Wimley W C, White S H
Department of Physiology and Biophysics and the Program in Macromolecular Structure, University of California at Irvine, Irvine, California 92697-4560, USA.
Biochemistry. 2000 Apr 18;39(15):4432-42. doi: 10.1021/bi992746j.
Direct measurement of the free energies of transfer of hydrophobic membrane-spanning alpha-helices from water to membranes is important for the determination of an accurate experiment-based hydrophobicity scale for membrane proteins. An important objective of such a scale is to account for the presently unknown thermodynamic cost of partitioning hydrogen-bonded peptide bonds into the membrane hydrocarbon core. We describe here the physical properties of a transmembrane (TM) peptide, TMX-1, designed to test the feasibility of engineering peptides that spontaneously insert across bilayers but that have the important property of measurable monomeric water solubility. TMX-1, Ac-WNALAAVAAAL-AAVAAALAAVAAGKSKSKS-NH(2), is a 31-residue sequence with a 21-residue nonpolar core, N- and C-caps to favor helix formation, and a highly polar C-terminus to improve solubility and to control directionality of insertion into lipid vesicles. TMX-1 appeared to be soluble in water up to a concentration of at least 1 mg/mL (0.3 mM). However, fluorescence spectroscopy, fluorescence quenching, and circular dichroism (CD) spectroscopy indicated that the high solubility was due to the formation of molecular aggregates that persisted at peptide concentrations down to at least 0.1 microM peptide. Nevertheless, aqueous TMX-1 partitioned strongly into membrane vesicles with apparent mole-fraction free-energy values of -7.1 kcal mol(-1) for phosphatidylcholine (POPC) vesicles and -8.2 kcal mol(-1) for phosphatidylglycerol (POPG) vesicles. CD spectroscopy of TMX-1 in oriented multilayers formed from either lipid disclosed a very strong preference for a transmembrane alpha-helical conformation. When TMX-1 was added to preformed vesicles, it was fully helical. A novel fluorescence resonance energy transfer (FRET) method demonstrated that at least 50% of the TMX-1 insered spontaneously across the vesicle membranes. Binding and insertion were found to be fully reversible for POPC vesicles but not POPG vesicles. TMX-1 was thus found to have many of the properties required for thermodynamic measurements of TM peptide insertion. Importantly, the results obtained delineate the experimental problems that must be considered in the design of peptides that can partition spontaneously and reversibly as monomers into and across membranes. Our success with TMX-1 suggests that these problems are not insurmountable.
直接测量疏水性跨膜α螺旋从水相转移到膜相的自由能，对于确定基于实验的准确膜蛋白疏水性标度至关重要。这样一个标度的一个重要目标是考虑目前未知的将氢键连接的肽键分配到膜烃核心的热力学成本。我们在此描述一种跨膜（TM）肽TMX-1的物理性质，该肽旨在测试设计能自发插入双层膜但具有可测量单体水溶性这一重要性质的肽的可行性。TMX-1，Ac-WNALAAVAAAL-AAVAAALAAVAAGKSKSKS-NH₂，是一个31个残基的序列，有一个21个残基的非极性核心、用于促进螺旋形成的N端和C端封端，以及一个高度极性的C端以提高溶解度并控制插入脂质体的方向性。TMX-1在浓度至少为1mg/mL（0.3mM）时似乎可溶于水。然而，荧光光谱、荧光猝灭和圆二色（CD）光谱表明，高溶解度是由于形成了分子聚集体，这些聚集体在肽浓度低至至少0.1μM时仍然存在。尽管如此，水相中的TMX-1强烈分配到膜泡中，对于磷脂酰胆碱（POPC）泡，其表观摩尔分数自由能值为-7.1kcal mol⁻¹，对于磷脂酰甘油（POPG）泡为-8.2kcal mol⁻¹。由任何一种脂质形成的定向多层膜中TMX-1的CD光谱显示出对跨膜α螺旋构象有非常强烈的偏好。当将TMX-1添加到预先形成的泡中时，它完全呈螺旋状。一种新颖的荧光共振能量转移（FRET）方法表明，至少50%的TMX-1自发插入泡膜中。发现对于POPC泡，结合和插入是完全可逆的，但对于POPG泡则不是。因此发现TMX-1具有TM肽插入热力学测量所需的许多性质。重要的是，所获得的结果描绘了在设计能够作为单体自发且可逆地分配到膜中并穿过膜的肽时必须考虑的实验问题。我们在TMX-1上的成功表明这些问题并非不可克服。
