The extremely slow‐exchanging core and acid‐denatured state of green fluorescent protein

Huang, Jie Rong; Hsu, Shang-Te Danny; Christodoulou, John; Jackson, Sophie

doi:10.2976/1.2976660

Cited by 17 publications

(36 citation statements)

References 64 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Indeed, the overall shape of the GFP 1-10 spectrum was also indicative of a predominantly b-strand structure. More importantly, the CD spectra for GFP [1][2][3][4][5][6][7][8][9][10] and CA-GFP are more similar to each other than to mature GFP. This suggests a higher degree of structural similarity between GFP 1-10 and CA-GFP than mature GFP.…”

Section: Resultsmentioning

confidence: 98%

“…Chromophore maturation in split GFP occurs with similar kinetics to CA-GFP. 4,15 We hypothesized that CA-GFP might adopt a stable, partially folded structure similar to that of GFP [1][2][3][4][5][6][7][8][9][10] [ Fig. 1(C)].…”

Section: Resultsmentioning

confidence: 99%

“…[6][7][8][9][10][11][12] It is now clear that folding of GFP occurs co-translationally and that proper folding into the GFP b-barrel only occurs once the entire protein (strands 1 through 11) has been translated. 13,14 Despite the fact that all 11 strands must be available for proper folding of GFP, it is possible to cut GFP following the 10th strand to make split GFP.…”

Section: Resultsmentioning

confidence: 99%

“…13,14 Despite the fact that all 11 strands must be available for proper folding of GFP, it is possible to cut GFP following the 10th strand to make split GFP. 4 In the split-GFP system, co-expression of GFP strands 1-10 (GFP [1][2][3][4][5][6][7][8][9][10] ) with the 11th strand allows reassociation of the 11th strand with GFP [1][2][3][4][5][6][7][8][9][10] and folding of the GFP b-barrel. Once the full GFP b-barrel is formed the chromophore is competent to mature, and split GFP becomes fluorescent.…”

Section: Resultsmentioning

confidence: 99%

“…In addition to the shape of the CD spectra, the thermal stability of CA-GFP also provides insight into the structure relative to mature GFP and GFP [1][2][3][4][5][6][7][8][9][10] . To assess stability, we collected spectra from 200 to 250 nm of each protein every 5 as the temperature was raised from 20 to 90 C [ Fig.…”

Section: Resultsmentioning

confidence: 99%

See 4 more Smart Citations

Structural basis of fluorescence quenching in caspase activatable‐GFP

Nicholls

Hardy

2013

Protein Science

View full text Add to dashboard Cite

Apoptosis is critical for organismal homeostasis and a wide variety of diseases. Caspases are the ultimate executors of the apoptotic programmed cell death pathway. As caspases play such a central role in apoptosis, there is significant demand for technologies to monitor caspase function. We recently developed a caspase activatable-GFP (CA-GFP) reporter. CA-GFP is unique due to its ''dark'' state, where chromophore maturation of the GFP is inhibited by the presence of a C-terminal peptide. Here we show that chromophore maturation is prevented because CA-GFP does not fold into the robust b-barrel of GFP until the peptide has been cleaved by active caspase. Both CA-GFP and GFP 1-10 , a split form of GFP lacking the 11th strand, have similar secondary structure, different from mature GFP. A similar susceptibility to proteolytic digestion indicates that this shared structure is not the robust, fully formed GFP b-barrel. We have developed a model that suggests that as CA-GFP is translated in vivo it follows the same folding path as wild-type GFP; however, the presence of the appended peptide does not allow CA-GFP to form the barrel of the fully matured GFP. CA-GFP is therefore held in a ''pro-folding'' intermediate state until the peptide is released, allowing it to continue folding into the mature barrel geometry. This new understanding of the structural basis of the dark state of the CA-GFP reporter will enable manipulation of this mechanism in the development of reporter systems for any number of cellular processes involving proteases and potentially other enzymes.

show abstract

Section: Resultsmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 3 more Smart Citations

Structural basis of fluorescence quenching in caspase activatable‐GFP

Nicholls

Hardy

2013

Protein Science

View full text Add to dashboard Cite

show abstract

Time, space, and disorder in the expanding proteome universe

2017

View full text Add to dashboard Cite

Proteins are highly dynamic entities. Their myriad functions require specific structures, but proteins’ dynamic nature ranges all the way from the local mobility of their amino acid constituents to mobility within and well beyond single cells. A truly comprehensive view of the dynamic structural proteome includes: (i) alternative sequences, (ii) alternative conformations, (iii) alternative interactions with a range of biomolecules, (iv) cellular localizations, (v) alternative behaviors in different cell types. While these aspects have traditionally been explored one protein at a time, we highlight recently emerging global approaches that accelerate comprehensive insights into these facets of the dynamic nature of protein structure. Computational tools that integrate and expand on multiple orthogonal data types promise to enable the transition from a disjointed list of static snapshots to a structurally explicit understanding of the dynamics of cellular mechanisms.

show abstract