Lew S, Ren J, London E
Department of Biochemistry and Cell Biology and Department of Chemistry, State University of New York at Stony Brook, Stony Brook, New York 11794-5215, USA.
Biochemistry. 2000 Aug 15;39(32):9632-40. doi: 10.1021/bi000694o.
To explore the influence of amino acid composition on the behavior of membrane-inserted alpha-helices, we examined the behavior of Lys-flanked polyleucyl (pLeu) helices containing a single polar/ionizable residue within their hydrophobic core. To evaluate the location of the helices within the membrane by fluorescence, each contained a Trp residue at the center of the sequence. When incorporated into dioleoylphosphatidylcholine (DOPC) model membrane vesicles, pLeu helices with or without a single Ser, Asn, Lys, or Asp residue in the hydrophobic core maintained a transmembrane state (named the N state) at neutral and acidic pH. In this state, the central Trp exhibited highly blue-shifted fluorescence, and fluorescence quenching by nitroxide-labeled lipids showed it located at the bilayer center. A state in which Trp fluorescence red-shifted by several nanometers (named the B state) was observed above pH 10-11. B state formation appears to result from deprotonation of the flanking Lys residues. Despite the red shift in Trp emission, fluorescence quenching showed that in the B state the Trp at most is only slightly shallower than in the N state, suggesting the B state also is a transmembrane or near-transmembrane structure. The B state is characterized by increased helix oligomerization, as shown by the dependence of Trp lambda(max) on the concentration of the peptide within the bilayer at high pH. The pLeu peptide with a Asp residue in the core underwent a pH-dependent transition at a lower pH than the other peptides (pH 8-9). At high pH, it exhibited both a more highly red-shifted fluorescence and shallower Trp location than the other peptides. This state (named the S state) did not exhibit a concentration-dependent Trp lambda(max). We attribute S state behavior to the formation of a charged Asp residue at high pH, and a consequent movement of the Asp toward the membrane surface, resulting in the formation of a nontransmembrane state. We conclude that a polar or ionizable residue can readily be tolerated in a single transmembrane helix, but that the charges on ionizable residues in the core and regions flanking the helix significantly modulate the stability of transmembrane insertion and/or helix-helix association.
为了探究氨基酸组成对膜插入α-螺旋行为的影响，我们研究了在其疏水核心内含有单个极性/可电离残基的赖氨酸侧翼聚亮氨酸（pLeu）螺旋的行为。为了通过荧光评估螺旋在膜内的位置，每个螺旋在序列中心都含有一个色氨酸残基。当整合到二油酰磷脂酰胆碱（DOPC）模型膜囊泡中时，在疏水核心中含有或不含有单个丝氨酸、天冬酰胺、赖氨酸或天冬氨酸残基的pLeu螺旋在中性和酸性pH下保持跨膜状态（称为N状态）。在这种状态下，中心色氨酸表现出高度蓝移的荧光，并且通过氮氧化物标记的脂质进行的荧光猝灭表明它位于双层中心。在pH 10 - 11以上观察到色氨酸荧光红移几纳米的状态（称为B状态）。B状态形成似乎是由于侧翼赖氨酸残基的去质子化。尽管色氨酸发射发生红移，但荧光猝灭表明在B状态下色氨酸最多只是比在N状态下稍微浅一点，这表明B状态也是一种跨膜或接近跨膜的结构。B状态的特征是螺旋寡聚化增加，如在高pH下色氨酸λ（max）对双层内肽浓度的依赖性所示。在核心中含有天冬氨酸残基的pLeu肽在比其他肽更低的pH（pH 8 - 9）下经历了pH依赖性转变。在高pH下，它表现出比其他肽更高的红移荧光和更浅的色氨酸位置。这种状态（称为S状态）没有表现出浓度依赖性的色氨酸λ（max）。我们将S状态行为归因于在高pH下形成带电荷的天冬氨酸残基，以及随后天冬氨酸向膜表面的移动，导致形成非跨膜状态。我们得出结论，单个跨膜螺旋中可以很容易地容忍一个极性或可电离残基，但核心中和螺旋侧翼区域中可电离残基上的电荷会显著调节跨膜插入和/或螺旋 - 螺旋缔合的稳定性。
