Function and structure of GFP-like proteins in the protein data bank

Abstract

The RCSB protein databank contains 266 crystal structures of green fluorescent proteins (GFP) and GFP-like proteins. This is the first systematic analysis of all the GFP-like structures in the pdb. We have used the pdb to examine the function of fluorescent proteins (FP) in nature, aspects of excited state proton transfer (ESPT) in FPs, deformation from planarity of the chromophore and chromophore maturation. The conclusions reached in this review are that (1) The lid residues are highly conserved, particularly those on the "top" of the β-barrel. They are important to the function of GFP-like proteins, perhaps in protecting the chromophore or in β-barrel formation. (2) The primary/ancestral function of GFP-like proteins may well be to aid in light induced electron transfer. (3) The structural prerequisites for light activated proton pumps exist in many structures and it's possible that like bioluminescence, proton pumps are secondary functions of GFP-like proteins. (4) In most GFP-like proteins the protein matrix exerts a significant strain on planar chromophores forcing most GFP-like proteins to adopt non-planar chromophores. These chromophoric deviations from planarity play an important role in determining the fluorescence quantum yield. (5) The chemospatial characteristics of the chromophore cavity determine the isomerization state of the chromophore. The cavities of highlighter proteins that can undergo cis/trans isomerization have chemospatial properties that are common to both cis and trans GFP-like proteins.

Spectral and structural comparison between bright and dim green fluorescent proteins in Amphioxus

“…The emission of increasingly brighter fluorescence indeed seems to strongly correlate with the enhanced stiffness of the encapsulated chromophore, by preventing dissipation of the excited state energy through isomerization of neighboring residues during the excited state144647. Enhanced stiffness of the chromophore, which is combined with a slight tilt in the bright bfloGFPa1, could also decrease the non-coplanarity of the residues, which is known to increase QE in GFP114849. This is also supported from studies on the photochromic GFP Padron in which the photoswitched transition between fluorescent and non-fluorescent states is associated with the combination of both tilting and twisting of the two chromophoric rings relative to one another50.…”

“…Since the publication of the landmark crystal structures from the Remington and Phillips groups, respectively45, more than 250 structures of engineered GFP variants from Aequorea victorea have been deposited into the Protein Data Bank (PDB), and are described in many publications (www.rcsb.org)11. This extensive catalog of three-dimensional structures coupled with biochemical characterization has enabled both structure-guided engineering of the wild-type Av GFP and the ability to decipher the effect of random mutations and directed amino acid substitutions on the proteins' optical characteristics11.…”

“…This extensive catalog of three-dimensional structures coupled with biochemical characterization has enabled both structure-guided engineering of the wild-type Av GFP and the ability to decipher the effect of random mutations and directed amino acid substitutions on the proteins' optical characteristics11. These engineering efforts have produced GFPs with widely varying solubilities, oligomerization states, pH optima, halide and temperature sensitivities, and a diversity of fluorescence intensity, brightness, absorption and emission spectra (e.g 121314…”

Identification and expressional analysis of green fluorescent protein genes in amphioxus

“…GFP 最早是 Shimomura et al (1962)在研究太平 洋维多利亚水母(Aequoreavitofia)的荧光素酶时意 外发现的一种能够自发释放出生物荧光的蛋白质。 Morise et al (1974)纯化了该蛋白质。在水母中分离 得到的这种 GFP 是由 238 个氨基酸组成的一种单体 蛋白质, 其中 GFP 荧光的产生主要归功于其分子内 65～67 位(SYG)的三个氨基酸所形成的生色团。随 后的研究发现，GFP 的晶体结构显示蛋白质中央是 一个水桶样结构, 由 11 个围绕中心 α 螺旋的反平行 β 折叠组成的一个完整结构, 这个桶状结构的完整 性是 GFP 产生荧光的前提条件。 GFP 家族的成员其 生色/荧光基团的形成完全是由多肽链序列本身决 定, 其成熟以及荧光发射除了氧气外，都不需要其 他任何辅助因子 (Ormö et al,1996;Tsien, 1998;Yang et al,1996;Zimmer, 2002) 。 自 从 1992 年 Douglas Prasher 在水母中克隆得到第一个 GFP 基因 avGFP 后, 许多新的 GFP 基因也陆续在其他物种得 到克隆 (Chalfie et al, 1994;Gurskaya et al, 2003;Labas et al, 2002;Matz et al, 1999;Shagin et al, 2004)。这些 GFP 基因主要来自后生动物的两大类： 刺细胞动物(Cnidarians)和桡足类(Copepods)，而在 已经测序的其他后生动物中却尚未找到 GFP 基因。 Deheyn et al (2007)在头索动物佛罗里达文昌 鱼(Branchiostoma floridae)中发现一个 GFP 基因, 而且在这个物种胚胎发育的各个时期及成体中都 发现了绿色荧光蛋白表达。随后进一步的系统发育 分析暗示， 文昌鱼 GFP 的起源符合先前关于两侧对 称动物的荧光蛋白是从单一的后生动物祖先的荧 光蛋白进化而来的推测, 而不是独立源于后口动物 的 G2FP 基元蛋白 (Shagin et al, 2004)。 目前在 RCSB 蛋白数据库中已收集的 27 个物种中发现有 266 个 GFP 基因家族的成员。该数据库对这些基因的空间 结构等方面有详细的描述, 人们对这些 GFP 基因进 行了系统分析, 并对 GFP 的功能作了一些推测；但 很遗憾，RCSB 数据库中没有包含头索动物文昌鱼 的 GFP 基因数据 (Ong et al, 2011), 而文昌鱼作为发 现内源 GFP 蛋白的最高等生物类群, 对其 GFP 的研 究有着重要意义。作为隶属脊索动物门(Chordata) 头索动物亚门(Cephalochordata)的文昌鱼, 在进化 上占有极其重要的地位, 是介于无脊椎动物和脊椎 动物之间的过渡类型 …”

Concerted Asynchronous Proton Transfer in H-Bonding Relay Model: An Implication of Green Fluorescent Protein

“…This non-planar deformation can accelerate ESPT process. 30 Anyhow, the main purpose is to figure out the proton motion, rather than obtain accurate energy barrier. To take the surrounding environment into account or get correct reaction rate, higher level simulation is needed, such as quantum dynamics/molecular dynamics (QDs/MSs) or quantum mechanical/molecular mechanics (AM/MMs).…”