Assessing the Structural Stability of Designed β‐Hairpin Peptides in the Cytoplasm of Live Cells

Cheng, Zihao; Campbell, Robert E.

doi:10.1002/cbic.200500540

Cited by 24 publications

(19 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Colonies were screen for red fluorescence using a previously described colony-imaging system. 32 Briefly, plates were illuminated with 535-550 nm excitation light and emission was collected by a monochromatic CCD camera and a 630-660 nm emission filter. If no red colonies were observed, the plates were allowed to sit on the bench top or 4 C fridge for several days.…”

Section: Plasmid Library Screeningmentioning

confidence: 99%

Circularly permuted monomeric red fluorescent proteins with new termini in the β‐sheet

2010

Self Cite

View full text Add to dashboard Cite

Circularly permuted fluorescent proteins (FPs) have a growing number of uses in live cell fluorescence biosensing applications. Most notably, they enable the construction of single fluorescent protein-based biosensors for Ca 21 and other analytes of interest. Circularly permuted FPs are also of great utility in the optimization of fluorescence resonance energy transfer (FRET)-based biosensors by providing a means for varying the critical dipole-dipole orientation. We have previously reported on our efforts to create circularly permuted variants of a monomeric red FP (RFP) known as mCherry. In our previous work, we had identified six distinct locations within mCherry that tolerated the insertion of a short peptide sequence. Creation of circularly permuted variants with new termini at the locations corresponding to the sites of insertion led to the discovery of three permuted variants that retained no more than 18% of the brightness of mCherry. We now report the extensive directed evolution of the variant with new termini at position 193 of the protein sequence for improved fluorescent brightness. The resulting variant, known as cp193g7, has 61% of the intrinsic brightness of mCherry and was found to be highly tolerant of circular permutation at other locations within the sequence. We have exploited this property to engineer an expanded series of circularly permuted variants with new termini located along the length of the 10th b-strand of mCherry. These new variants may ultimately prove useful for the creation of single FP-based Ca 21 biosensors.

show abstract

Section: Plasmid Library Screeningmentioning

confidence: 99%

Circularly permuted monomeric red fluorescent proteins with new termini in the β‐sheet

2010

Self Cite

View full text Add to dashboard Cite

show abstract

“…Recent studies in this field have used FRET based assays both in vivo and in vitro to study structure and stability of small peptides. 4,8 However, the attachment of large proteins to either end of the b-hairpin may influence the enzymatic cleavage, particularly with exopeptidases. Thus, we have used HPLC based experiments that remove the need for bulky GFP-derived fluorophores that may interfere with the enzyme binding and function.…”

Section: Introductionmentioning

confidence: 99%

The structure of well‐folded β‐hairpin peptides promotes resistance to peptidase degradation

Cline

Waters

2009

Biopolymers

View full text Add to dashboard Cite

To investigate the effect of peptide secondary structure on proteolytic resistance, we have synthesized a series of peptides based on a well-folded beta-hairpin, WKWK. Mutations were made within the peptide which either decreased or increased the propensity to form beta-hairpin structures and one scrambled sequence was used as an unstructured control. The peptides were incubated with three different enzymes, alpha-chymotrypsin, trypsin, and pronase E, which represented both specific and non-specific proteases. The reactions were quenched at varying time points and analyzed with RP-HPLC to determine the rate of degradation for each of the peptides. We found that an increase in structure correlates well with an increase in resistance to degradation. We have shown that having both strong side chain interactions and a rigid D-Pro-Gly beta-turn resulted in the most proteolytic resistant peptides. The results from this study suggest that beta-hairpin structure is a viable way to provide added protease resistance to a peptide.

show abstract

“…[6][7][8] We have recently described an approach that enables us to asses the ability of peptides to fold into b-hairpins in the cytoplasm of live cells. [9] Our strategy entails the recombinant expression of a peptide gene fused in frame between flanking genes encoding a cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP). If a particular peptide sequence adopts a folded structure, CFP and YFP are brought into closer proximity and exhibit a higher efficiency of fluorescence resonance energy transfer (FRET).…”

mentioning

confidence: 99%

“…[11][12][13] We have proposed that trpzip-type peptides (or tandem fusions of such peptides) could serve as a minimal protein scaffold for molecular recognition in the cytoplasm of live cells. [9, 14] With this goal in mind, we sought to employ our screening strategy to identify a candidate trpziptype peptide with high fold stability in vivo. A similar approach has previously been used to increase the thermal stability of engineered immunoglobulin V L domains.…”

mentioning

confidence: 99%

“…We have previously demonstrated the ability of this imaging system to reliably distinguish colonies that express FRET constructs of various FRET efficiencies. [9] The fluorescent brightness of each colony, considered commensurate with peptide solubility, [16] was determined by direct excitation and imaging of YFP. Five individual colonies that exhibited both a high ratio of YFP-to-CFP fluorescence emission and high brightness were cultured overnight, and the plasmid DNA was purified.…”

mentioning

confidence: 99%

See 1 more Smart Citation

In Vivo Screening Identifies a Highly Folded β‐Hairpin Peptide with a Structured Extension

2007

Self Cite

View full text Add to dashboard Cite

The conventional approach for developing folded peptides involves chemical synthesis of systematically modified peptide variants and individual characterization by circular dichroism and NMR spectroscopy. [1][2][3][4][5] The use of synthetic peptides and reliance on low-throughput characterization techniques necessarily restricts the sequence diversity that can be explored, though efforts have been made to overcome this limitation. [6][7][8] We have recently described an approach that enables us to asses the ability of peptides to fold into b-hairpins in the cytoplasm of live cells.[9] Our strategy entails the recombinant expression of a peptide gene fused in frame between flanking genes encoding a cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP). If a particular peptide sequence adopts a folded structure, CFP and YFP are brought into closer proximity and exhibit a higher efficiency of fluorescence resonance energy transfer (FRET). Higher FRET efficiency enhances the YFP (acceptor) fluorescence at the expense of the CFP (donor) fluorescence. Imaging of plates harboring colonies of transformed bacteria provides the YFP and CFP fluorescence emission intensities for each colony, and thus peptides that are highly folded in vivo can be distinguished from those that are not.As will be described in this manuscript, this FRET-based approach also provides a versatile method for screening large libraries of peptide sequences for highly structured variants. We have applied this strategy to the development of a version of a "tryptophan zipper" (trpzip)-type b-hairpin [1] with a structured extension. Trpzips are a class of highly folded b-hairpins that are defined by the presence of cross-strand diagonally oriented Trp/Trp pairs on one face of the hairpin. [1,5,10] The minimal and highly stable trpzip structure has emerged as a preferred model system for computational and experimental studies of protein folding. [11][12][13] We have proposed that trpzip-type peptides (or tandem fusions of such peptides) could serve as a minimal protein scaffold for molecular recognition in the cytoplasm of live cells. [9, 14] With this goal in mind, we sought to employ our screening strategy to identify a candidate trpziptype peptide with high fold stability in vivo. A similar approach has previously been used to increase the thermal stability of engineered immunoglobulin V L domains. [15] The template for our initial "extended trpzip" library was a 20-mer version of the highly folded 16-mer trpzip HP5W4.[10]The 20-mer contained two additional pairs of residues, one random and one threonine, genetically inserted after what would otherwise have been the second and 14th residues of HP5W4. ; here X represents all 20 amino acids, and the residues inserted relative to HP5W4 are underlined. Assuming b-strand conformation, the randomized positions would be directed towards the face of HP5W4 that harbors the interdigitated Trp side chains.Escherichia coli was transformed with the gene library, and 6 10 3 colonies on 10 Petri dis...

show abstract

Assessing the Structural Stability of Designed β‐Hairpin Peptides in the Cytoplasm of Live Cells

Cited by 24 publications

References 21 publications

Circularly permuted monomeric red fluorescent proteins with new termini in the β‐sheet

Circularly permuted monomeric red fluorescent proteins with new termini in the β‐sheet

The structure of well‐folded β‐hairpin peptides promotes resistance to peptidase degradation

In Vivo Screening Identifies a Highly Folded β‐Hairpin Peptide with a Structured Extension

Contact Info

Product

Resources

About