Solid-State NMR Derived Structure: Membrane-Bound Polypetides to Protein

9603935交叉从Graicidin研究中获得的知识现在将用于应对与生物合成制备的蛋白质-M2蛋白质相关的挑战，M2蛋白质被研究为脂双层中的一种稀释物。灵敏度的提高将来自样品几何形状的微调、磁场强度的增加和实验方案的改进。M2样品中的线宽主要由样品的不均一性所主导，通过调整样品制备技术可以解决这一问题，也有合理的预期，线宽(以ppm为单位)至少在某些情况下将降低增加的场强。数学上的挑战将涉及膜蛋白中普遍存在的不完美α-螺旋的特征。这项工作将利用这种利用取向而不是距离限制的固态核磁共振方法，并将其应用于“真正的”蛋白质系统。包括核磁共振波谱在内的高科技方法将被开发来解决大分子的三维结构。通常情况下，这种结构是由应用于结晶样品的衍射法确定的，但有许多重要的大分子是不可结晶的。这种独特的核磁共振方法使用的是均匀排列的样品，换句话说，它们是一维取向的，而晶体涉及三维顺序。用于演示这些方法的分子系统是在膜中形成离子通道的蛋白质。获得的核磁共振数据将通过新的数学方法进行分析，这种方法将普遍适用于许多分子体系。总体而言，这种结构测定方法的发展将为许多类型的不易结晶的分子开辟新的研究途径。这项工作将为研究生和博士后提供很好的培训机会，在这个跨学科的环境中，分子生物学、合成化学、核磁共振光谱学、核磁共振硬件开发、分子建模和数学分析都在本研究工作的范围内。

9603935 Cross The knowledge gained from the gramicidin studies will now be used to face the challenges associated with a biosynthetically prepared protein, M2 protein, studied as a dilute species in a lipid bilayer. Improvements in sensitivity will come from modications in the sample geometry, increased field strength and improvements in the experimental protocols. Linewidths in the M2 samples are primarily dominated by sample heterogeneity which through adjustments in sample preparation techniques can be addressed and there is also reasonable expectations that linewidths (in ppm) at least in some cases will decrease the increasing field strength. The mathematical challenges will involve the characterization of imperfect alpha-helices that are prevalent in membrane proteins. The work will take this solid state NMR method that utilizes orientational rather than distance constraints and apply it to a "real" protein system. High technology methods involving Nuclear Magnetic resonance (NMR) spectroscopy will be developed to solve the three-dimensional structures of macromolecules. Typically such structures are determined by diffraction methods applied to crystallized samples, but there are many important macromolecules that are not crystallizable. This unique NMR approach uses samples that are uniformly aligned, in other words they are oriented in one dimension, while crystals involve three-dimensional order. The molecular system that will be used for demonstrating these methods is protein that forms an ion channel in membranes. The NMR data obtained will be analyzed by new mathematical approaches that will be generally applicable to many molecular systems. Overall, the development of this structure determination approach will open new avenues of research for a great many types of molecules that are not easily crystallized. The work will provide great training opportunities for graduate students and postdocs in an interdisciplinary environment where molecular biology, synthetic chemistr y, NMR spectroscopy, NMR hardware development, molecular modeling and mathematical analysis occur in the confines of this research effort.