RESUMEN
We have developed a sensor concept capable of discriminating environments that induce proteins to enter unfolding intermediate states. Such a sensor detects the presence of environmental stressors such as chemical agents in aqueous media, thermal stress or the presence of ionizing or non-ionizing radiation by monitoring the conformation state of a "sensor protein". In this paper, we demonstrate the concept by using surface plasmon resonance to monitor binding of thermally and chemically stressed sensor proteins to a chaperone, alpha-crystallin, bound to the sensor surface. Citrate synthase and insulin were used as example sensor proteins to detect the presence of thermal stress and chemical stress, respectively. It was shown that alpha-crystallin retained its chaperone action after immobilization on the Biacore sensor chip. The binding of early and late unfolding intermediates of citrate synthase was discriminated using the association and dissociation behaviour of the binding. The sensor is therefore capable of assessing the severity of an environmental stress.
Asunto(s)
Técnicas Biosensibles/métodos , Citrato (si)-Sintasa/química , Citrato (si)-Sintasa/ultraestructura , Cristalografía/métodos , Insulina/química , Chaperonas Moleculares/química , Resonancia por Plasmón de Superficie/métodos , Sitios de Unión , Unión Proteica , Desnaturalización Proteica , Pliegue de Proteína , Propiedades de Superficie , TemperaturaRESUMEN
We study the effect of microwaves at 2,450 MHz on protein unfolding using surface plasmon resonance sensing. Our experimental method makes use of the fact that unfolding proteins tend to bind to chaperones on their unfolding pathway and this attachment is readily monitored by surface plasmon resonance. We use the protein citrate synthase (CS) for this study as it shows strong binding to the chaperone alpha crystallin when stressed by exposure to excess temperature. The results of microwave heating are compared with the effect of ambient heating and a combination of ambient and microwave heating to the same final temperature. We study the temperature distributions during the heating process. We show that microwaves cause a significantly higher degree of unfolding than conventional thermal stress for protein solutions heated to the same maximum temperature.