Structural consequences of chromophore formation and exploration of conserved lid residues amongst naturally occurring fluorescent proteins

Zimmer, Matthew; Li, Binsen; Shahid, Ramza S.; Peshkepija, Paola

doi:10.1016/j.chemphys.2013.11.015

Cited by 8 publications

(14 citation statements)

References 71 publications

(82 reference statements)

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“…Accumulation of processed tFT fragments was reduced to below the detection limit with all circular permutations except cp3 ( Figure 5B). This is consistent with higher mobility of the β 7 -β 11 sheets in the β-barrel fold and the tendency of GFP to start unfolding from β 7 -β 11 (Huang et al, 2007;Zimmer et al, 2014). As seen in the case of Clover (Figure 4,C and D), reduced accumulation of processed tFT fragments correlated with a reduced relative difference in mCherry/sfGFP ratios between the stable and unstable Ubi-X-mCherry-greenFP fusions ( Figure 5C).…”

Section: Resultssupporting

confidence: 83%

Incomplete proteasomal degradation of green fluorescent proteins in the context of tandem fluorescent protein timers

Khmelinskii

Meurer

et al. 2015

Preprint

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Tandem fluorescent protein timers (tFTs) report on protein age through time-dependent change in color, which can be exploited to study protein turnover and trafficking. Each tFT, composed of two fluorescent proteins (FPs) that differ in maturation kinetics, is suited to follow protein dynamics within a specific time range determined by the maturation rates of both FPs. So far, tFTs have been constructed by combining slower-maturing red fluorescent proteins (redFPs) with the faster-maturing superfolder green fluorescent protein (sfGFP). Toward a comprehensive characterization of tFTs, we compare here tFTs composed of different faster-maturing green fluorescent proteins (greenFPs) while keeping the slower-maturing redFP constant (mCherry). Our results indicate that the greenFP maturation kinetics influences the time range of a tFT. Moreover, we observe that commonly used greenFPs can partially withstand proteasomal degradation due to the stability of the FP fold, which results in accumulation of tFT fragments in the cell. Depending on the order of FPs in the timer, incomplete proteasomal degradation either shifts the time range of the tFT toward slower time scales or precludes its use for measurements of protein turnover. We identify greenFPs that are efficiently degraded by the proteasome and provide simple guidelines for the design of new tFTs. INTRODUCTIONFluorescent proteins (FPs) become fluorescent only upon correct folding of the polypeptide and formation of the fluorophore through a series of chemical reactions within the β-barrel fold. This maturation process takes place on time scales from minutes to hours, depending on the FP and the environment (Tsien, 1998;Shaner et al., 2005). The time delay between protein synthesis and completion of the maturation process is exploited in tandem fluorescent protein timers (tFTs) to measure the dynamics of various cellular processes (Khmelinskii et al., 2012). A tFT is a fusion of two FPs that differ in maturation kinetics and spectral properties (i.e., color)-for example, a slower-maturing red fluorescent protein (redFP) and a fastermaturing green fluorescent protein (greenFP; Figure 1A). The greenFP is typically selected to be as rapidly maturing as possible and thus reports on protein localization and abundance. Because of the difference in maturation kinetics between the greenFP and redFP moieties, the color of a tFT (defined as the redFP/greenFP ratio of fluorescence intensities) changes over time ( Figure 1A). When a tFT is fused to a protein of interest, its color provides a measure of protein age that can be used to follow protein degradation, trafficking, and segregation during cell division (Khmelinskii et al., 2012).The range of protein ages that can be analyzed with a tFT, that is, its time range, is strongly influenced by the maturation kinetics of the slower-maturing FP in the pair (Khmelinskii et al., 2012). Therefore, in the tFTs described thus far (Khmelinskii et al., 2012;Khmelinskii and Knop, 2014;Dona et al., 2013), different slowermaturin...

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Section: Resultssupporting

confidence: 83%

Incomplete proteasomal degradation of green fluorescent proteins in the context of tandem fluorescent protein timers

Khmelinskii

Meurer

et al. 2015

Preprint

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“…A previous analysis of the structure of more than 250 GFP-like proteins in the Protein Databank identified 23 highly conserved amino acids ( Ong et al 2011 ), 21 of which are present in the GFP protein used in this study ( Figure S1 ). Many of the most highly conserved residues are situated at the lids on the top and bottom of the β-barrel and at bends between the β-sheets ( Ong et al 2011 ; Zimmer et al 2014 ). The purpose of the lids is unclear, but the low variability displayed by these regions in GFP-like proteins suggests an important role in folding, stability, or other aspect of GFP function ( Ong et al 2011 ; Stepanenko et al 2013 ; Zimmer et al 2014 ).…”

Section: Resultsmentioning

confidence: 99%

“…Many of the most highly conserved residues are situated at the lids on the top and bottom of the β-barrel and at bends between the β-sheets ( Ong et al 2011 ; Zimmer et al 2014 ). The purpose of the lids is unclear, but the low variability displayed by these regions in GFP-like proteins suggests an important role in folding, stability, or other aspect of GFP function ( Ong et al 2011 ; Stepanenko et al 2013 ; Zimmer et al 2014 ). Our screen retrieved mutations resulting in substitutions of 11 of the 21 highly conserved amino acids present in the GFP protein used in this study ( Table 1 ).…”

Section: Resultsmentioning

confidence: 99%

“…For the remaining substitutions, no GFP protein was detected by Western blotting under the conditions used. Lid residues at the N and C termini (G91 and G127) and the opposite side (G20), which is referred to as the "top" of the barrel ( Zimmer et al 2014 ), are highlighted in blue.…”

Section: Resultsmentioning

confidence: 99%

“…We recovered substitutions in three highly conserved "lid" residues in our screen: Gly91 is situated at the lid of the N and C termini, which are in close proximity at the same end of the barrel ( Figure 1 ), whereas Gly20 and Gly127 are located on the opposite lid, which is referred to as the "top" of the barrel ( Ong et al 2011 ; Zimmer et al 2014 ). Consistent with the fact that glycines can reside in parts of proteins that are unable to accommodate other amino acids, such as tight turns in structures ( Betts and Russell, 2003 ), these three lid residues as well as Gly40, also identified in our screen, are all present in the vicinity of bends representing transitions from β-strands to loops in the folding pattern of GFP ( Figure 1 ) ( Zimmer et al 2014 ).…”

Section: Resultsmentioning

confidence: 99%

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GFP Loss-of-Function Mutations inArabidopsis thaliana

Kanno

Liang

et al. 2015

G3 Genes|Genomes|Genetics

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Green fluorescent protein (GFP) and related fluorescent proteins are widely used in biological research to monitor gene expression and protein localization in living cells. The GFP chromophore is generated spontaneously in the presence of oxygen by a multi-step reaction involving cyclization of the internal tripeptide Ser65 (or Thr65)-Tyr66-Gly67, which is embedded in the center of an 11-stranded β-barrel structure. Random and site-specific mutagenesis has been used to optimize GFP fluorescence and create derivatives with novel properties. However, loss-of-function mutations that would aid in understanding GFP protein folding and chromophore formation have not been fully cataloged. Here we report a collection of ethyl methansulfonate–induced GFP loss-of-function mutations in the model plant Arabidopsis thaliana. Mutations that alter residues important for chromophore maturation, such as Arg96 and Ser205, greatly reduce or extinguish fluorescence without dramatically altering GFP protein accumulation. By contrast, other loss-of-fluorescence mutations substantially diminish the amount of GFP protein, suggesting that they compromise protein stability. Many mutations in this category generate substitutions of highly conserved glycine residues, including the following: Gly67 in the chromogenic tripeptide; Gly31, Gly33, and Gly35 in the second β-strand; and Gly20, Gly91, and Gly127 in the lids of the β-barrel scaffold. Our genetic analysis supports conclusions from structural and biochemical studies and demonstrates a critical role for multiple, highly conserved glycine residues in GFP protein stability.

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Twenty years of exceptional success: The molecular education and research consortium in undergraduate computational chemistry (MERCURY)

Shields

2020

Int J of Quantum Chemistry

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The molecular education and research consortium in undergraduate computational chemistry (MERCURY) consortium, established in 2000, has contributed greatly to the scientific development of faculty and undergraduates. The MERCURY faculty peer-reviewed publication rate from 2001 to 2019 of 1.7 papers/faculty/year is 3.4 times the rate of the physical science faculty at primarily undergraduate institutions. We have worked with over 1000 students on research projects since 2001, and 75% of our undergraduate research students have been under-represented in chemistry, either female or students of color. Approximately half of our alumni attend graduate school for the purpose of obtaining advanced degrees in STEM fields, and two-thirds are female and/or students of color. We have had more than 1600 attendees at 18 MERCURY conferences, including 111 invited speakers, 61 of whom have been female and/or faculty of color. In this paper, the research accomplishments, transformational outcomes, and scientific productivity of the MERCURY faculty are highlighted.

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Structural consequences of chromophore formation and exploration of conserved lid residues amongst naturally occurring fluorescent proteins

Cited by 8 publications

References 71 publications

Incomplete proteasomal degradation of green fluorescent proteins in the context of tandem fluorescent protein timers

Incomplete proteasomal degradation of green fluorescent proteins in the context of tandem fluorescent protein timers

GFP Loss-of-Function Mutations inArabidopsis thaliana

Twenty years of exceptional success: The molecular education and research consortium in undergraduate computational chemistry (MERCURY)

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