Probing Protein Folding Using Site-Specifically Encoded Unnatural Amino Acids as FRET Donors with Tryptophan

Miyake-Stoner, Shigeki J.; Miller, Andrew M.; Hammill, Jared T.; Peeler, Jennifer C.; Hess, Kenneth R.; Mehl, Ryan A.; Brewer, Scott H.

doi:10.1021/bi900426d

Cited by 121 publications

(163 citation statements)

References 54 publications

(83 reference statements)

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“…[13][14] Expression of a GFP gene interrupted by an amber codon at site 150 in the presence of pDule-Tet2.0 was efficient and dependent on the presence of Tet-v2.0 (Fig. 1C).…”

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confidence: 99%

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Ideal Bioorthogonal Reactions Using A Site-Specifically Encoded Tetrazine Amino Acid

et al. 2015

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Bioorthogonal reactions for labeling biomolecules in live cells have been limited by slow reaction rates or low component selectivity and stability. Ideal bioorthogonal reactions with high reaction rates, high selectivity, and high stability would allow for stoichiometric labeling of biomolecules in minutes and eliminate the need to wash out excess labeling reagent. Currently, no general method exists for controlled stoichiometric or substoichiometric labeling of proteins in live cells. To overcome this limitation, we developed a significantly improved tetrazine-containing amino acid (Tet-v2.0) and genetically encoded Tet-v2.0 with an evolved aminoacyl-tRNA synthetase/tRNA(CUA) pair. We demonstrated in cellulo that protein containing Tet-v2.0 reacts selectively with cyclopropane-fused trans-cyclooctene (sTCO) with a bimolecular rate constant of 72,500 ± 1660 M(-1) s(-1) without reacting with other cellular components. This bioorthogonal ligation of Tet-v2.0-protein reacts in cellulo with substoichiometric amounts of sTCO-label fast enough to remove the labeling reagent from media in minutes, thereby eliminating the need to wash out label. This ideal bioorthogonal reaction will enable the monitoring of a larger window of cellular processes in real time.

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“…[13][14] Expression of a GFP gene interrupted by an amber codon at site 150 in the presence of pDule-Tet2.0 was efficient and dependent on the presence of Tet-v2.0 (Fig. 1C).…”

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confidence: 99%

“…13 Ninety-six colonies assessed for Tet-v2.0-dependent expression of GFP, contained seven clones that had significant GFP-Tet-v2.0 expression in the presence of Tet-v2.0 and no detectable GFP fluorescence over background in the absence of Tet-v2.0 (Sup. Fig.…”

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confidence: 99%

Ideal Bioorthogonal Reactions Using A Site-Specifically Encoded Tetrazine Amino Acid

et al. 2015

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“…The kinetics of formation of specific key sub-domain structures should be probed directly, in situ, on the background of the rest of the chain, where mutual influences exist. The possible significance of the loop closure effect in folding in vivo is an intriguing question that might be addressed with currently available FRET and timeresolved fluorescence methods [77,[211][212][213][214]. Co-translational folding of nascent polypeptides on the ribosome is a subject of a major field of current research.…”

Section: Resultsmentioning

confidence: 99%

Fast closure of long loops at the initiation of the folding transition of globular proteins studied by time-resolved FRET-based methods

Orevi

Rahamim

Shemesh

et al. 2014

Bio-Algorithms and Med-Systems

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The protein folding problem would be considered "solved" when it will be possible to "read genes", i.e., to predict the native fold of proteins, their dynamics, and the mechanism of fast folding based solely on sequence data. The long-term goal should be the creation of an algorithm that would simulate the stepwise mechanism of folding, which constrains the conformational space and in which random search for stable interactions is possible. Here, we focus attention on the initial phases of the folding transition starting with the compact disordered collapsed ensemble, in search of the initial sub-domain structural biases that direct the otherwise stochastic dynamics of the backbone. Our studies are designed to test the "loop hypothesis", which suggests that fast closure of long loop structures by non-local interactions between clusters of mainly non-polar residues is an essential conformational step at the initiation of the folding transition of globular proteins. We developed and applied experimental methods based on time-resolved resonance excitation energy transfer (trFRET) measurements combined with fast mixing methods and studied the initial phases of the folding of Escherichia coli adenylate kinase (AK). A series of AK mutants were prepared, in which the ends of selected backbone segments that form long closed loops or secondary structure elements were labeled by donors and acceptors of excitation energy. The end-to-end distance distributions of such segments were determined under equilibrium and during the fast folding transitions. These experiments show that three out of seven long loops that were labeled in the AK molecule are closed very early in the transition. The N terminal 26-residue loop (loop I) is closed in < 200 μs after the initiation of folding, while the β strand included in loop I is still disordered. The closure of the second 44-residue loop (loop II, which starts at the end of loop I) is also complete within < 300 μs. Four other loops as well as five secondary structures of the CORE domain of AK (an α helix and four β strands) are formed at a late step, at a rate of 0.5 ± 0.3 s -1 , the rate of the cooperative folding of the molecule. These experiments reveal a hierarchically ordered pathway of folding of the AK molecule, ranging from microseconds to seconds. The results reviewed here, obtained mainly from studying a small number of model proteins, support the counterintuitive mechanism whereby non-local interactions are effective in the initiation of the folding pathways. The experiments presented demonstrate the importance of mapping the rates of sub-domain structural transitions along the folding transition, in situ, in the context of the other sections of the chain, whether folded or disordered. These experiments also show the power of the time-resolved FRET measurements in achieving this goal. A large body of data obtained by theoretical and experimental studies that support, or can accommodate, the loop hypothesis is reviewed. We suggest that mapping multiple sub-domain structural tr...

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“…Miyake-Stoner et al reported a smFRET method to probe the disruption of hydrophobic core upon protein unfolding [71]. More recently, Woori Bae et al demonstrated a smFRET approach to unveil the kinetics of multiple protein-protein interactions occurring far below the diffraction limit [72].…”

Section: Detection Of Protein Foldingmentioning

confidence: 99%

Single-molecule FRET for Ultrasensitive Detection of Biomolecules

Yang¹,

Yang²,

Zhang³

2014

NanoBioImaging

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Single-molecule Förster resonance energy transfer (sm-FRET) has been widely employed to detect biomarkers and to probe the structure and dynamics of biomolecules. By monitoring the biological reaction in a spatio-temporal manner, smFRET can reveal the transient intermediates of biological processes that cannot be obtained by conventional ensemble measurements. This review provides an overview of singlemolecule FRET and its applications in ultrasensitive detection of biomolecules, including the major techniques and the molecular probes used for smFRET as well as the biomedical applications of smFRET. Especially, the combination of sm-FRET with new technologies might expand its applications in clinical diagnosis and biomedical research.

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Probing Protein Folding Using Site-Specifically Encoded Unnatural Amino Acids as FRET Donors with Tryptophan

Cited by 121 publications

References 54 publications

Ideal Bioorthogonal Reactions Using A Site-Specifically Encoded Tetrazine Amino Acid

Ideal Bioorthogonal Reactions Using A Site-Specifically Encoded Tetrazine Amino Acid

Fast closure of long loops at the initiation of the folding transition of globular proteins studied by time-resolved FRET-based methods

Single-molecule FRET for Ultrasensitive Detection of Biomolecules

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