Augustijns P F, Brown S C, Willard D H, Consler T G, Annaert P P, Hendren R W, Bradshaw T P
Biophysical and Structural Chemistry Department, Molecular Sciences Department and Biopharmaceutical Development, Glaxo Wellcome Inc., RTP, North Carolina 27709, USA.
Biochemistry. 2000 Jun 27;39(25):7621-30. doi: 10.1021/bi9929709.
Cyclosporin A is a cyclic peptide believed to exist as multiple conformers in aqueous solution. Two major conformations, distinguished by a single cis-trans isomerization and the presence of four either intramolecular or intermolecular hydrogen bonds, have been confirmed depending on whether CsA is characterized in organic solvents or bound in aqueous complex with cyclophilin. The relationship between CsA conformation and its ability to penetrate biological membranes is currently unknown. Using Caco-2 cell monolayers, we documented a remarkable increase (more than 2 orders of magnitude) in the membrane permeation of the peptide as temperature was increased from 5 to 37 degrees C. The solubility of CsA was 72 microM at 5 degrees C, but decreased by more than an order of magnitude at 37 degrees C. Moreover, CsA partitioned into non-hydrogen bond donating solvents linearly as a function of increasing temperature, suggestive of a significant conformational change. However, while NMR spectra of CsA confirmed the previously predicted presence of multiple conformers in aqueous solution, the equilibrium between the two major species was not affected by changes in temperature. These NMR data indicated that the observed temperature-dependent changes in the membrane permeability of CsA do not originate from changes in the peptide backbone conformation. Sedimentation equilibrium analysis revealed that CsA behaves in a highly nonideal manner over the temperature range tested. We interpret this behavior as a change in the hydration state with a smaller (or weaker) hydration shell surrounding the peptide at higher temperatures. Such a change would result in lower peptide desolvation energy, thereby promoting partitioning into cellular membranes. We contend that changes in membrane penetration result from alterations in the hydration state of CsA and are not related to the interconversion of the defined conformations.
环孢素A是一种环状肽，据信在水溶液中以多种构象存在。根据环孢素A是在有机溶剂中表征还是与亲环蛋白在水性复合物中结合，已确认了两种主要构象，它们由单个顺反异构化以及四个分子内或分子间氢键的存在来区分。目前尚不清楚环孢素A构象与其穿透生物膜能力之间的关系。使用Caco-2细胞单层，我们记录到随着温度从5℃升高到37℃，该肽的膜渗透率显著增加（超过2个数量级）。环孢素A在5℃时的溶解度为72μM，但在37℃时下降了超过一个数量级。此外，环孢素A随着温度升高线性地分配到不提供氢键的溶剂中，这表明存在显著的构象变化。然而，虽然环孢素A的核磁共振光谱证实了先前预测的在水溶液中存在多种构象，但两种主要构象之间的平衡不受温度变化的影响。这些核磁共振数据表明，观察到的环孢素A膜渗透率随温度的变化并非源于肽主链构象的变化。沉降平衡分析表明，环孢素A在测试的温度范围内表现出高度非理想的行为。我们将这种行为解释为水合状态的变化，即在较高温度下围绕肽的水合壳较小（或较弱）。这种变化将导致较低的肽去溶剂化能，从而促进其分配到细胞膜中。我们认为膜穿透的变化是由环孢素A水合状态的改变引起的，与确定构象的相互转化无关。
