这些连接性暗示了跨螺旋界面的侧链具有相对明确的排列方式。在计算结构中观察到g位和e'(+1)位残基的官能团之间形成了部分氢键。NMR pH滴定实验揭示了谷氨酸δ-羧基的pKa值：4.14±0.02（E(1)）、4.82±0.07（E(6)）、4.52±0.01（E(8)）、4.37±0.03（E(13)）、4.11±0.02（E(15)）、4.41±0.07（E(20)）、4.82±0.03（E(22)）、4.65±0.04（E(27)）、4.63±0.03（E(29)）、4.22±0.02（E(1)'）。通过与未折叠肽中谷氨酸的pKa（约4.3±0.1）进行比较，我们的pKa数据表明带电荷的谷氨酸对AB拉链稳定性的贡献很小甚至是不利的。由于不带电荷的谷氨酸疏水性增加，因此在链界面处与非极性残基的堆积更好，在谷氨酸质子化时，螺旋间离子对获得的静电能可能会被疏水能量项超过。
