Investigations of Protein Folding by Solid State NMR

通过固态核磁共振研究蛋白质折叠

• 批准号: 7593511
• 负责人: ROBERT TYCKO
• 金额: $ 23万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7593511
• 关键词: Accounting; Address; Amyloid Fibrils; Biological Process; Caliber; Characteristics; Chemicals; Computer Simulation; Condition; Crystallography; Data; Dependence; Development; Disease; Equilibrium; Freezing; Glass; Glycerol; Goals; Heating; Helix (Snails); Investigation; Kinetics; Label; Manuscripts; Measurement; Methods; Modeling; Molecular Conformation; Names; Optics; Peptide Synthesis; Phase; Play; Population; Preparation; Process; Property; Proteins; Pump; Residual state; Resolution; Role; SH3 Domains; Signal Transduction; Site; Solid; Solutions; Solvents; Spectrum Analysis; Staging; Standards of Weights and Measures; Structural Models; Structure; System; Techniques; Temperature; Tertiary Protein Structure; Thermodynamics; Time; Torsion; Transition Temperature; Valine; Vertebral column; Work; alpha Spectrin; cold temperature; ear helix; interest; isopentane; polyproline; pressure; protein folding; research study; solid state; water solution

In previous work in our group (Havlin and Tycko, PNAS 2005), we carried out the first solid state NMR studies of protein folding. These studies focussed on the 35-residue protein domain HP35, which is known to contain three alpha-helical segments in its folded state and to be thermally stable up to approximately 70 C. HP35 was chosen because it can be readily synthesized by standard solid-phase peptide synthesis methods and because it has been the subject of numerous previous studies by experimental techniques and by computer modelling. In this work, we examined the dependence of solid state NMR signals from selectively isotopically-labeled HP35 on chemical denaturant (GdnHCl) concentration in frozen glycerol/water solutions (glass transition temperature of roughly -70 C). At low denaturant concentrations, 13C NMR signals characteristic of a helical protein were observed, as expected. At higher denaturant concentrations, two quite unexpected observations were made: (1) In the "fully unfolded" state (7 M GdnHCl), NMR signals from the three helical segments were markedly different, indicating a high level of disorder in the third helical segment, a mixture of residual helix content and disorder in the second helical segment, and an apparently low degree of disorder but no helix content in the first helical segment. This contradicts the simple assumption that the unfolded state is a uniform random coil; (2) Near the unfolding midpoint (4.5 M GdnHCl), the three helical segments appeared to have progressed to different stages along their respective unfolding paths, i.e., denaturation of HP35 cab not be described by a simple two-state model in which only the fully-folded and fully-unfolded states coexist. This work demonstrated that solid state NMR measurements can indeed provide new information about protein folding. Of particular interest was the observation that the first helical segment of HP35 was apparently highly ordered (but nonhelical) in the unfolded state. This raises the question of what the conformation of this segment actually is in the unfolded state. We have addressed this question in FY2007 by carrying out solid state NMR measurements that directly probe backbone phi and psi torsion angles for a particular site (Valine-50) in the first helical segment. These measurements employ three techniques developed previously in our group, with abbreviated names CT-DQFD, DQCSA, and 2DEXMAS. Without going into details of these techniques, suffice it to say that each technique provides independent constraints on backbone phi and psi torsion angles. Multiple independent constraints allow us to fit the data to simple models for the distribution of phi and psi populations (i.e., simple representations of the structural disorder). When these techniques are applied to HP35 in its folded state, the data are fit best by a single conformation, very close to the helical phi and psi angles determined by crystallography for folded HP35. In the unfolded state (7 M GdnHCl), the combined data can not be fit by a single conformation. Instead, the data are well described by significant populations near two phi,psi pairs, namely -75,155 degrees and -115,75 degrees. The first of these (with approximately 33% of the population) corresponds to the polyproline II conformation that has been suggested by other groups to be a dominant conformation in unfolded proteins. The second (with approximately 67% of the population) is in the "transition region" between alpha-helical and beta-strand conformations, and was not anticipated. Interestingly, ab initio calculations of 13C chemical shifts indicate that these two conformations have similar chemical shifts, accounting for the observation of relatively sharp lines in solid state NMR spectra, which had suggested a high degree of structural order. The work described in the above paragraph represents the first application of quantitative solid state NMR methods for determination of site-specific structural parameters in an unfolded state. A manuscript describing this work is in preparation. Experiments described above probe a "thermodynamically unfolded" state. We are currently attempting to study "kinetically unfolded" states, i.e., structural states that are trapped out of equilibrium by rapid change of solvent conditions, followed by rapid freeze-quenching. HP35 is a challenging case, because the main folding transition is believed to occur in approximately 10 microseconds, too fast to be freeze-quenched. Nonetheless, we have constructed an apparatus that permits freeze-quenching in approximately 100 microseconds (by spraying a protein solution, initially heated above the unfolding temperature, into cold isopentane, using a 50-micron-diameter spray nozzle and high pressure pumps to create a very fine, high-velocity jet of solution). With this apparatus, we are currently investigating whether folding of HP35 may occur in two stages, consisting of an initial formation of secondary structure on the 10 microsecond time scale, followed by a slower stage in which tertiary contacts form and sidechain packing is optimized. Such a slow stage might be invisible to optical techniques that have been used to characterize HP35 folding kinetics, but may be visible in solid state NMR. We also plan to carry out kinetic folding studies of slower-folding proteins, including the 67-residue alpha-spectrin SH3 domain, which we have recently succeeded in synthesizing directly by solid-phase methods and which is therefore amenable to selective isotopic labeling.

在我们小组以前的工作中（Havlin和Tycko，PNAS 2005），我们进行了第一次蛋白质折叠的固态NMR研究。 这些研究集中在35个残基的蛋白质结构域HP 35上，已知其在折叠状态下含有三个α-螺旋片段，并且在高达约70 ℃下是热稳定的。 选择HP 35是因为它可以通过标准固相肽合成方法容易地合成，并且因为它已经是通过实验技术和通过计算机建模的许多先前研究的主题。 在这项工作中，我们研究了固态NMR信号的依赖性，从选择性同位素标记的HP 35对化学变性剂（盐酸钆）浓度在冷冻甘油/水溶液（玻璃化转变温度约-70 ℃）。 在低变性剂浓度下，如预期的那样，观察到螺旋蛋白质的13 C NMR信号特征。 在更高的变性剂浓度下，进行了两个非常出乎意料的观察：（1）在“完全展开”状态下（7 M GdnHCl）时，来自三个螺旋段的NMR信号明显不同，表明第三螺旋段中的高度无序，第二螺旋段中的残留螺旋含量和无序的混合物，以及在第一螺旋段中明显低的无序度但没有螺旋含量。 这与展开状态是均匀无规卷曲的简单假设相矛盾;（2）在展开中点（4.5 M GdnHCl）附近，三个螺旋段似乎已经沿着它们各自的展开路径沿着进展到不同的阶段，即，HP 35的变性不能用简单的两态模型来描述，其中只有完全折叠和完全未折叠的状态共存。 这项工作表明，固态NMR测量确实可以提供有关蛋白质折叠的新信息。 特别令人感兴趣的是观察到的第一螺旋段的HP 35显然是高度有序的（但非螺旋）在未折叠状态。 这就提出了一个问题，即在未折叠状态下，该片段的构象实际上是什么。 我们在2007财年通过进行固态NMR测量解决了这个问题，该测量直接探测第一螺旋片段中特定位点（缬氨酸-50）的骨架phi和psi扭转角。 这些测量采用了我们小组以前开发的三种技术，缩写为CT-DQFD，DQCSA和2DEXMAS。 无需深入这些技术的细节，只要说每种技术提供对主干phi和psi扭转角的独立约束就足够了。 多个独立的约束允许我们将数据拟合到用于phi和psi群体分布的简单模型（即，结构紊乱的简单表示）。 当将这些技术应用于处于折叠状态的HP 35时，数据通过单一构象最佳拟合，非常接近于通过结晶学确定的折叠HP 35的螺旋phi和psi角。 在未折叠状态下（7 M盐酸钆），合并的数据不能用单一构象拟合。 相反，数据很好地描述了两个phi，psi对附近的显着群体，即-75，155度和-115，75度。 其中第一种（约占总人口的33%）对应于聚脯氨酸II构象，其他研究组已建议将其作为未折叠蛋白质中的主要构象。 第二种（约占总数的67%）位于α-螺旋和β-链构象之间的“过渡区”，这是没有预料到的。 有趣的是，13 C化学位移的从头计算表明，这两种构象具有相似的化学位移，占观察到的相对尖锐的线在固态NMR光谱，这表明了高度的结构顺序。 上段所述的工作代表了定量固态NMR方法用于确定未折叠状态下的位点特异性结构参数的首次应用。 描述这项工作的手稿正在编写中。 上述实验探测了“螺旋展开”状态。 我们目前正试图研究“动力学展开”状态，即，通过溶剂条件的快速变化，随后通过快速冷冻淬灭而被捕获脱离平衡的结构状态。 HP 35是一个具有挑战性的情况，因为主要的折叠转变被认为发生在大约10微秒内，太快而不能被冷冻猝灭。 尽管如此，我们已经构建了一种允许在大约100微秒内冷冻猝灭的装置（通过将最初加热到展开温度以上的蛋白质溶液喷射到冷的异戊烷中，使用50微米直径的喷嘴和高压泵来产生非常精细的高速溶液射流）。 使用该装置，我们目前正在研究折叠的HP 35是否可能发生在两个阶段，包括在10微秒的时间尺度上的二级结构的初始形成，随后由一个较慢的阶段，其中三级接触形式和侧链包装优化。 这样的缓慢阶段对于用于表征HP 35折叠动力学的光学技术来说可能是不可见的，但在固态核磁共振中可能是可见的。 我们还计划进行缓慢折叠蛋白质的动力学折叠研究，包括67个残基的α-血影蛋白SH 3结构域，我们最近成功地直接通过固相方法合成，因此适合选择性同位素标记。