Probing the folding pathway of a β-clam protein with single-tryptophan constructs

Clark, Patricia L.; Weston, Benjamin F.; Gierasch, Lila M.

doi:10.1016/s1359-0278(98)00053-4

Cited by 65 publications

(88 citation statements)

References 36 publications

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“…Escherichia coli (E. coli) BL21(DE3) cells transformed with CRABP or tetra-Cys CRABP plasmids (in all cases the CRABP harbors a stabilizing mutation, Arg131Gln, and is thus a 'wild-type*' variant 7,8 ) were cultured at 378C in LB medium with 100 g/ml ampicillin. Protein expression was induced at OD 600 ¼ 1.0 by adding isopropyl--D-thiogalactoside (IPTG) to a final concentration of 0.4 mM for 4 h, and the tetra-Cys CRABP was purified from the soluble fraction of the cell extract.…”

Section: Protein Expressionmentioning

confidence: 99%

“…Protein expression was induced at OD 600 ¼ 1.0 by adding isopropyl--D-thiogalactoside (IPTG) to a final concentration of 0.4 mM for 4 h, and the tetra-Cys CRABP was purified from the soluble fraction of the cell extract. 8 Although it partitions nearly equally between the soluble and insoluble cell fractions, we isolated P39A tetra-Cys CRABP from the soluble fraction to insure the homogeneity of the samples used for in vitro studies. The concentration of the purified samples was determined spectrophotometrically using the " 280 value of 21750 M À1 cm À1 .…”

Section: Protein Expressionmentioning

confidence: 99%

“…We have used a predominantly -sheet protein, cellular retinoic acid-binding protein I (CRABP), whose in vitro folding we have explored in detail, [6][7][8][9] to study folding and aggregation in vivo. We have incorporated a binding site (tetra-cysteine motif) for the fluorescent dye, 4 0 ,5 0 -bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH), 10,11 into a loop in both wild type CRABP and a slow-folding, aggregation prone mutant, P39A CRABP ( Figure 1).…”

Section: Introductionmentioning

confidence: 99%

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From the test tube to the cell: Exploring the folding and aggregation of a β‐clam protein

Ignatova¹,

Krishnan²,

Bombardier³

et al. 2007

Biopolymers

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106

126

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A crucial challenge in present biomedical research is the elucidation of how fundamental processes like protein folding and aggregation occur in the complex environment of the cell. Many new physico‐chemical factors like crowding and confinement must be considered, and immense technical hurdles must be overcome in order to explore these processes in vivo. Understanding protein misfolding and aggregation diseases and developing therapeutic strategies to these diseases demand that we gain mechanistic insight into behaviors and misbehaviors of proteins as they fold in vivo. We have developed a fluorescence approach using FlAsH labeling to study the thermodynamics of folding of a model β‐rich protein, cellular retinoic acid binding protein (CRABP) in Escherichia coli cells. The labeling approach has also enabled us to follow aggregation of a modified version of CRABP and chimeras between CRABP and huntingtin exon 1 with its glutamine repeat tract. In this article, we review our recent results using FlAsH labeling to study in‐vivo folding and present new observations that hint at fundamental differences between the thermodynamics and kinetics of protein folding in vivo and in vitro. © 2007 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 88: 157–163, 2007. This article was originally published online as an accepted preprint. The ‘Published Online’ date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

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Section: Protein Expressionmentioning

confidence: 99%

Section: Protein Expressionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

From the test tube to the cell: Exploring the folding and aggregation of a β‐clam protein

Ignatova¹,

Krishnan²,

Bombardier³

et al. 2007

Biopolymers

Self Cite

106

126

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“…Nucleotide binding at this concentration gives Ͻ10% saturation but yields the highest signal to noise ratio. Nucleotide binding to hexahistidine-tagged cellular retinoic acid-binding protein (21) purified from E. coli using Ni-NTA chromatography was used as background reference.…”

Section: Toc33/34 Mutations and Attoc159mentioning

confidence: 99%

The Roles of Toc34 and Toc75 in Targeting the Toc159 Preprotein Receptor to Chloroplasts

Wallas

Smith

Sánchez‐Nieto³

et al. 2003

Journal of Biological Chemistry

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The Toc complex at the outer envelope of chloroplasts initiates the import of nuclear-encoded preproteins from the cytosol into the organelle. The core of the Toc complex is composed of two receptor GTPases, Toc159 and Toc34, as well as Toc75, a ␤-barrel membrane channel. Toc159 is equally distributed between a soluble cytoplasmic form and a membrane-inserted form, suggesting that assembly of the Toc complex is dynamic. In the present study, we used the Arabidopsis thaliana orthologs of Toc159 and Toc34, atToc159 and atToc33, respectively, to investigate the requirements for assembly of the trimeric Toc complex. In addition to its intrinsic GTPase activity, we demonstrate that integration of atToc159 into the Toc complex requires atToc33 GTPase activity. Additionally, we show that the interaction of the two GTPase domains stimulates association of the membrane anchor of atToc159 with the translocon. Finally, we employ reconstituted proteoliposomes to demonstrate that proper insertion of the receptor requires both Toc75 and Toc34. Collectively these data suggest that Toc34 and Toc75 act sequentially to mediate docking and insertion of Toc159 resulting in assembly of the functional translocon.

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“…Our early studies have focused on a heterologous protein expressed in E. coli, and we chose to use cellular retinoic acid-binding protein I (CRABP I) initially because of our extensive experience with the folding of this b-rich protein in vitro. [51][52][53][54][55][56] A clear advantage of using a non-E. coli protein is that folding can be studied without altering directly the cellular physiology by virtue of the function of the protein under study. CRABP I is also delicately balanced between successful folding and formation of aggregates, both in vitro and in vivo.…”

Section: The Full Monty: Exploring Protein Folding In Intact Cellsmentioning

confidence: 99%

A career pathway in protein folding: From model peptides to postreductionist protein science

Gierasch

2011

Protein Science

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This review discusses the inherent challenge of linking ''reductionist'' approaches to decipher the information encoded in protein sequences with burgeoning efforts to explore protein folding in native environments-''postreductionist'' approaches. Because the invitation to write this article came as a result of my selection to receive the 2010 Dorothy Hodgkin Award of the Protein Society, I use examples from my own work to illustrate the evolution from the reductionist to the postreductionist perspective. I am incredibly honored to receive the Hodgkin Award, but I want to emphasize that it is the combined effort, creativity, and talent of many students, postdoctoral fellows, and collaborators over several years that has led to any accomplishments on which this selection is based. Moreover, I do not claim to have unique insight into the topics discussed here; but this writing opportunity allows me to illustrate some threads in the evolution of protein folding research with my own experiences and to point out to those embarking on careers how the twists and turns in anyone's scientific path are influenced and enriched by the scientific context of our research. The path my own career has taken thus far has been shaped by the timing of discoveries in the field of protein science; together with our contemporaries, we become part of a knowledge evolution. In my own case, this has been an epoch of great discovery in protein folding and I feel very fortunate to have participated in it.

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Probing the folding pathway of a β-clam protein with single-tryptophan constructs

Cited by 65 publications

References 36 publications

From the test tube to the cell: Exploring the folding and aggregation of a β‐clam protein

From the test tube to the cell: Exploring the folding and aggregation of a β‐clam protein

The Roles of Toc34 and Toc75 in Targeting the Toc159 Preprotein Receptor to Chloroplasts

A career pathway in protein folding: From model peptides to postreductionist protein science

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