کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
5914052 | 1162719 | 2014 | 14 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Improving coiled coil stability while maintaining specificity by a bacterial hitchhiker selection system
ترجمه فارسی عنوان
بهبود پایداری کویل در حالی که حفظ ویژگی با یک سیستم انتخاب مجدد باکتری است
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کلمات کلیدی
ITCMITFIM10HIT - اصابتTAT - تاتtwin-arginine translocase - ترنسپاز دوقلو آرژنینcircular dichroism - رنگ تابی دورانیleucine zipper - زیپ لوسینbasic helix-loop-helix leucine zipper - زیپ لوکسی اسلیمی اسلحه پایهMicrophthalmia-associated transcription factor - فاکتور رونویسی مرتبط با میکرو فتالمیIsothermal titration calorimetry - کالری سنجی تیتاسیون ایزوترمالcoiled coils - کویل کویلCoiled coil - کویل کویل شده
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شناسی مولکولی
چکیده انگلیسی
The design and selection of peptides targeting cellular proteins is challenging and often yields candidates with undesired properties. Therefore we deployed a new selection system based on the twin-arginine translocase (TAT) pathway of Escherichia coli, named hitchhiker translocation (HiT) selection. A pool of α-helix encoding sequences was designed and selected for interference with the coiled coil domain (CC) of a melanoma-associated basic-helix-loop-helix-leucine-zipper (bHLHLZ) protein, the microphthalmia associated transcription factor (MITF). One predominant sequence (iM10) was enriched during selection and showed remarkable protease resistance, high solubility and thermal stability while maintaining its specificity. Furthermore, it exhibited nanomolar range affinity towards the target peptide. A mutation screen indicated that target-binding helices of increased homodimer stability and improved expression rates were preferred in the selection process. The crystal structure of the iM10/MITF-CC heterodimer (2.1 Ã
) provided important structural insights and validated our design predictions. Importantly, iM10 did not only bind to the MITF coiled coil, but also to the markedly more stable HLHLZ domain of MITF. Characterizing the selected variants of the semi-rational library demonstrated the potential of the innovative bacterial selection approach.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Structural Biology - Volume 186, Issue 3, June 2014, Pages 335-348
Journal: Journal of Structural Biology - Volume 186, Issue 3, June 2014, Pages 335-348
نویسندگان
Tim Kükenshöner, Daniel Wohlwend, Christoph Niemöller, Padmarupa Dondapati, Janina Speck, Adebola V. Adeniran, Anita Nieth, Stefan Gerhardt, Oliver Einsle, Kristian M. Müller, Katja M. Arndt,