Exploiting Elements of Transcriptional Machinery to Enhance Protein Stability

Barakat, Nora H.; Barakat, Nesreen H.; Carmody, Lisa; Love, John J.

doi:10.1016/j.jmb.2006.10.091

Cited by 10 publications

(11 citation statements)

References 54 publications

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“…Genes for GB1 A and GB1 B were constructed by inverse-PCR mutagenesis and separately expressed in Escherichia coli and purified using standard procedures. The expression level for GB1 A was similar to that for GB1 (;40 mg/L), but the yield of GB1 B was ;10-fold lower (;4 mg/L), consistent with thermal melting temperatures of >100°C and ;37°C for GB1 A and GB1 B , respectively (data published previously as part of a stability study) (Barakat et al 2007). The thermal stability for GB1 is ;83°C.…”

Section: Resultssupporting

confidence: 82%

A de novo designed protein–protein interface

2007

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As an approach to both explore the physical/chemical parameters that drive molecular self-assembly and to generate novel protein oligomers, we have developed a procedure to generate protein dimers from monomeric proteins using computational protein docking and amino acid sequence design. A fast Fourier transform-based docking algorithm was used to generate a model for a dimeric version of the 56-amino-acid b1 domain of streptococcal protein G. Computational amino acid sequence design of 24 residues at the dimer interface resulted in a heterodimer comprised of 12-fold and eightfold variants of the wild-type protein. The designed proteins were expressed, purified, and characterized using analytical ultracentrifugation and heteronuclear NMR techniques. Although the measured dissociation constant was modest (;300 mM), 2D-[ 1 H, 15 N]-HSQC NMR spectra of one of the designed proteins in the absence and presence of its binding partner showed clear evidence of specific dimer formation.

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Section: Resultssupporting

confidence: 82%

A de novo designed protein–protein interface

2007

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“…Thermal unfolding curves for the Gβ1 variants monitored at 218 nm confirmed previous findings in relationship to the importance of position 45 for the overall stability of Gβ1-WT (Figure 2a and Table 1). 41 CD spectra collected on free SpA B -WT and five variants also indicate that the overall helical composition is maintained and that there are no obvious structural perturbations for the SpA B test variants. Except for the significantly destabilized double mutant (i.e., tSpA B L20A/L23A), the CD spectra are highly similar (Supporting Information).…”

Section: ■ Resultsmentioning

confidence: 83%

Characterizing Substrate Selectivity of Ubiquitin C-Terminal Hydrolase-L3 Using Engineered α-Linked Ubiquitin Substrates

et al. 2014

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The ubiquitin-proteasome system (UPS) is highly complex and entails the concerted actions of many enzymes that function to ubiquitinate proteins targeted to the proteasome as well as enzymes that remove and recycle ubiquitin for additional rounds of proteolysis. Ubiquitin C-terminal hydrolase-L3 (UCH-L3) is a human cytosolic deubiquitinase whose precise biological function is not known. It is believed to hydrolyze small peptides or chemical adducts from the C-terminus of ubiquitin that may be remnant from proteasomal processing. In addition, UCH-L3 is a highly effective biotechnological tool that is used to produce small or unstable peptides/proteins recalcitrant to production in Escherichia coli expression systems. Previous research, which explored the substrate selectivity of UCH-L3, demonstrated a substrate size limitation for proteins/peptides expressed as α-linked C-terminal fusions to ubiquitin and also suggested that an additional substrate property may affect UCH-L3 hydrolysis [ Larsen , C. N. et al. (1998) Biochemistry 37 , 3358 - 3368 ]. Using a series of engineered protein substrates, which are similar in size yet differ in secondary structure, we demonstrate that thermal stability is a key factor that significantly affects UCH-L3 hydrolysis. In addition, we show that the thermal stabilities of the engineered substrates are not altered by fusion to ubiquitin and offer a possible mechanism as to how ubiquitin affects the structural and unfolding properties of natural in vivo targets.

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“…However, they are end-point assays based on the proteolytic lability of the fusion construct when the POI is unstable and therefore cannot readily yield an estimate of folding free energy. A related in vivo screen flanks the POI between a DNA-binding domain and a transcriptional activation domain [78] (Figure 3c). Only when the POI is sufficiently flexible can the flanking domains effectively activate transcription of a reporter gene.…”

Section: Promising New Methods For Study Of In Vivo Protein Foldingmentioning

confidence: 99%

“…As in (a), this correlation is presumably based on the enhanced proteolytic susceptibility of the fusion protein when the POI is unstable. (c) Construct designed such that flexibility of the POI controls transcription of the β-lactamase gene [78,79]. When the POI is flanked by the N-terminal DNA-binding domain of bacteriophage λ, which binds to the λ operator, and the RNA polymerase α subunit, which activates transcription of the β-lactamase gene, flexible POIs lead to greater transcriptional activation and higher antibiotic resistance.…”

Section: Figuresmentioning

confidence: 99%

Protein folding in the cell: challenges and progress

Gershenson

Gierasch

2011

Current Opinion in Structural Biology

148

135

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It is hard to imagine a more extreme contrast than that between the dilute solutions used for in vitro studies of protein folding and the crowded, compartmentalized, sticky, spatially inhomogeneous interior of a cell. This review highlights recent research exploring protein folding in the cell with a focus on issues that are generally not relevant to in vitro studies of protein folding, such as macromolecular crowding, hindered diffusion, co-translational folding, molecular chaperones, and evolutionary pressures. The technical obstacles that must be overcome to characterize protein folding in the cell are driving methodological advances, and we draw attention to several examples, such as fluorescence imaging of folding in cells and genetic screens for incell stability.

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Exploiting Elements of Transcriptional Machinery to Enhance Protein Stability

Cited by 10 publications

References 54 publications

A de novo designed protein–protein interface

A de novo designed protein–protein interface

Characterizing Substrate Selectivity of Ubiquitin C-Terminal Hydrolase-L3 Using Engineered α-Linked Ubiquitin Substrates

Protein folding in the cell: challenges and progress

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