Optical spectroscopy for biomolecular interactions

生物分子相互作用的光谱

• 批准号: BB/F011199/1
• 负责人: Alison Rodger
• 金额: $ 12.23万
• 依托单位: University of Warwick
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2008
• 资助国家: 英国
• 起止时间: 2008 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FF011199%2F1
• 关键词: Optical; spectroscopy; biomolecular; interactions

The applicants wish to purchase spectroscopic equipment totalling £139,710 (including service contracts) to enable fluorescence detected linear dichroism measurements (FDLD) and the interpretation of the FDLD data using fluorescence and absorption data. The spectrometers will also be used for linear dichroism (LD), circular dichroism (CD), fluorescence, fluorescence polarisation anisotropy and absorbance measurements as stand-alone experiments as well as in conjunction with the new technique of FDLD. Jasco UK Ltd. are contributing £49,219 to the project in the form of £6,000 of staff time for training and supporting workshops to be run at Warwick and the remainder in discounts on the purchase of the instruments. Jasco wish to establish Warwick as the Jasco Centre of excellence in Polarised Spectroscopy which will further enhance the collaboration between the applicants and Jasco UK Ltd. Crystal Precision Optics will contribute £4,000 of staff time to support design and development of new equipment emanating from this project. A small amount of PI time will be required to oversee the procurement, installation, and establishing the equipment as a multi-user facility. Rationale underlying proposal: All biological processes are fundamentally interactions between molecules, mainly between macromolecules or between macromolecules and small molecules. Despite significant advances in our ability to characterise such interactions and the 'single-molecule' revolution that is taking place, we still struggle to measure key properties of molecular interactions. This is particularly true for the significant classes of biological molecules that have proved difficult to study by established structural techniques such as crystallography and NMR. These include long polymeric structures: DNA; DNA-ligand complexes; fibres including fibrous proteins; and also membrane proteins. Wishing to probe the time dependence of interactions including enzyme kinetics, fibre assembly or protein insertion into membranes introduces an added dimension of complexity. The aim of this proposal is to establish the newly invented technique of FDLD so the UK community can benefit from its advantages. The core FDLD instrument will be supported by standard fluorescence and absorption instruments to enable data interpretation. All three instruments will also be used for independent LD, CD, fluorescence, polarised fluorescence and measurements of interacting biomolecular systems. Over the last few years Warwick has become the national and indeed international hub for innovation based on the technique of flow linear dichroism (LD), which is the difference in absorbance of light polarised parallel and perpendicular to an orientation axis. LD can be used to deduce kinetic and structural information about a wide range of systems, the only requirements being that they have absorbance spectroscopy and they can be oriented. Flow LD requires samples to be long enough to be oriented by shear forces in solution and is ideally suited to DNA and fibrous proteins. We have also had significant success in orienting membrane systems as the flow-distortion of liposomes creates an orientation axis. In the context of ligand binding, the key attraction of LD is that it is selective only for those molecules bound to the long system. Thus, e.g., only PCR products are detected, not the background population of free nucleotides. One of the new techniques we have developed is fluorescence detected linear dichroism (FDLD). This is the focus of the current application. FDLD captures the advantages for intermolecular interactions of (i) LD (namely detecting only oriented samples) and (ii) fluorescence (namely only detecting fluorophores and with higher sensitivity than absorption-based techniques). In the proof of concept paper for FDLD we showed how FDLD can be used selectively to study the orientation on DNA of ligands whose spectroscopy lies under the DNA absorbance bands.

申请人希望购买总计139,710 GB的光谱设备(包括服务合同)，以实现荧光检测线性二向色性测量(FDLD)和利用荧光和吸收数据解释FDLD数据。光谱仪还将用于线性二色性(LD)、圆二色性(CD)、荧光、荧光偏振各向异性和吸光度的测量，作为独立实验以及与FDLD的新技术结合使用。JASCO UK Ltd以6,000 GB工作人员时间的形式向该项目提供49,219 GB的资金，用于在Warwick举办培训和支助讲习班，其余部分用于购买仪器的折扣。JASCO希望将Warwick建立为Jasco偏振光谱领域的卓越中心，这将进一步加强申请者与Jasco UK Ltd之间的合作。Crystal Precision Optics将贡献4000 GB的员工时间来支持该项目产生的新设备的设计和开发。将需要少量的PI时间来监督设备的采购、安装和建立多用户设施。基本原理：所有生物过程基本上都是分子之间的相互作用，主要是大分子之间或大分子和小分子之间的相互作用。尽管我们在描述这种相互作用的能力和正在发生的“单分子”革命方面取得了重大进展，但我们仍然难以测量分子相互作用的关键属性。对于那些已被证明难以用现有的结构技术，如结晶学和核磁共振来研究的重要的生物分子来说，情况尤其如此。这些结构包括长聚合物结构：DNA；DNA-配体复合体；纤维，包括纤维蛋白；以及膜蛋白。希望探索相互作用的时间依赖性，包括酶动力学、纤维组装或蛋白质插入膜中，引入了额外的复杂性维度。这项提议的目的是建立新发明的FDLD技术，以便英国社区能够从其优势中受益。核心FDLD仪器将得到标准荧光和吸收仪器的支持，以便能够进行数据解释。所有这三种仪器还将用于独立的LD、CD、荧光、偏振荧光和相互作用的生物分子系统的测量。在过去的几年里，华威已经成为基于流线性二色性(LD)技术的全国乃至国际创新中心，LD是平行于和垂直于取向轴的偏振光的吸光度的差异。LD可以用来推断各种体系的动力学和结构信息，唯一的要求是它们具有吸收光谱，并且它们可以定向。Flow LD要求样品足够长，可以通过溶液中的剪切力进行定向，非常适合DNA和纤维蛋白。我们在膜系统的定向方面也取得了重大成功，因为脂质体的流动扭曲产生了定向轴。在配体结合的背景下，LD的关键吸引力在于它只对那些与长体系结合的分子具有选择性。因此，例如，仅检测到聚合酶链式反应产物，而不是游离核苷酸的背景群体。我们开发的新技术之一是荧光检测线性二色性(FDLD)。这是当前应用的重点。FDLD抓住了(I)LD分子间相互作用的优点(即只检测定向样品)和(Ii)荧光(即只检测荧光团，并且比基于吸收的技术具有更高的灵敏度)。在FDLD的概念证明文件中，我们展示了FDLD如何选择性地用于研究其光谱位于DNA吸收带下的配体在DNA上的取向。

项目成果

Calculations of flow-induced orientation distributions for analysis of linear dichroism spectroscopy

• DOI: 10.1039/c3sm27419e
• 发表时间: 2013-01-01
• 期刊: SOFT MATTER
• 影响因子: 3.4
• 作者: McLachlan, James R. A.;Smith, David J.;Rodger, Alison
• 通讯作者: Rodger, Alison

Infrared absorbance spectroscopy of aqueous proteins: Comparison of transmission and ATR data collection and analysis for secondary structure fitting

• DOI: 10.1002/chir.23002
• 发表时间: 2018-07-20
• 期刊: Chirality
• 影响因子: 2
• 作者: Corujo MP;Sklepari M;Ang DL;Millichip M;Reason A;Goodchild SC;Wormell P;Amarasinghe DP;Lindo V;Chmel NP;Rodger A
• 通讯作者: Rodger A

Breaking the 200 nm limit for routine flow linear dichroism measurements using UV synchrotron radiation.

使用紫外同步加速器辐射突破常规流动线性二色性测量的 200 nm 限制。

• DOI: 10.1529/biophysj.108.139964
• 发表时间: 2008
• 期刊: Biophysical journal
• 影响因子: 3.4
• 作者: Dicko C

Polyproline as a Minimal Antifreeze Protein Mimic That Enhances the Cryopreservation of Cell Monolayers.

• DOI: 10.1002/anie.201706703
• 发表时间: 2017-12-11
• 期刊: Angewandte Chemie (International ed. in English)
• 作者: Graham B;Bailey TL;Healey JRJ;Marcellini M;Deville S;Gibson MI
• 通讯作者: Gibson MI

Biological Insights from a Simulation Model of the Critical FtsZ Accumulation Required for Prokaryotic Cell Division.

原核细胞分裂所需的关键 FtsZ 积累模拟模型的生物学见解。

• DOI: 10.1021/acs.biochem.5b00261
• 发表时间: 2015
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Dow CE