Selective Labeling of Proteins by Using Protein Farnesyltransferase

Duckworth, Benjamin P.; Zhang, Zhiyuan; Hosokawa, Ayako; Distefano, Mark D.

doi:10.1002/cbic.200600340

Cited by 103 publications

(113 citation statements)

References 36 publications

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“…3A shows the FRET histogram when 3.7-kb TxY-DNA is packaged in the GFP portal. From this measurement, the donor-to-acceptor distance (r) is estimated to be 5 nm, given a Förster distance of 4 nm for a GFP-Tx donor-acceptor pair (29).…”

Section: Gfp-gene 20 Fusion Protein Portals Can Form Proheads With Bomentioning

confidence: 99%

Dynamics of the T4 Bacteriophage DNA Packasome Motor

Dixit

Ray

Lakowicz

et al. 2011

Journal of Biological Chemistry

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Conserved bacteriophage ATP-based DNA translocation motors consist of a multimeric packaging terminase docked onto a unique procapsid vertex containing a portal ring. DNA is translocated into the empty procapsid through the portal ring channel to high density. In vivo the T4 phage packaging motor deals with Y-or X-structures in the replicative concatemer substrate by employing a portal-bound Holliday junction resolvase that trims and releases these DNA roadblocks to packaging. Here using dye-labeled packaging anchored 3.7-kb Y-DNAs or linear DNAs, we demonstrate FRET between the dye-labeled substrates and GFP portal-containing procapsids and between GFP portal and single dye-labeled terminases. We show using FRET-fluorescence correlation spectroscopy that purified T4 gp49 endonuclease VII resolvase can release DNA compression in vitro in prohead portal packaging motor anchored and arrested Y-DNA substrates. In addition, using active terminases labeled at the N-and C-terminal ends with a single dye molecule, we show by FRET distance of the N-terminal GFP-labeled portal protein containing prohead at 6.9 nm from the N terminus and at 5.7 nm from the C terminus of the terminase. Packaging with a C-terminal fluorescent terminase on a GFP portal prohead, FRET shows a reduction in distance to the GFP portal of 0.6 nm in the arrested Y-DNA as compared with linear DNA; the reduction is reversed by resolvase treatment. Conformational changes in both the motor proteins and the DNA substrate itself that are associated with the power stroke of the motor are consistent with a proposed linear motor employing a terminal-to-portal DNA grip-and-release mechanism.The nucleic acid packaging machinery of double-stranded DNA and RNA bacteriophages and of herpesviruses and adenoviruses fills empty preassembled capsid precursors by broadly conserved ATP-driven motor mechanisms (1-3). In most of the large tailed double-stranded DNA bacteriophages, a prohead is filled to near liquid crystalline DNA density (ϳ500 mg/ml) from a replicative genomic concatemer in vivo by a powerful packasome motor (4). The motor consists of a multimeric terminase enzyme docked onto a unique capsid vertex containing the DNA entrance and exit channel-containing portal dodecamer. In conjunction with high ATP consumption, the terminase-portal motor translocates DNA through the prohead portal, and the terminase also cuts the genomic concatemer to form the mature viral particle DNA ends upon receiving a headful signal from the portal, hence the name terminase (5, 6).The essential components of the packasome motor appear to share many structural and mechanistic features among intensively studied double-stranded DNA phages T4, lambda, P22, SPP1, Phi29, and others (7). Accordingly, a number of general mechanisms for DNA translocation have been proposed, although some can now apparently be eliminated by more recent experiments employing a variety of genetic, biochemical, biophysical, and structural approaches. Thus, genetic and biophysical evidence suggests that the D...

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Section: Gfp-gene 20 Fusion Protein Portals Can Form Proheads With Bomentioning

confidence: 99%

Dynamics of the T4 Bacteriophage DNA Packasome Motor

Dixit

Ray

Lakowicz

et al. 2011

Journal of Biological Chemistry

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“…4 enzymes such as farnesyl transferase, 13 N-myristroyltransferase 15 or microbial transglutaminase, 10 which can then react in a 'click' reaction with a complementary azide-or alkyne-containing fluorophore, respectively. Other variants make use of other functional groups on enzyme substrate tags 11, 14,16 or may directly append the fluorophore to the tag by enzymatic catalysis.…”

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confidence: 99%

Fluorogenic protein labelling: a review of photophysical quench mechanisms and principles of fluorogen design

2015

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Fluorescent labelling of specific proteins in complex biological systems remains an important challenge in chemical biology. One promising approach comprises the use of small molecules designed to react specifically with a targeted protein of interest and to increase in fluorescent intensity following this reaction. This kind of fluorogenic reaction generally derives from fluorescence quenching in the unreacted probe that is abrogated over the course of the reaction.Herein, we review the mechanistic principles of three major photophysical quenching mechanisms, involving Förster resonance energy transfer (FRET), through-bond energy transfer (TBET), and photoinduced electron transfer (PeT). We then present design principles for novel fluorogenic probes based on an understanding of these quench mechanisms, with emphasis on the emerging utility of density functional theory (DFT) calculations in the design process. DedicationThe authors dedicate this manuscript to Prof. R. S. Brown, the doctoral supervisor of JWK. Prof.Brown's acclaimed research continues to inspire the scientific community, notably through the rigour and depth of its mechanistic investigations, followed by its creative and relevant practical applications of that knowledge.

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“…24–26 Based on these data, we hypothesized that different FPP analogs would alter the peptide selectivity of FTase based on the size of the residue in the a 2 position. To test this hypothesis, four alkyne-modified FPP analogs, analog 1 27 , 2 and 3 28 , and 4 14 (Figure 1), were synthesized, as previously reported. The affect of these analogs on FTase peptide sequence specificity was then determined by comparing their in vitro kinetic activity to that of FPP for catalyzing farnesylation of a library of peptides with varying CaaX sequences.…”

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confidence: 99%

Analogs of farnesyl diphosphate alter CaaX substrate specificity and reactions rates of protein farnesyltransferase

Jennings

Danowitz

Wang

et al. 2016

Bioorganic & Medicinal Chemistry Letters

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Attempts to identify the prenyl-proteome of cells or changes in prenylation following drug treatment have used “clickable” alkyne-modified analogs of the lipid substrates farnesyl- and geranylgeranyl-diphosphate (FPP and GGPP). We characterized the reactivity of four alkyne-containing analogs of FPP with purified protein farnesyltransferase and a small library of dansylated peptides using an in vitro continuous spectrofluorimetric assay. These analogs alter prenylation specificity and reactivity suggesting that in vivo results obtained using these FPP analogs should be interpreted cautiously.

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Selective Labeling of Proteins by Using Protein Farnesyltransferase

Cited by 103 publications

References 36 publications

Dynamics of the T4 Bacteriophage DNA Packasome Motor

Dynamics of the T4 Bacteriophage DNA Packasome Motor

Fluorogenic protein labelling: a review of photophysical quench mechanisms and principles of fluorogen design

Analogs of farnesyl diphosphate alter CaaX substrate specificity and reactions rates of protein farnesyltransferase

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