J. Appl. Cryst.の発刊に際して

One of the fastest moving and most exciting interfaces of nanotechnology is the use of quantum dots (QDs) in biology. The unique optical properties of QDs make them appealing as in vivo and in vitro fluorophores in a variety of biological investigations, in which traditional fluorescent labels based on organic molecules fall short of providing long-term stability and simultaneous detection of multiple signals. The ability to make QDs water soluble and target them to specific biomolecules has led to promising applications in cellular labelling, deep-tissue imaging, assay labelling and as efficient fluorescence resonance energy transfer donors. Despite recent progress, much work still needs to be done to achieve reproducible and robust surface functionalization and develop flexible bioconjugation techniques. In this review, we look at current methods for preparing QD bioconjugates as well as presenting an overview of applications. The potential of QDs in biology has just begun to be realized and new avenues will arise as our ability to manipulate these materials improves.

Quantum dot bioconjugates for imaging, labelling and sensing

Fluorescent calcium sensors are widely used to image neural activity. Using structure-based mutagenesis and neuron-based screening, we developed a family of ultrasensitive protein calcium sensors (GCaMP6) that outperformed other sensors in cultured neurons and in zebrafish, flies and mice in vivo. In layer 2/3 pyramidal neurons of the mouse visual cortex, GCaMP6 reliably detected single action potentials in neuronal somata and orientation-tuned synaptic calcium transients in individual dendritic spines. The orientation tuning of structurally persistent spines was largely stable over timescales of weeks. Orientation tuning averaged across spine populations predicted the tuning of their parent cell. Although the somata of GABAergic neurons showed little orientation tuning, their dendrites included highly tuned dendritic segments (5-40-µm long). GCaMP6 sensors thus provide new windows into the organization and dynamics of neural circuits over multiple spatial and temporal scales.

/pdf/ultrasensitive-fluorescent-proteins-for-imaging-neuronal-1f4lmx12cf.pdf

Ultrasensitive fluorescent proteins for imaging neuronal activity.

Cell Death: Critical Control Points

Fluorescent proteins are genetically encoded, easily imaged reporters crucial in biology and biotechnology. When a protein is tagged by fusion to a fluorescent protein, interactions between fluorescent proteins can undesirably disturb targeting or function. Unfortunately, all wild-type yellow-to-red fluorescent proteins reported so far are obligately tetrameric and often toxic or disruptive. The first true monomer was mRFP1, derived from the Discosoma sp. fluorescent protein "DsRed" by directed evolution first to increase the speed of maturation, then to break each subunit interface while restoring fluorescence, which cumulatively required 33 substitutions. Although mRFP1 has already proven widely useful, several properties could bear improvement and more colors would be welcome. We report the next generation of monomers. The latest red version matures more completely, is more tolerant of N-terminal fusions and is over tenfold more photostable than mRFP1. Three monomers with distinguishable hues from yellow-orange to red-orange have higher quantum efficiencies.

/pdf/improved-monomeric-red-orange-and-yellow-fluorescent-3xexx3emc3.pdf

Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.

All coelenterate fluorescent proteins cloned to date display some form of quaternary structure, including the weak tendency of Aequorea green fluorescent protein (GFP) to dimerize, the obligate dimerization of Renilla GFP, and the obligate tetramerization of the red fluorescent protein from Discosoma (DsRed). Although the weak dimerization of Aequorea GFP has not impeded its acceptance as an indispensable tool of cell biology, the obligate tetramerization of DsRed has greatly hindered its use as a genetically encoded fusion tag. We present here the stepwise evolution of DsRed to a dimer and then either to a genetic fusion of two copies of the protein, i.e., a tandem dimer, or to a true monomer designated mRFP1 (monomeric red fluorescent protein). Each subunit interface was disrupted by insertion of arginines, which initially crippled the resulting protein, but red fluorescence could be rescued by random and directed mutagenesis totaling 17 substitutions in the dimer and 33 in mRFP1. Fusions of the gap junction protein connexin43 to mRFP1 formed fully functional junctions, whereas analogous fusions to the tetramer and dimer failed. Although mRFP1 has somewhat lower extinction coefficient, quantum yield, and photostability than DsRed, mRFP1 matures >10 times faster, so that it shows similar brightness in living cells. In addition, the excitation and emission peaks of mRFP1, 584 and 607 nm, are ≈25 nm red-shifted from DsRed, which should confer greater tissue penetration and spectral separation from autofluorescence and other fluorescent proteins.

/pdf/a-monomeric-red-fluorescent-protein-22nkyufygq.pdf

A monomeric red fluorescent protein

Many proteins associated with the plasma membrane are known to partition into submicroscopic sphingolipid- and cholesterol-rich domains called lipid rafts, but the determinants dictating this segregation of proteins in the membrane are poorly understood. We suppressed the tendency of Aequorea fluorescent proteins to dimerize and targeted these variants to the plasma membrane using several different types of lipid anchors. Fluorescence resonance energy transfer measurements in living cells revealed that acyl but not prenyl modifications promote clustering in lipid rafts. Thus the nature of the lipid anchor on a protein is sufficient to determine submicroscopic localization within the plasma membrane.

/pdf/partitioning-of-lipid-modified-monomeric-gfps-into-membrane-ujca4k909c.pdf

Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells.

http://www.tsienlab.ucsd.edu/Publications/Griesbeck%202001%20JBC%20-%20Reducing%20the%20Environmental%20Sensitivity.pdf

Reducing the Environmental Sensitivity of Yellow Fluorescent Protein MECHANISM AND APPLICATIONS

DsRed is a recently cloned 28-kDa fluorescent protein responsible for the red coloration around the oral disk of a coral of the Discosoma genus. DsRed has attracted tremendous interest as a potential expression tracer and fusion partner that would be complementary to the homologous green fluorescent protein from Aequorea, but very little is known of the biochemistry of DsRed. We now show that DsRed has a much higher extinction coefficient and quantum yield than previously reported, plus excellent resistance to pH extremes and photobleaching. In addition, its 583-nm emission maximum can be further shifted to 602 nm by mutation of Lys-83 to Met. However, DsRed has major drawbacks, such as strong oligomerization and slow maturation. Analytical ultracentrifugation proves DsRed to be an obligate tetramer in vitro, and fluorescence resonance energy transfer measurements and yeast two-hybrid assays verify oligomerization in live cells. Also, DsRed takes days to ripen fully from green to red in vitro or in vivo, and mutations such as Lys-83 to Arg prevent the color change. Many potential cell biological applications of DsRed will require suppression of the tetramerization and acceleration of the maturation.

/pdf/biochemistry-mutagenesis-and-oligomerization-of-dsred-a-red-4n86g2frwh.pdf

Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral

Many areas of biology and biotechnology have been revolutionized by the ability to label proteins genetically by fusion to the Aequorea green fluorescent protein (GFP). In previous fusions, the GFP has been treated as an indivisible entity, usually appended to the amino or carboxyl terminus of the host protein, occasionally inserted within the host sequence. The tightly interwoven, three-dimensional structure and intricate posttranslational self-modification required for chromophore formation would suggest that major rearrangements or insertions within GFP would prevent fluorescence. However, we now show that several rearrangements of GFPs, in which the amino and carboxyl portions are interchanged and rejoined with a short spacer connecting the original termini, still become fluorescent. These circular permutations have altered pKa values and orientations of the chromophore with respect to a fusion partner. Furthermore, certain locations within GFP tolerate insertion of entire proteins, and conformational changes in the insert can have profound effects on the fluorescence. For example, insertions of calmodulin or a zinc finger domain in place of Tyr-145 of a yellow mutant (enhanced yellow fluorescent protein) of GFP result in indicator proteins whose fluorescence can be enhanced severalfold upon metal binding. The calmodulin graft into enhanced yellow fluorescent protein can monitor cytosolic Ca2+ in single mammalian cells. The tolerance of GFPs for circular permutations and insertions shows the folding process is surprisingly robust and offers a new strategy for creating genetically encodable, physiological indicators.

/pdf/circular-permutation-and-receptor-insertion-within-green-4k9qgua32v.pdf

David A. Zacharias

Papers

A monomeric red fluorescent protein

Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells.

Reducing the Environmental Sensitivity of Yellow Fluorescent Protein MECHANISM AND APPLICATIONS

Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral

Circular permutation and receptor insertion within green fluorescent proteins