Site-specific protein labeling by Sfp phosphopantetheinyl transferase

Yin, Jun; Lin, Alison J.; Golan, David E.; Walsh, Christopher T.

doi:10.1038/nprot.2006.43

Cited by 279 publications

(355 citation statements)

References 18 publications

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“…AlexaFluor488-maleimide and AlexaFluor594-maleimide were purchased from Invitrogen. The fluorophorecoenzyme A (CoA) conjugates (Alexa 488-CoA and Alexa 594-CoA) were prepared as described previously (22). The C-terminal S6 peptide was labeled in a reaction mixture containing 1.5 M Gag-S6, 5 M Alexa 488-CoA, 1 M Sfp (a phosphopantetheinyl transferase from Bacillus subtilis), and Labeling Buffer (50 mM HEPES [pH 7.5], 10 mM MgCl 2 , 1 M NaCl, 1% glycerol, 0.1% Igepal CA-630).…”

Section: Methodsmentioning

confidence: 99%

A Conformational Transition Observed in Single HIV-1 Gag Molecules during In Vitro Assembly of Virus-Like Particles

Munro

Nath

Farber

et al. 2014

J Virol

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The conformational changes within single HIV-1 Gag molecules that occur during assembly into immature viruses are poorly understood. Using an in vitro assembly assay, it has been proposed that HIV-1 Gag undergoes a conformational transition from a compact conformation in solution to an extended rod-like conformation in virus-like particles (VLPs). Here we used singlemolecule Förster resonance energy transfer (smFRET) to test this model by directly probing the conformation of single HIV-1 Gag molecules. We demonstrate that monomeric HIV-1 Gag lacking the p6 domain and the N-terminal myristoyl moiety is found in solution predominantly in a compact conformation. Gag in this conformation, and in the presence of nucleic acid, assembles into 30-nm-diameter particles. However, with the addition of inositol hexakisphosphate, Gag adopts a linear conformation and assembles into full-sized ϳ100-to-150-nm-diameter VLPs. Parallel fluorescence correlation spectroscopy measurements show that this conformational transition occurs early in the assembly process when Gag oligomers are small, perhaps as early as upon dimerization. Thus, smFRET measurements confirm that HIV-1 Gag transitions from a compact to a linear conformation during the formation of VLPs. Our results are consistent with a model whereby binding of HIV-1 Gag to phosphoinositides at the plasma membrane stabilizes an extended conformation and promotes oligomerization into the radially aligned immature capsid.

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Section: Methodsmentioning

confidence: 99%

A Conformational Transition Observed in Single HIV-1 Gag Molecules during In Vitro Assembly of Virus-Like Particles

Munro

Nath

Farber

et al. 2014

J Virol

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“…Instead, we used a previously described enzymatic labeling protocol (22) to attach Alexa Fluor 647 (A647) to a 11 amino acid peptide tag (termed ybbR) fused to the N-terminus of DDX1. The DNA sequence encoding the ybbR tag (GATTCTCTTGAATTTA TTGCTAGTAAGCTTGCG) was inserted into a plasmid containing the DDX1 gene (also including hexahistidine affinity tag) using the QuikChange II protocol (Stratagene) and the forward and reverse primers shown in Supplementary Table S1.…”

Section: Preparation Of Wt Ddx1 and Ddx1 Mutants With Ybbr Tagmentioning

confidence: 99%

A Dead-Box Protein Acts through RNA to Promote HIV-1 Rev-RRE Assembly

Lamichhane

Hammond

Pauszek

et al. 2017

Biophysical Journal

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The HIV-1 Rev protein activates nuclear export of unspliced and partially spliced viral RNA transcripts, which encode the viral genome and the genes encoding viral structural proteins, by binding to and oligomerizing on the Rev Response Element (RRE). The human DEAD-box protein 1 (DDX1) enhances the RNA export activity of Rev through an unknown mechanism. Using a single-molecule assembly assay and various DDX1 mutants, we show that DDX1 acts through the RRE RNA to specifically accelerate the nucleation step of the Rev-RRE assembly process. Single-molecule Förster resonance energy transfer (smFRET) experiments using donor-labeled Rev and acceptor-labeled DDX1 show that both proteins can associate with a single RRE molecule. However, simultaneous interaction is only observed in a subset of binding events and does not explain the extent to which DDX1 promotes the nucleation step of Rev-RRE assembly. Together, these results are consistent with a model wherein DDX1 acts as an RNA chaperone, remodeling the RRE into a conformation that is pre-organized to bind the first Rev monomer, thereby promoting the overall Rev-RRE assembly process.

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“…The conjugations between these proteins and their substrates exhibit high selectivity and high affinity, even within cells and cell extracts [24]. The second technology is a series of 'enzyme-mediated' labeling strategies such as biotin ligase-mediated [31], phosphopantetheine transferase-mediated [32], lipoic acid ligase-mediated [33], and formylglycine-generating enzyme-mediated [34,35] approaches. These techniques involve the recognition of specific amino-acid motifs of cellular proteins that are then modified with remarkably high specificity.…”

Section: Box 1 Labeling Proteins With Organic Fluorophores Within Celmentioning

confidence: 99%

Bringing single-molecule spectroscopy to macromolecular protein complexes

Joo

Fareh

Kim

2013

Trends in Biochemical Sciences

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Single-molecule fluorescence spectroscopy offers real-time, nanometer-resolution information. Over the past two decades, this emerging single-molecule technique has been rapidly adopted to investigate the structural dynamics and biological functions of proteins. Despite this remarkable achievement, single-molecule fluorescence techniques must be extended to macromolecular protein complexes that are physiologically more relevant for functional studies. In this review, we present recent major breakthroughs for investigating protein complexes within cell extracts using single-molecule fluorescence. We outline the challenges, future prospects and potential applications of these new single-molecule fluorescence techniques in biological and clinical research. Single-molecule protein studiesSingle-molecule techniques have become potent tools for the discovery of novel protein mechanisms by allowing high spatiotemporal resolution. Sub-nanometer resolution, the ultimate scale of biological systems, has been reached with single-molecule fluorescence microscopy[1] and spectroscopy [2]; single-molecule force and torque spectroscopy [3,4]; atomic force microscopy [5] and spectroscopy [6]; and nanopores [7]. Measurements can be carried out on the biologically relevant time scales of microseconds to minutes.Among these techniques, single-molecule fluorescence spectroscopy has enabled researchers to unveil the action mechanism of a protein by imaging protein activity in real time. Benefitting from advances in general microscopy[1], detection devices [8], and fluorophore physics [9,10], single-molecule fluorescence spectroscopy has reached sub-nanometer spatial resolution [11] and microsecond temporal resolution [12,13]. Single-molecule fluorescence imaging is carried out primarily with total internal reflection, confocal, and zero-mode waveguide microscopy [2]. Among these, total internal reflection fluorescence microscopy has been employed by several research teams in the development of novel techniques described in this review [14][15][16][17] Single-molecule observations using cell extractsUnlike purified recombinant proteins, crude cell extracts provide more biologically relevant environment in which molecules of interest can interact not only with their partners but also with other cellular components. It was anticipated to combine this approach with single molecule techniques and observe the assembly and function of macromolecular complexes in real time. However, technical hurdles related to the specificity and efficiency of labeling molecules of interest within crude cell extracts had been reported [22][23][24]. In order to overcome this limitation, several groups recently developed original approaches. Hoskins et al. used protein complexes specifically tagged with fluorophores [14,19], and Yardimci et al. immunostained reaction products using specific fluorescent antibodies [15,20]. Real-time observation of spliceosome assemblyDespite the relatively small number of genes in the human genome, we have extraordin...

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Site-specific protein labeling by Sfp phosphopantetheinyl transferase

Cited by 279 publications

References 18 publications

A Conformational Transition Observed in Single HIV-1 Gag Molecules during In Vitro Assembly of Virus-Like Particles

A Conformational Transition Observed in Single HIV-1 Gag Molecules during In Vitro Assembly of Virus-Like Particles

A Dead-Box Protein Acts through RNA to Promote HIV-1 Rev-RRE Assembly

Bringing single-molecule spectroscopy to macromolecular protein complexes

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