ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTDetection of a Stable Intermediate in the Thermal Unfolding of a Cysteine-Free Form of Dihydrofolate Reductase from Escherichia ColiJ. Luo, M. Iwakura, and C. R. MatthewsCite this: Biochemistry 1995, 34, 33, 10669–10675Publication Date (Print):August 22, 1995Publication History Published online1 May 2002Published inissue 22 August 1995https://pubs.acs.org/doi/10.1021/bi00033a043https://doi.org/10.1021/bi00033a043research-articleACS PublicationsRequest reuse permissionsArticle Views147Altmetric-Citations21LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
Utilizing surface plasmon resonance (SPR), we have developed novel methodology for the detection of conformational change(s) in immobilized proteins. A genetically altered E. coli dihydrofolate reductase (DHFR-ASC) was attached to a carboxymethyldextran matrix layer covering the sensor surface of an SPR biosensor through a disulfide linkage at the engineered protein's C-terminus. The DHFR-ASC-immobilized surface exhibited a larger response to acid treatment than reference surfaces lacking immobilized proteins. The SPR signal of the tethered protein and the molar ellipticity of DHFR-ASC in solution responded similarly to pH changes, consistent with the interpretation that changes in the SPR signal reflect conformational changes occurring during acid denaturation. A pH shift observed between the SPR signal and ellipticity changes may reflect a difference between surface and bulk pH. The tethered protein sensor surface was stable to repeated acid treatment using solutions in the pH range of 0.12−7.80 and yielded reproducible measurements. This is the first demonstration of detection of conformational changes in an immobilized protein using an SPR biosensor. This technique has potential for developing novel sensors and/or switching devices in response to protein conformational changes.
We recently isolated an aptamer that binds to the Tat protein of HIV-1 with extremely high affinity and specificity (Yamamoto, R.; et al. Genes Cells 2000, 5, 371.). In the present study, we exploited this strong binding to develop a novel coupling method that links genotype with phenotype. To strengthen the original RNA-protein interaction still further, we connected three units of the aptamer in tandem and three units of a peptide derived from Tat that interacted with the aptamer. The binding of the resultant RNA, which consisted of three units of the aptamer, to the resultant peptide, which consisted of three units of the peptide, was extremely strong. In fact, the RNA-protein interaction was one of the strongest ever reported, with an apparent K(d) below 16 pM. This strong interaction was attempted for the selection of functional proteins, namely, dihydrofolate reductase (DHFR) or streptavidin, which we chose as an example, and we succeeded in the expected selection, although to a limited extent, of the target protein. The noncovalent but strong interaction described above should be useful as a novel tool for the future selection of functional proteins from pools of random sequences of amino acids.
Journal Article Effects of Mutation at Methionine-42 of Escherichia coli Dihydrofolate Reductase on Stability and Function: Implication of Hydrophobic Interactions Get access Eiji Ohmae, Eiji Ohmae *To whom correspondence should be addressed. Fax: +81-82-424-7387, E-mail: gekko@sci.hiroshima-u.ac.jp Search for other works by this author on: Oxford Academic PubMed Google Scholar Yukari Fukumizu, Yukari Fukumizu Search for other works by this author on: Oxford Academic PubMed Google Scholar Masahiro Iwakura, Masahiro Iwakura Search for other works by this author on: Oxford Academic PubMed Google Scholar Kunihiko Gekko Kunihiko Gekko Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Biochemistry, Volume 137, Issue 5, May 2005, Pages 643–652, https://doi.org/10.1093/jb/mvi079 Published: 01 May 2005 Article history Received: 07 January 2005 Accepted: 14 March 2005 Published: 01 May 2005
Amino acid sequences can be described as foldable or un-foldable depending on the nature of the tertiary structures they produce. Thus, understanding what makes a sequence foldable or un-foldable is crucial not only for classifying the huge number of sequences being produced by the various genome projects but also for understanding how amino acid sequence determines tertiary structure, that is, solving the protein folding problem. Through systematic circular permutation analysis of a small globular protein, dihydrofolate reductase, an idea of folding element has been introduced and led us to the conclusion that a complete set of folding elements, which leads to a collapse of the molecule in the early stages of folding, is required for a protein to be foldable.
NADP-linked malic enzyme from Escherichia coli W contains 7 cysteinyl residues per enzyme subunit. The reactivity of sulfhydryl (SH) groups of the enzyme was examined using several SH reagents, including 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM). 1. Two SH groups in the native enzyme subunit reacted with DTNB (or NEM) with different reaction rates, accompanied by a complete loss of the enzyme activity. The second-order modification rate constant of the "fast SH group" with DTNB coincided with the second-order inactivation rate constant of the enzyme by the reagent, suggesting that modification of the "fast SH group" is responsible for the inactivation. When the enzyme was denatured in 4 M guanidine HCl, all the SH groups reacted with the two reagents. 2. Althoug the inactivation rate constant was increased by the addition of Mg2+, an essential cofactor in the enzyme reaction, the modification rate constant of the "fast SH group" was unaffected. The relationship between the number of SH groups modified with DTNB or NEM and the residual enzyme activity in the absence of Mg2+ was linear, whereas that in the presence of Mg2+ was concave-upwards. These results suggest that the Mg2+-dependent increase in the inactivation rate constant is not the result of an increase in the rate constant of the "fast FH group" modification. 3. The absorption spectrum of the enzyme in the ultraviolet region was changed by addition of Mg2+. The dissociation constant of the Mg2+-enzyme complex obtained from the Mg2+- dependent increment of the difference absorption coincided with that obtained from the Mg2+- dependent enhancement of NEM inactivation. 4. Both the inactivation rate constant and the modification rate constant of the "fast SH group" were decreased by the addition of NADP+. The protective effect of NADP+ was increased by the addition of Mg2+. Based on the above results, the effects of Mg2+ on the SH-group modification are discussed from the viewpoint of conformational alteration of the enzyme.
