Fitness landscapes1,2, depictions of how genotypes manifest at the phenotypic level, form the basis for our understanding of many areas of biology2–7 yet their properties remain elusive. Studies addressing this issue often consider specific genes and their function as proxy for fitness2,4, experimentally assessing the impact on function of single mutations and their combinations in a specific sequence2,5,8–15 or in different sequences2,3,5,16–18. However, systematic high-throughput studies of the local fitness landscape of an entire protein have not yet been reported. Here, we chart an extensive region of the local fitness landscape of the green fluorescent protein from Aequorea victoria (avGFP) by measuring the native function, fluorescence, of tens of thousands of derivative genotypes of avGFP. We find that its fitness landscape is narrow, with half of genotypes with two mutations showing reduced fluorescence and half of genotypes with five mutations being completely non-fluorescent. The narrowness is enhanced by epistasis, which was detected in up to 30% of genotypes with multiple mutations arising mostly through the cumulative impact of slightly deleterious mutations causing a threshold-like decrease of protein stability and concomitant loss of fluorescence. A model of orthologous sequence divergence spanning hundreds of millions of years predicted the extent of epistasis in our data, indicating congruence between the fitness landscape properties at the local and global scales. The characterization of the local fitness landscape of avGFP has important implications for a number of fields including molecular evolution, population genetics and protein design.
Photoinduced reactions play an important role in the photocycle of fluorescent proteins from the green fluorescent protein (GFP) family. Among such processes are photoisomerization, photooxidation/photoreduction, breaking and making of covalent bonds, and excited-state proton transfer (ESPT). Many of these transformations are initiated by electron transfer (ET). The quantum yields of these processes vary significantly, from nearly 1 for ESPT to 10-10 for ET. Importantly, even when quantum yields are relatively small, at the conditions of repeated illumination the overall effect is significant. Depending on the task at hand, fluorescent protein photochemistry is regarded either as an asset facilitating new applications or as a nuisance leading to the loss of optical output. The phenomena arising due to phototransformations include (i) large Stokes shifts, (ii) photoconversions, photoactivation, and photoswitching, (iii) phototoxicity, (iv) blinking, (v) permanent bleaching, and (vi) formation of long-lived intermediates. The focus of this review is on the most recent experimental and theoretical work on photoinduced transformations in fluorescent proteins. We also provide an overview of the photophysics of fluorescent proteins, highlighting the interplay between photochemistry and other channels (fluorescence, radiationless relaxation, and intersystem crossing). The similarities and differences with photochemical processes in other biological systems and in dyes are also discussed.
During development of the nervous system, axons and growth cones contain mRNAs, such as β-actin, cofilin, and RhoA, that are locally translated in response to guidance cues. Intra-axonal translation of these mRNAs results in local morphological responses, however other functions of intra-axonal mRNA translation remain unknown. Here we show that axons of developing mammalian neurons contain mRNA encoding the cAMP-responsive element (CRE)-binding protein (CREB). CREB is translated within axons in response to NGF and is retrogradely trafficked to the cell body. In neurons that are selectively deficient in axonal CREB transcripts, increases in nuclear pCREB, CRE-mediated transcription, and neuronal survival elicited by axonal application of NGF are abolished, indicating a signalling function for axonally-synthesised CREB. These studies identify a signalling role for axonally-derived CREB, and indicate that signaldependent synthesis and retrograde trafficking of transcription factors enables specific transcriptional responses to signalling events at distal axons.An important mechanism by which the protein composition at specific subcellular sites is regulated is by the precise intracellular targeting and translation of certain mRNAs 1 . This mechanism particularly evident in axons of developing neurons, which contain mRNAs, ribosomes, and other translational machinery 2 . Most known axonal mRNAs encode proteins that are involved in cytoskeletal regulation, and are involved in mediating the response to guidance cues [3][4][5] . Roles for axonal mRNA translation in other processes have not been established.One critical function of axons during development is to detect signals in the extracellular environment and to transduce these signals into changes in gene expression in the nucleus [6][7][8][9] . In developing sympathetic and sensory axons, growth cones detect nerve growth factor (NGF) synthesised by target cells, resulting in the generation of a retrograde signal that is conveyed to the soma that promotes neuronal survival 10 .Here we describe a role for axonal mRNA translation in the regulation of neuronal survival and nuclear transcription elicited by axonal application of NGF. We find NGF triggers axonal protein synthesis, which is required for NGF-mediated retrograde survival. A cDNA library prepared from the axons of developing sensory neurons reveals that CREB mRNA is an axonally-localised transcript. We find that CREB is selectively translated in axons in response to NGF and retrogradely trafficked to the cell body. Furthermore, selective 3 Correspondence should be addressed to S.R.J. (srj2003@med.cornell.edu). NIH Public Access Author ManuscriptNat Cell Biol. Author manuscript; available in PMC 2011 August 9. knockdown of axonal CREB mRNA reveals that axonally-synthesised CREB is required for NGF at axons to promote the accumulation of pCREB in the nucleus, transcription of a CRE-containing reporter gene, and neuronal survival. These data identify a role for axonally-synthesised CREB and identify a ...
The green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its fluorescent homologs from Anthozoa corals have become invaluable tools for in vivo imaging of cells and tissues. Despite spectral and chromophore diversity, about 100 cloned members of the GFP-like protein family possess common structural, biochemical and photophysical features. Anthozoa GFP-like proteins are available in colors and properties unlike those of A. victoria GFP variants and thus provide powerful new fluorophores for molecular labeling and intracellular detection. Although Anthozoa GFP-like proteins provide some advantages over GFP, they also have certain drawbacks, such as obligate oligomerization and slow or incomplete fluorescence maturation. In the past few years, effective approaches for eliminating some of these limitations have been described. In addition, several Anthozoa GFP-like proteins have been developed into novel imaging agents, such as monomeric red and dimeric far-red fluorescent proteins, fluorescent timers and photoconvertible fluorescent labels. Future studies on the structure of this diverse set of proteins will further enhance their use in animal tissues and as intracellular biosensors.
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