Hi there!
I'm trying to unfold R-phycoerythrin reversibly. I can't seem to find any mild denaturing agent without any loss of protein photoactivity. I've tried to use organic (Gdm/ClH, Urea) and inorganic (LiBr) agents. None of them worked reversibly. Either the protein would collapse or would be only slightly unfolded.
Is there somebody here to give me a good advice?

thanks

bennie

This reference may be of some help to you...


Gaigalas A, Gallagher T, Cole KD, Singh T, Wang L, Zhang YZ. A multistate model for the fluorescence response of R-phycoerythrin. Photochem Photobiol. 2006 May-Jun;82(3):635-44.

Abstract:

Although strong fluorescence makes the R-phycoerythrin (R-PE) proteins increasingly useful in biological and clinical assays, they are subject to nonlinear effects including transitions to collective dark states and photodegradation, which complicate quantitative applications. We report measurements of R-PE fluorescence intensity as a function of incident power, duration of illumination and temperature. Emission intensity in the band at 570 nm is proportional to incident power for low power levels. At higher incident power, the emission at 570 nm is smaller than expected from a linear dependence on power. We propose that R-PE undergoes both reversible emission cessation on a millisecond time scale attributed to transitions to a collective dark state, and irreversible photodegradation on a time scale of minutes. Singlet oxygen scavengers such as dithiothreitol and n-propyl gallate have protective effects against the latter effect but not the former. Electrophoretic analysis of irradiated solutions of R-PE indicates that significant noncovalent aggregation is correlated with photodegradation. A multistate model based on fluorescence measurements and geometric analysis is proposed for the fluorophores in R-PE. The phycobilin fluorophores are partitioned into three groups: the phycourobilins (PUB) absorbing at 490 nm, one group of phycoerythobilins (PEB) absorbing at 530 nm (PEB-530) and another group of PEB absorbing at 560 nm (PEB-560). The two processes that result in the loss of fluorescence intensity are most likely associated with the PEB-560 group.

You may want to investigate thermal affects of reversible unfolding. However, without knowing your downstream application, I'm not sure this would be applicable. Anyways, here are some good references for you to look into...


Vaidya S, Orta-Ramirez A, Smith DM, Ofoli RY. Effect of heat on phycoerythrin fluorescence: Influence of thermal exposure on the fluorescence emission of R-phycoerythrin. Biotechnol Bioeng. 2003 Aug 20;83(4):465-73.

Abstract:
The goal of this work was to measure and model the effect of thermal exposure on the fluorescence emission of R-phycoerythrin (R-PE). The long-term objective of our work is to assess the feasibility of encapsulating R-PE for use as the critical component of a time-temperature integrator (TTI) for ascertaining the degree of inactivation of food pathogens such as Salmonella. In this article we present a study to measure and model the thermally induced fluorescence emission decay of R-PE in several isothermal experiments. We used the isothermal data to determine the kinetic parameters, based on a general n(th) order reaction, and evaluated the utility of the resulting model by using it to predict R-PE fluorescence emission decay for several nonisothermal experiments based on published USDA safe harbor guidelines for cooked beef products. The transient experiments were conducted over the same temperature range used in the isothermal study. Very good agreement was obtained between theory and experiment at temperatures of 62.8 degrees C and above, although the model slightly underpredicted the extent of fluorescence emission decay at 60 degrees C. Our results indicate that R-PE fluorescence emission decay kinetics is well behaved and that the protein is a strong candidate for use as a time-temperature integrator.



Tang XC, Pikal MJ. Measurement of the kinetics of protein unfolding in viscous systems and implications for protein stability in freeze-drying. Pharm Res. 2005 Jul;22(7):1176-85. Epub 2005 Jul 22.

Abstract:
PURPOSE: The aim of the study is to determine the degree of coupling between protein unfolding rate and system viscosity at low temperatures in systems relevant to freeze-drying.

METHODS: The cold denaturation of both phosphoglycerate kinase (PGK) and beta-lactoglobulin were chosen as models for the protein unfolding kinetics study. The system viscosity was enhanced by adding stabilizers (such as sucrose), and denaturant (guanidine hydrochloride or urea) was added to balance the stabilizing effect of sucrose to maintain the cold denaturation temperature roughly constant. The protein unfolding kinetics were studied by both temperature-controlled tryptophan emission fluorescence spectroscopy and isothermal high-sensitivity modulated differential scanning calorimetry (MDSC) (Tzero). Viscometers were used to determine the system viscosity. To verify the predictions of structure based on protein unfolding dynamics, protein formulations were freeze-dried above the glass transition temperatures, and the protein structures in dry products were determined by fluorescence spectroscopy of reconstituted solids by extrapolation of the solution data to the time of reconstitution.

RESULTS: Empirical equations describing the effect of sucrose and denaturant (urea and guanidine hydrochloride) on protein cold denaturation were developed based on DSC observations [X. C. Tang and M. J. Pikal. The Effects of Stabilizers and Denaturants on the Cold Denaturation Temperature of Proteins and Implications for Freeze-Drying. Pharm. Res. Submitted (2004)]. It was found that protein cold denaturation temperature can be maintained constant in system of increasing sucrose concentration by simultaneous addition of denaturants (urea and guanidine hydrochloride) using the empirical equations as a guide. System viscosities were found to increase dramatically with increasing sucrose concentration and decreasing temperature. The rate constants of protein unfolding (or the half-life of unfolding) below the cold denaturation temperature were determined by fitting the time dependence of either fluorescence spectroscopy peak position shift or DSC heat capacity increase to a first-order reversible kinetic model. The half-life of unfolding did slow considerably as system viscosity increased. The half-life of PGK unfolding, which was only 3.5 min in dilute buffer solution at -10 degrees C, was found to be about 200 min in 37% sucrose at the same temperature. Kinetics of protein unfolding are identical as measured by tryptophan fluorescence emission spectroscopy and by high-sensitivity modulated DSC. The coupling between protein unfolding kinetics and system viscosity for both proteins was significant with a stronger coupling with PGK than with beta-lactoglobulin. The half-lives of PGK and beta-lactoglobulin unfolding are estimated to be 5.5 x 10(11) and 2.2 years, respectively, even when they are freeze-dried in sucrose formulations 20 degrees C above Tg'. Thus, freeze-drying below Tg' should not be necessary to preserve the native conformation. In support of this conclusion, native PGK was obtained after the freeze-drying of PGK at a temperature more than 60 degrees C above the system Tg' in a thermodynamically unstable system during freeze-drying.

CONCLUSIONS: Protein unfolding kinetics is highly coupled with system viscosity in high viscosity systems, and the coupling coefficients are protein dependent. Protein unfolding is very slow on the time scale of freeze-drying, even when the system is freeze-dried well above Tg'. Thus, it is not always necessary to freeze-dry protein formulations at temperature below Tg' to avoid protein unfolding. That is, protein formulations could be freeze-dried at product temperature far above the Tg', thereby allowing much shorter freeze-drying cycle times, with dry cake structure being maintained by the simultaneous use of a bulking agent and a disaccharide stabilizer.



Orta-Ramirez A, Merrill JE, Smith DM. Sucrose, sodium dodecyl sulfate, urea, and 2-mercaptoethanol affect the thermal inactivation of R-phycoerythrin. J Food Prot. 2001 Nov;64(11):1806-11.

Abstract:
Thermal inactivation kinetics (D- and z-values) of the algal protein, R-phycoerythrin (R-PE), were studied under different buffer conditions (pH 4.0, 7.0, and 10.0) and concentrations of sucrose, sodium dodecyl sulfate (SDS), urea, and 2-mercaptoethanol (ME). R-PE solutions were heated in capillary tubes at temperatures between 40 and 90 degrees C depending on buffer conditions. Thermal inactivation parameters for R-PE, calculated on the basis of fluorescence loss, were modified by addition of chemicals. Overall, sucrose and ME had a thermostabilizing effect, while SDS and urea decreased thermal stability of R-PE. The z-values ranged from 5.9 degrees C in 50 mM NaCl, 20 mM glycine buffer, pH 10.0, to 37.8 degrees C in 60% sucrose, 50 mM NaCl, 20 mM phosphate buffer, pH 7.0. The z-values obtained for R-PE closely matched the z-values of some target microorganisms in food processes, suggesting R-PE might be used as a time-temperature integrator to verify thermal processing adequacy.



Smith SE, Orta-Ramirez A, Ofoli RY, Ryser ET, Smith DM. R-phycoerythrin as a time-temperature integrator to verify the thermal processing adequacy of beef patties. J Food Prot. 2002 May;65(5):814-9.

Abstract:
The objective of this study was to relate R-phycoerythrin (PE) fluorescence decay to the inactivation of Salmonella in beef patties cooked using adequate and inadequate thermal processes as defined by the U.S. Department of Agriculture (USDA) safe harbor requirements and lethality standards. Ground beef containing 4.8 or 19.1% fat was inoculated with an eight-strain cocktail of Salmonella and formed into 113-g patties. Capillary tubes containing PE in borate buffer at pH 9.0 were attached to a thermocouple and inserted horizontally into the patties. Patties (n = 43) were cooked on a grill maintained at 177 degrees C for 6 to 13 min and reached internal temperatures ranging from 57 to 77 degrees C. Patties were analyzed for Salmonella survivors and for fluorescence decay of PE. The thermal lethality of each process was calculated at a reference temperature of 65 degrees C. Twenty-four of the 43 high-fat patties met the USDA safe harbor regulations, with thermal lethalities of >66 s, whereas only 20 of these patties met the proposed 5-log10 lethality standard. Three of the 20 low-fat patties that met USDA regulations did not meet the proposed lethality standard. A normalized PE fluorescence value of about 0.3 (confidence interval = 99%) indicated that patties had been processed sufficiently to reduce Salmonella by 5 log10 cycles. PE has the potential for use as a marker to verify processing adequacy in food-processing plants and in other settings in which the use of the target pathogen is inappropriate.

Thanks for the references ;)

I've seen some of them previously, but some are new to me and should be of good help.

...knew it was a good idea to join scientistsolutions

There are many references in which trifluoroethanol (TFE) and hexafluoroisopropanol (HFIP) are used for this type of application