Evans J S, Chan S I
Arthur Amos Noyes Laboratory for Chemical Physics, California Institute of Technology, Pasadena 91125.
Biopolymers. 1994 Apr;34(4):507-27. doi: 10.1002/bip.360340407.
The protein folding behavior of a polyelectrolyte protein, bovine dentine phosphophoryn (BDPP), in the pH range of 1.82-11.0 has been investigated. One- and two-dimensional nmr spectroscopy has been utilized to obtain proton spin assignments for amino acid residues in D2O and in H2O. One-dimensional 31P-nmr experiments verify the existence of three separate classes of O-phosphoserine (PSer) resonances in BDPP (alpha, beta, chi), representing three distinct PSer residue populations at pH 6.94. By means of pH titration and 1H-nmr, five populations of Asp residues can be identified. Three of these populations exhibit secondary inflection points on their pH titration curves that correspond to an observed pKa of 6.17-6.95. The presence or absence of secondary inflection points for Asp populations and the 31P-nmr chemical shift dispersion for the three PSer residue populations indicate that BDPP may be comprised of homologous (Asp-Asp)n. (PSer-PSer)n, and heterologous (PSer-Asp)n sequences arranged into polyelectrolyte cluster regions. The pH titration also revealed that certain populations of Ser, Gly, and Pro residues in BDPP exhibit pH-dependent resonance frequency shifts. The "apparent" pKa for the transition points of these frequency shifts corresponds to either the pK1a of Pser monophosphate ester and/or the pKa of Asp COOH group of BDPP polyelectrolyte regions. On the basis of these transition points, we can assign four types of Ser, Gly, or Pro-containing "intervening" regions in BDPP, based on their sensitivity to protonation and deprotonation events occurring at (Asp)n, (PSer)n, or (PSer-Asp)n anionic cluster regions that flank the intervening regions. Our 1H-nmr experiments also reveal that BDPP assumes a folded conformation at low pH. As the pH increases, this conformation undergoes several unfolding transitions as the BDPP molecule assumes more open conformations in response to increased electrostatic repulsion between polyelectrolyte anionic regions in the protein. These folding-unfolding transitions are mediated by the intervening regions, which act as "hinges" to allow the polyelectrolyte regions to fold relative to one another.
研究了聚电解质蛋白牛牙本质磷蛋白（BDPP）在1.82 - 11.0 pH范围内的蛋白质折叠行为。利用一维和二维核磁共振光谱来确定D₂O和H₂O中氨基酸残基的质子自旋归属。一维³¹P - 核磁共振实验证实了BDPP中存在三类不同的O - 磷酸丝氨酸（PSer）共振（α、β、χ），代表了在pH 6.94时三种不同的PSer残基群体。通过pH滴定和¹H - 核磁共振，可以鉴定出五类天冬氨酸（Asp）残基。其中三类群体在其pH滴定曲线上呈现二级拐点，对应观察到的pKa为6.17 - 6.95。Asp群体二级拐点的有无以及三类PSer残基群体的³¹P - 核磁共振化学位移分散表明，BDPP可能由排列成聚电解质簇区域的同源（Asp - Asp）n、（PSer - PSer）n和异源（PSer - Asp）n序列组成。pH滴定还表明，BDPP中某些丝氨酸（Ser）、甘氨酸（Gly）和脯氨酸（Pro）残基群体表现出pH依赖性共振频率变化。这些频率变化转变点处的“表观”pKa对应于PSer单磷酸酯的pK₁a和/或BDPP聚电解质区域中天冬氨酸COOH基团的pKa。基于这些转变点，我们可以根据BDPP中含Ser、Gly或Pro的“间隔”区域对其侧翼（Asp）n、（PSer）n或（PSer - Asp）n阴离子簇区域发生的质子化和去质子化事件的敏感性，将其分为四种类型。我们的¹H - 核磁共振实验还表明，BDPP在低pH下呈现折叠构象。随着pH升高，这种构象会经历几次去折叠转变，因为BDPP分子会因蛋白质中聚电解质阴离子区域之间静电排斥增加而呈现出更开放的构象。这些折叠 - 去折叠转变由间隔区域介导，间隔区域起到“铰链”的作用，使聚电解质区域能够相对彼此折叠。
