Fluorescence and circular dichroism stopped-flow have been widely used to determine the kinetics of protein folding including folding rates and possible folding pathways. Yet, these measurements are not able to provide spatial information of protein folding/unfolding. Especially, conformations of denatured states cannot be elaborated in detail. In this study, we apply the method of fluorescence energy transfer with a stopped-flow technique to study global structural changes of the staphylococcal nuclease (SNase) mutant K45C, where lysine 45 is replaced by cysteine, during folding and unfolding. By labeling the thiol group of cysteine with TNB (5,5'-dithiobis-2-nitrobenzoic acid) as an energy acceptor and the tryptophan at position 140 as a donor, distance changes between the acceptor and the donor during folding and unfolding are measured from the efficiency of energy transfer. Results indicate that the denatured states of SNase are highly compact regardless of how the denatured states (pH-induced or GdmCl-induced) are induced. The range of distance changes between two probes is between 25.6 and 25.4 A while it is 20.4 A for the native state. Furthermore, the folding process consists of three kinetic phases while the unfolding process is a single phase. These observations agree with our previous sequential model: N(0) left arrow over right arrow D(1) left arrow over right arrow D(2) left arrow over right arrow D(3) (Chen et al., J Mol Biol 1991;220:771-778). The efficiency of protein folding may be attributed to initiating the folding process from these compact denatured structures.
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http://dx.doi.org/10.1002/prot.21985 | DOI Listing |
Acta Histochem
December 2024
Instituto de Ciencias Ambientales y Salud (ICAS), Fundación Pro Salud y Medio Ambiente (PROSAMA), Paysandú 752, Buenos Aires CP1405, Argentina; Centro Integrativo de Biología y Química Aplicada (CIBQA), Universidad Bernardo O'Higgins, General Gana 1702, Santiago 8370854, Chile. Electronic address:
Many fluorophores display interesting features that make them useful biological labels and dyes, particularly in Cell Biology and Cytogenetics. Changes in the absorption-emission spectra (ortho- and metachromasia) are accounted among them. Acridine orange (AO) is one of such fluorochromes with an exemplary orthochromatic vs.
View Article and Find Full Text PDFJ Biol Chem
December 2024
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, 37232; Department of Medicine, Division of Diabetes, Endocrinology and Metabolism, Vanderbilt University Medical Center, Nashville, TN, 37232. Electronic address:
Mechanistic Target of Rapamycin (mTOR) binds the small metabolite inositol hexakisphosphate (IP) as shown in structures of mTOR, however it remains unclear if IP, or any other inositol phosphate species, function as an integral structural element(s) or catalytic regulator(s) of mTOR. Here, we show that multiple, exogenously added inositol phosphate species can enhance the ability of mTOR and mTORC1 to phosphorylate itself and peptide substrates in in vitro kinase reactions, with the higher order phosphorylated species being more potent (IP=IP>IP>>IP). IP increased the V and decreased the apparent K of mTOR for ATP.
View Article and Find Full Text PDFFood Chem
December 2024
Institute for Egg Science and Technology, School of Food and Biological Engineering, Chengdu University, Chengdu 610106, Sichuan, China. Electronic address:
Egg white protein's functional properties and digestibility can be significantly affected by processing methods. In this study, a novel combined ultrasound-heating (UH) pretreatment protocol was developed, where liquid egg white was subjected to ultrasonic treatment (0.4 W/mL, 10 min) followed by mild heating (72 °C, 10 min) prior to drying processes.
View Article and Find Full Text PDFFood Chem
December 2024
School of Food Science and Technology, Dalian Polytechnic University, Dalian 116034, China; State Key Laboratory of Marine Food Processing & Safety Control, Dalian Polytechnic University, Dalian 116034, China; Liaoning Key Laboratory of Food Nutrition and Health, Dalian Polytechnic University, Dalian 116034, China; National Engineering Research Center of Seafood. Electronic address:
There has been a growing demand for the development of high protein beverages in the food industry. However, during thermal processing, high-protein beverages undergo protein aggregation and gelation. In this study, thermostable soy protein was prepared by disulfide bond cleavage combined with preheating treatment.
View Article and Find Full Text PDFJ Biol Inorg Chem
December 2024
Department of Chemistry, Babes-Bolyai University, 11 Arany Janos Str., 400028, Cluj-Napoca, Romania.
The ferryl state in globins has previously been reported to undergo a protonation event below pH 5, as assessed using pH jump experiments with stopped-flow UV-Vis spectroscopy. This protonation entails hypsochromic shifts in the α and β bands (~ 20 to 40 nm) and an ~ 10 nm reduction in the energy difference between these two bands. We now report that in Mb this event is also characterized by a hypsochromic shift in the Soret band (~ 5 nm).
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