Dronpa is a novel GFP-like fluorescent protein with exceptional light-controlled switching properties. It may be reversibly switched between a fluorescent on-state and a nonfluorescent off-state by irradiation with light. To elucidate the molecular basis of the switching mechanism, we generated reversibly switchable Dronpa protein crystals. Using these crystals we determined the elusive dark-state structure of Dronpa at 1.95-Å resolution. We found that the photoswitching results in a cis-trans isomerization of the chromophore accompanied by complex structural rearrangements of four nearby amino acid residues. Because of this cascade of intramolecular events, the chromophore is exposed to distinct electrostatic surface potentials, which are likely to influence the protonation equilibria at the chromophore. We suggest a comprehensive model for the light-induced switching mechanism, connecting a cascade of structural rearrangements with different protonation states of the chromophore. The recently discovered reversibly switchable FPs (RSFPs) are a further powerful class of FPs for cell biology and beyond. Other than the photoactivatable FPs, RSFPs may be repeatedly and reversibly switched by irradiation between a fluorescent and a nonfluorescent state. Hence they exhibit unique advantages for protein tracking applications, subdiffraction microscopy, and several novel applications that had not been addressable previously (8-13). Dronpa (14) and asFP595 (asulCP, asCP) (15) are the most prominent RSFPs. Like all FPs, they exhibit a GFP-like fold, namely a ␤-barrel enclosing an ␣-helix containing the autocatalytically formed chromophore. Because of its low quantum yield, the tetrameric asFP595 is only of limited use for cell biology applications, whereas Dronpa has been successfully used for several protein tracking studies (14,16,17). Dronpa is monomeric, displays favorable switching properties, and shows bright fluorescence with a remarkable fluorescence quantum yield of 0.85. Furthermore, several new Dronpa variants with accelerated switching kinetics have been described (18).Despite its tremendous potential for many applications, little is known about the molecular basis of the switching in Dronpa. Thus far competing models discussing either the light-driven regulation of the chromophoric protonation state by the surrounding protein matrix (19, 20) or postulating a cis-trans isomerization of the chromophore indirectly determining its own protonation state (18) have been suggested.To unravel this problem we generated Dronpa protein crystals that were reversibly switchable with visible light at ambient conditions. After switching the whole Dronpa crystals we determined its thus far elusive off-state structure by x-ray crystallography. We found that the primary event of the switching is a light-activated cis-trans isomerization of the chromophore together with a cascade of residue rearrangements. Because the electrostatic surface potentials in the chromophoric cis and trans cavities differ substantially, we postulate ...

Proteins that can be reversibly photoswitched between a fluorescent and a nonfluorescent state bear enormous potential in diverse fields, such as data storage, in vivo protein tracking, and subdiffraction resolution light microscopy. However, these proteins could hitherto not live up to their full potential because the molecular switching mechanism is not resolved. Here, we clarify the molecular photoswitching mechanism of asFP595, a green fluorescent protein (GFP)-like protein that can be transferred from a nonfluorescent ''off'' to a fluorescent ''on'' state and back again, by green and blue light, respectively. To this end, we establish reversible photoswitching of fluorescence in whole protein crystals and show that the switching kinetics in the crystal is identical with that in solution. Subsequent x-ray analysis demonstrated that upon the absorption of a green photon, the chromophore isomerizes from a trans (off) to a cis (on) state. Molecular dynamics calculations suggest that isomerization occurs through a bottom hula twist mechanism with concomitant rotation of both bonds of the chromophoric methine ring bridge. This insight into the switching mechanism should facilitate the targeted design of photoswitchable proteins. Reversible photoswitching of the protein chromophore system within intact crystals also constitutes a step toward the use of fluorescent proteins in three-dimensional data recording.photoisomerization ͉ asCP ͉ photochromism ͉ optical bistability ͉ asulCP F luorescent proteins have been widely used as genetically encodable tags to monitor protein localizations and dynamics in live cells (1-3). Recently, novel GFP-like fluorescent proteins have been discovered (4-6) that can be reversibly photoswitched between a fluorescent (on) and nonfluorescent (off) state, that is, they are optically bistable and fluorescent. This feature is remarkable, because the reversible photoswitching occurring in photochromic organic compounds, such as in fulgides and diarylethenes, is usually not accompanied by fluorescence (7). Therefore, not surprisingly, these proteins hold great promise in many areas of science reaching out far beyond their prominent use as triggerable protein markers in live cells. For example, the reversible photoswitching of fluorescent markers should provide nanoscale resolution in fluorescence microscopy by using lenses and regular illumination, which was hardly conceivable only a few years ago (8-10). As fluorescence can be sensitively read out from a bulky crystal, the prospect of erasable three-dimensional data storage is equally intriguing.The GFP-like protein asFP595 (asCP or asulCP) from the sea anemone Anemonia sulcata is such a protein: It can be transferred by green light from a nonfluorescent off into a fluorescent on state from which it reverts back eventually, but this transition can also be promptly stimulated by gentle irradiation with blue light (6). The ''on-off'' cycle can be repeated many times. However, with its low quantum yield (Ͻ0.001, ref. 6) and comparatively slo...

RSFPs (reversibly switchable fluorescent proteins) may be repeatedly converted between a fluorescent and a non-fluorescent state by irradiation and have attracted widespread interest for many new applications. The RSFP Dronpa may be switched with blue light from a fluorescent state into a non-fluorescent state, and back again with UV light. To obtain insight into the underlying molecular mechanism of this switching, we have determined the crystal structure of the fluorescent equilibrium state of Dronpa. Its bicyclic chromophore is formed spontaneously from the Cys62-Tyr63-Gly64 tripeptide. In the fluorescent state, it adopts a slightly non-coplanar cis conformation within the interior of a typical GFP (green fluorescent protein) b-can fold. Dronpa shares some structural features with asFP595, another RSFP whose chromophore has previously been demonstrated to undergo a cis-trans isomerization upon photoswitching. Based on the structural comparison with asFP595, we have generated new Dronpa variants with an up to more than 1000-fold accelerated switching behaviour. The mutations which were introduced at position Val157 or Met159 apparently reduce the steric hindrance for a cis-trans isomerization of the chromophore, thus lowering the energy barrier for the blue light-driven on-to-off transition. The findings reported in the present study support the view that a cis-trans isomerization is one of the key events common to the switching mechanism in RSFPs.

The human enzyme that joins transfer RNA exons together is discovered.

The peptide-loading complex (PLC) is a transient, multisubunit membrane complex in the endoplasmic reticulum that is essential for establishing a hierarchical immune response. The PLC coordinates peptide translocation into the endoplasmic reticulum with loading and editing of major histocompatibility complex class I (MHC-I) molecules. After final proofreading in the PLC, stable peptide-MHC-I complexes are released to the cell surface to evoke a T-cell response against infected or malignant cells. Sampling of different MHC-I allomorphs requires the precise coordination of seven different subunits in a single macromolecular assembly, including the transporter associated with antigen processing (TAP1 and TAP2, jointly referred to as TAP), the oxidoreductase ERp57, the MHC-I heterodimer, and the chaperones tapasin and calreticulin. The molecular organization of and mechanistic events that take place in the PLC are unknown owing to the heterogeneous composition and intrinsically dynamic nature of the complex. Here, we isolate human PLC from Burkitt's lymphoma cells using an engineered viral inhibitor as bait and determine the structure of native PLC by electron cryo-microscopy. Two endoplasmic reticulum-resident editing modules composed of tapasin, calreticulin, ERp57, and MHC-I are centred around TAP in a pseudo-symmetric orientation. A multivalent chaperone network within and across the editing modules establishes the proofreading function at two lateral binding platforms for MHC-I molecules. The lectin-like domain of calreticulin senses the MHC-I glycan, whereas the P domain reaches over the MHC-I peptide-binding pocket towards ERp57. This arrangement allows tapasin to facilitate peptide editing by clamping MHC-I. The translocation pathway of TAP opens out into a large endoplasmic reticulum lumenal cavity, confined by the membrane entry points of tapasin and MHC-I. Two lateral windows channel the antigenic peptides to MHC-I. Structures of PLC captured at distinct assembly states provide mechanistic insight into the recruitment and release of MHC-I. Our work defines the molecular symbiosis of an ABC transporter and an endoplasmic reticulum chaperone network in MHC-I assembly and provides insight into the onset of the adaptive immune response.