Brodie Nicholas I, Makepeace Karl A T, Petrotchenko Evgeniy V, Borchers Christoph H
University of Victoria - Genome British Columbia Proteomics Centre, University of Victoria, #3101-4464 Markham Street, Vancouver Island Technology Park, Victoria V8Z7X8, Canada.
University of Victoria - Genome British Columbia Proteomics Centre, University of Victoria, #3101-4464 Markham Street, Vancouver Island Technology Park, Victoria V8Z7X8, Canada; Department of Biochemistry & Microbiology, University of Victoria, Petch Building Room 207, 3800 Finnerty Rd., Victoria, BC V8P 5C2, Canada.
J Proteomics. 2015 Apr 6;118:12-20. doi: 10.1016/j.jprot.2014.08.012. Epub 2014 Sep 2.
The resolution and the fidelity of a protein structural model, constructed using crosslinking data, is dependent on the crosslinking distance constraints. Most of the popular amine-reactive NHS-ester crosslinkers are limited in their capacity to provide short distance constraints because of the rarity of lysine residues occurring in close proximity in the protein structure. To solve this problem, hetero-bifunctional crosslinkers containing both a photo-reactive functional group and an NHS-ester group can be used to enable non-specific crosslinking within the proximity of these lysine residues. Here we develop three such isotopically-coded hetero-bifunctional photo-reactive crosslinkers, bearing azido, diazirine or benzophenone photo-reactive groups (azido-benzoic-acid-succinimide (ABAS)-(12)C6/(13)C6, succinimidyl-diazirine (SDA)-(12)C5/(13)C5, and carboxy-benzophenone-succinimide (CBS)-(12)C6/(13)C6, respectively). These crosslinkers were validated using several model proteins/peptides and were then applied to study the structure of the native α-synuclein protein. In that case the ABAS crosslinker proved to be the most suitable, with 10 crosslinks being found in the native α-synuclein structure.
Structural proteomics can be used for studying protein structures which may be difficult to examine by traditional structural biology methods such as NMR or X-ray crystallography. Crosslinking in particular is used to provide distance constraints for molecular modeling of individual proteins and protein complexes. The shortest distance constraints are most valuable for the modeling process. To be able to provide such short distance constraints, non-specific photo-reactive chemistry can be used for crosslinking reactions. However, detection of such non-specific crosslinks is difficult because the signal from any particular crosslink is low due to the broad reactivity of the crosslinking reagents. To overcome this problem, we have employed isotopic labeling of these crosslinkers. In this paper, we have demonstrated their effectiveness for studying the native α-synuclein protein structure. The non-specific reactivity, in combination with isotopic coding of these crosslinkers, allowed for the formation and detection of short-range crosslinks, targeting a variety of amino acids. These reagents may prove useful for future applications to a variety of protein structural problems. This article is part of a Special Issue entitled: Protein dynamics in health and disease. Guest Editors: Pierre Thibault and Anne-Claude Gingras.
使用交联数据构建的蛋白质结构模型的分辨率和保真度取决于交联距离限制。大多数常用的胺反应性NHS酯交联剂在提供短距离限制方面能力有限，因为在蛋白质结构中靠近的赖氨酸残基很少见。为了解决这个问题，可以使用含有光反应性功能基团和NHS酯基团的异双功能交联剂，以在这些赖氨酸残基附近实现非特异性交联。在此，我们开发了三种这样的同位素编码异双功能光反应性交联剂，分别带有叠氮基、重氮丙啶或二苯甲酮光反应基团（分别为叠氮基苯甲酸琥珀酰亚胺（ABAS）-(12)C6/(13)C6、琥珀酰亚胺基重氮丙啶（SDA）-(12)C5/(13)C5和羧基二苯甲酮琥珀酰亚胺（CBS）-(12)C6/(13)C6）。这些交联剂用几种模型蛋白质/肽进行了验证，然后用于研究天然α-突触核蛋白的结构。在这种情况下，ABAS交联剂被证明是最合适的，在天然α-突触核蛋白结构中发现了10个交联。
结构蛋白质组学可用于研究可能难以通过传统结构生物学方法（如核磁共振或X射线晶体学）检查的蛋白质结构。特别是交联用于为单个蛋白质和蛋白质复合物的分子建模提供距离限制。最短距离限制对建模过程最有价值。为了能够提供这样的短距离限制，可以使用非特异性光反应化学进行交联反应。然而，检测这种非特异性交联很困难，因为由于交联试剂的广泛反应性，任何特定交联的信号都很低。为了克服这个问题，我们对这些交联剂采用了同位素标记。在本文中，我们证明了它们在研究天然α-突触核蛋白结构方面的有效性。这些交联剂的非特异性反应性与同位素编码相结合，使得能够形成和检测针对多种氨基酸的短程交联。这些试剂可能对未来应用于各种蛋白质结构问题有用。本文是名为：健康与疾病中的蛋白质动力学的特刊的一部分。客座编辑：皮埃尔·蒂博和安妮 - 克劳德·金格拉斯。
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