کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2828501 1162718 2014 10 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Affinity maturation of a computationally designed binding protein affords a functional but disordered polypeptide
ترجمه فارسی عنوان
بلوغ یک پروتئین اتصال دهنده طراحی محاسباتی باعث می شود یک پلیپپتید کارآمد اما اختلال ایجاد کند
کلمات کلیدی
طراحی محاسباتی، تکامل هدایت، پروتئین های ناسازگار ذاتی. گلوبال گلوله، تعاملات پروتئین-پروتئین، رابط گیرنده پروتئین، نمایش سطح مخمر
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شناسی مولکولی
چکیده انگلیسی

Computational methods have been recently applied to the design of protein–protein interfaces. Using this approach, a 61 amino acid long protein called Spider Roll was engineered to recognize the kinase domain of the human p21-activated kinase 1 (PAK1) with good specificity but modest affinity (KD = 100 μM). Here we show that this artificial protein can be optimized by yeast surface display and fluorescence-activated cell sorting. After three rounds of mutagenesis and screening, a diverse set of tighter binding variants was obtained. A representative binder, MSR7, has a >102-fold higher affinity for PAK1 when displayed on yeast and a 6 to 11-fold advantage when produced free in solution. In contrast to the starting Spider Roll protein, however, MSR7 unexpectedly exhibits characteristics typical of partially disordered proteins, including lower α-helical content, non-cooperative thermal denaturation, and NMR data showing peak broadening and poor signal dispersion. Although conformational disorder is increasingly recognized as an important property of proteins involved in cellular signaling and regulation, it is poorly modeled by current computational methods. Explicit consideration of structural flexibility may improve future protein designs and provide deeper insight into molecular events at protein–protein interfaces.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Structural Biology - Volume 185, Issue 2, February 2014, Pages 168–177
نویسندگان
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