The 1.6 Å resolution structure of a FRET-optimized Cerulean fluorescent protein.

Genetically encoded cyan fluorescent proteins (CFPs) bearing a tryptophan-derived chromophore are commonly used as energy-donor probes in Förster resonance energy transfer (FRET) experiments useful in live cell-imaging applications. In recent years, significant effort has been expended on eliminating the structural and excited-state heterogeneity of these proteins, which has been linked to undesirable photophysical properties. Recently, mCerulean3, a descendant of enhanced CFP, was introduced as an optimized FRET donor protein with a superior quantum yield of 0.87. Here, the 1.6 Šresolution X-ray structure of mCerulean3 is reported. The chromophore is shown to adopt a planar trans configuration at low pH values, indicating that the acid-induced isomerization of Cerulean has been eliminated. ß-Strand 7 appears to be well ordered in a single conformation, indicating a loss of conformational heterogeneity in the vicinity of the chromophore. Although the side chains of Ile146 and Leu167 appear to exist in two rotamer states, they are found to be well packed against the indole group of the chromophore. The Ser65 reversion mutation allows improved side-chain packing of Leu220. A structural comparison with mTurquoise2 is presented and additional engineering strategies are discussed.

Mechanistic diversity of red fluorescence acquisition by GFP-like proteins.

This review aims to summarize our current state of knowledge of several post-translational modification mechanisms known to yield red fluorescence in the family of GFP-like (green fluorescent protein-like) proteins. We begin with a brief review of the maturation mechanism that leads to green fluorescence in GFPs. The main body of this article is focused on a series of main chain redox and beta-elimination reactions mediated by light and O(2), ultimately yielding a red-emitting chromophore. In all GFP-like proteins, a tyrosine-derived phenolic group constitutes an essential building block of the chromophore's skeleton. Two major classes of red-emitting species have been identified in naturally occurring fluorescent proteins. In the DsRed type, an acylimine moiety is found to be conjugated to the GFP-like chromophore. Recent evidence has suggested that two mechanistic pathways, a green branch and a red branch, diverge from an early cyclic intermediate that bears a standard tyrosine side chain. Therefore, the long-standing notion that all FP colors originate from modifications of the GFP-like chromophore may need to be revised. In the Kaede-type green-to-red photoconvertible class of FPs, a light-mediated main chain elimination reaction partakes in the formation of a three-ring chromophore that involves the incorporation of a histidine residue into the conjugated system. A mechanistic role for photoexcitation of the GFP-like chromophore is undisputed; however, the nature of associated proton transfer steps and the charge state of the critical imidazole group remain controversial. In addition to the two major classes of red fluorescent proteins, we briefly describe yellow fluorescence arising from modifications of DsRed-type intermediates, and the less well understood photoactivated oxidative redding phenomenon.

A synthetic protein selected for ligand binding affinity mediates ATP hydrolysis.

How primitive enzymes emerged from a primordial pool remains a fundamental unanswered question with important practical implications in synthetic biology. Here we show that a de novo evolved ATP binding protein, selected solely on the basis of its ability to bind ATP, mediates the regiospecific hydrolysis of ATP to ADP when crystallized with 1 equiv of ATP. Structural insights into this reaction were obtained by growing protein crystals under saturating ATP conditions. The resulting crystal structure refined to 1.8 A resolution reveals that this man-made protein binds ATP in an unusual bent conformation that is metal-independent and held in place by a key bridging water molecule. Removal of this interaction using a null mutant results in a variant that binds ATP in a normal linear geometry and is incapable of ATP hydrolysis. Biochemical analysis, including high-resolution mass spectrometry performed on dissolved protein crystals, confirms that the reaction is accelerated in the crystalline environment. This observation suggests that proteins with weak chemical reactivity can emerge from high affinity ligand binding sites and that constrained ligand-binding geometries could have helped to facilitate the emergence of early protein enzymes.