کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8305703 | 1538431 | 2014 | 7 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Tryptophan-scanning mutagenesis of the ligand binding pocket in Thermotoga maritima arginine-binding protein
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کلمات کلیدی
AF4TCSPCPBPQELSAsymmetric flow field flow fractionation - تقسیم جریان جریان میدان نامتقارنTime correlated single photon counting - شمارش تک فوتون همبستگیAnisotropy decay - فروپاشی انحصاریFluorescence - فلورسنسSlim - لاغرQuasi-elastic light scattering - پراکندگی نور نیمه الاستیکPeriplasmic binding protein - پروتئین اتصال پروپی پلاسمی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
The Thermotoga maritima arginine binding protein (TmArgBP) is a member of the periplasmic binding protein superfamily. As a highly thermostable protein, TmArgBP has been investigated for the potential to serve as a protein scaffold for the development of fluorescent protein biosensors. To establish a relationship between structural dynamics and ligand binding capabilities, we constructed single tryptophan mutants to probe the arginine binding pocket. Trp residues placed around the binding pocket reveal a strong dependence on fluorescence emission of the protein with arginine for all but one of the mutants. Using these data, we calculated dissociation constants of 1.9-3.3 μM for arginine. Stern-Volmer quenching analysis demonstrated that the protein undergoes a large conformational change upon ligand binding, which is a common feature of this protein superfamily. While still active at room temperature, time-resolved intensity and anisotropy decay data suggest that the protein exists as a highly rigid structure under these conditions. Interestingly, TmArgBP exists as a dimer at room temperature in both the presence and absence of arginine, as determined by asymmetric flow field flow fractionation (AF4) and supported by native gel-electrophoresis and time-resolved anisotropy. Our data on dynamics and stability will contribute to our understanding of hyperthermophilic proteins and their potential biotechnological applications.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Biochimie - Volume 99, April 2014, Pages 208-214
Journal: Biochimie - Volume 99, April 2014, Pages 208-214
نویسندگان
Lindsay J. Deacon, Hilbert Billones, Anne A. Galyean, Teraya Donaldson, Anna Pennacchio, Luisa Iozzino, Sabato D'Auria, Jonathan D. Dattelbaum,