Department of Anatomy and Structural Biology, and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York, USA.
Nat Methods. 2011 Jul 31;8(9):771-7. doi: 10.1038/nmeth.1664.
We report a photoswitchable monomeric Orange (PSmOrange) protein that is initially orange (excitation, 548 nm; emission, 565 nm) but becomes far-red (excitation, 636 nm; emission, 662 nm) after irradiation with blue-green light. Compared to its parental orange proteins, PSmOrange has greater brightness, faster maturation, higher photoconversion contrast and better photostability. The red-shifted spectra of both forms of PSmOrange enable its simultaneous use with cyan-to-green photoswitchable proteins to study four intracellular populations. Photoconverted PSmOrange has, to our knowledge, the most far-red excitation peak of all GFP-like fluorescent proteins, provides diffraction-limited and super-resolution imaging in the far-red light range, is optimally excited with common red lasers, and can be photoconverted subcutaneously in a mouse. PSmOrange photoswitching occurs via a two-step photo-oxidation process, which causes cleavage of the polypeptide backbone. The far-red fluorescence of photoconverted PSmOrange results from a new chromophore containing N-acylimine with a co-planar carbon-oxygen double bond.
我们报告了一种光可切换的单体橙色（PSmOrange）蛋白，它最初是橙色（激发波长 548nm，发射波长 565nm），但在蓝光绿光照射下会变成远红色（激发波长 636nm，发射波长 662nm）。与母体橙色蛋白相比，PSmOrange 的亮度更高、成熟更快、光转化对比度更高、光稳定性更好。两种形式的 PSmOrange 的红移光谱使其能够与青绿色光可切换蛋白同时使用，以研究四个细胞内群体。据我们所知，光转化后的 PSmOrange 具有所有 GFP 样荧光蛋白中最远的红色激发峰，可在远红光范围内提供衍射限制和超分辨率成像，用常见的红色激光最佳激发，并且可在小鼠的皮下进行光转化。PSmOrange 的光开关通过两步光氧化过程发生，导致多肽主链的断裂。光转化后的 PSmOrange 的远红色荧光源自含有 N-酰亚胺的新发色团，其中含有共面的碳-氧双键。
