Improving Rate/Quality Limitations in Membrane Protein Structure Determination

描述(申请人提供)：本提案旨在解决可用作药物靶标和医学用途的整体膜蛋白高分辨率结构生成速度慢的问题。该小组结合了膜蛋白结构生物学方面的成熟专业知识，这导致了蛋白质数据库中存放的16个结构，以及方法开发方面的前沿进展。经验表明，缺乏高分辨率的膜蛋白结构是结构确定管道中几个可识别的瓶颈的结果，包括：(I)异亚基原核生物亚基的共表达 (2)真核膜蛋白的表达；(3)稳定性和功能性的测定；(4)认识(A)脂类在结构和结晶速度中的作用；(B)蛋白降解，从而排除活性蛋白的纯化。将针对(I)和(Ii)开发一套新的表达方法，并在(Ii)中补充分析方法。问题(一-四)导致形成衍射良好的晶体的不确定性，这可能需要几周或几个月的时间才能产生。及早发现晶体生长将大大减少晶体筛选所需的时间，并允许进入更大的结晶条件相空间。我们描述了一种创新的方法，SONICC(手性晶体的二阶非线性光学成像)，用于灵敏和选择性地检测在体积低至0.5皮升的筛选平台中制备的小至100 nm的初始蛋白质晶体。它对麦芽糖转运蛋白和细胞色素b6f复合体的膜蛋白晶体的区分能力已经得到证实。这种方法将大大加快在SURFO和MESOO中产生衍射级质量的3维和2维晶体的速度。已经选择了一系列完整的膜蛋白靶标，重点是ABC和异寡聚体蛋白；(I)ABC：麦芽糖和核糖转运蛋白，ABCB1，ABCG2，和 非ABC：双精氨酸转位酶；KDP-ATPase；NADH脱氢酶。与公共健康相关：完整的膜蛋白控制细胞间的所有交通，膜本身的组装，细胞营养物质的供应，细胞的能量，以及细胞与细胞外环境的通讯。膜蛋白的原子结构对于理解人类疾病的分子基础以及开发抗击疾病或改善其后果的药物至关重要。

DESCRIPTION (provided by applicant): This proposal is directed to solving the problem of the slow rate of generation of high resolution structures of integral membrane proteins that can be useful as drug targets and in medicine. The group combines established expertise in membrane protein structural biology, which has led to 16 structures deposited in the Protein Data Bank, as well as cutting-edge advances in methods development. Experience has shown that the paucity of high resolution membrane protein structure is a consequence of several recognizable bottlenecks in the structure determination pipeline, including: (i) co-expression of the subunits of hetero-subunit prokaryotic proteins; (ii) expression of eukaryotic membrane proteins; (iii) determination of stability and functionality; (iv) recognition of (a) the role of lipid in the structure and in the rate of crystallization and (b) proteolytic degradation that can preclude purification of active protein. A suite of new expression approaches will be developed targeting (i) and (ii), with complementary analysis approaches in (ii). Problems (i-iv) lead to uncertainties in the formation of well-diffracting crystals, which can take weeks or months to generate. Early detection of crystal growth would greatly decrease the time required for crystal screening and allow access to a greater phase-space of crystallization conditions. We describe an innovative methodology, SONICC (second order nonlinear optical imaging of chiral crystals), for sensitive and selective detection of incipient protein crystals as small as 100 nm, prepared in screening platforms with volumes as low as 0.5 picoliter. Its ability to distinguish membrane protein crystals of the maltose transporter and cytochrome b6f complex has been confirmed. This approach will greatly speed generation of diffraction-quality 3-dimensional and 2-dimensional crystals, generated both in surfo and in meso. A range of integral membrane protein targets has been selected, with an emphasis on ABC and hetero-oligomeric proteins; (i) ABC: maltose and ribose transporters, ABCBl, ABCG2, and Sur-Kir6.2; (ii) non-ABC: twin-arginine translocase; Kdp-ATPase; NADH dehydrogenase. PUBLIC HEALTH RELEVANCE: Integral membrane proteins control all traffic between cell compartments, assembly of the membranes themselves, the supply of nutrients to the cell, its energization, and communication of the cell with the extracellular environment. The atomic structures of membrane proteins is crucial for an understanding of the molecular basis of human diseases and the development of drugs to combat them or ameliorate their consequences.