Laera, S., Ceccone, G., Rossi, F., Gilliland, D., Hussain, R., Siligardi, G. & Calzolai, L. Measuring protein structure and stability of protein-nanoparticle systems with synchrotron radiation circular dichroism. Nano Lett. 11, 4480-4 (2011)
Nanoparticles (NP) are used in different applications such as cosmetics and medicine. To assess potential toxic effects and to design NP-based drug delivery systems it is critical to understand what happens to proteins upon interaction with these special particles. This information is difficult to obtain, but for the first time we have shown that using the B23 beamline, it is possible to detect and analyze structural changes of proteins in protein-metallic nanoparticle complexes1. By using synchrotron radiation circular dichroism (SRCD) we have measured the structure and stability changes of proteins upon their interaction with nanoparticles at nanomolar concentration. In particular, we measured a decrease of 6°C in the thermal unfolding of human serum albumin upon interaction with silver nanoparticles. This effect does not emerge with gold nanoparticles. SRCD allows the measurement of critical parameters for protein-nanoparticle interactions by using only a few micrograms of proteins, providing the relative stability of key proteins. This information will help understanding and predicting the potential toxicity of nanomaterials. In addition it may contribute to the design of the next generation of non toxic nanoparticle-based drug delivery systems.
Nanotechnology is gaining more and more interest in many industrial activity fields. Nanoparticles are already extensively used in widely different applications such as cosmetic sunscreens, in diagnostics and in medicine as drug delivery systems2. When nanoparticles enter a biological system, they become coated with a complex mixture of proteins (the so-called protein corona3) and this interaction can both alter the properties of the nanoparticles and of the interacting proteins. In particular it has been shown that this interaction can change enzymatic activity, alter protein conformation, expose previously hidden epitopes4, and all these alterations can induce unexpected biological reactions and lead to toxicity5. In order to investigate possible toxic effects of nanoparticles and to design the next generation of drug delivery systems, it is essential to understand what happens to the structure and stability of relevant proteins upon interaction with it. Unfortunately this kind of information is very difficult to obtain due to the very complex nature of the system (solid/liquid interface) and the experimental constrains, usually the amount of protein- NP is very low.
Circular dichroism (CD) is an excellent and sensitive method for rapidly evaluating the secondary structure, folding and binding properties of proteins, and recently has also been used to detect structural changes of proteins interacting with nanoparticles6. The use of synchrotron radiation to perform CD experiments presents several advantages with respect to the conventional CD technique. The major advantage is the high flux provided by synchrotron radiation that allows CD data to be measured both with very low amounts of proteins and in the presence of highly absorbing chemicals such as suspensions of gold and silver nanoparticles. It enables the detection of the secondary structure of proteins with only a few hundred nanograms in the spectral range of 195-250 nm.
|Figure 1: CD spectra of HSA 20nM (black line) and HSA 20nM+AgNP (red line).|
Using beamline B23 at Diamond Light Source we have shown that, using a 10 cm cell with total volume under 0.8 ml, it is possible to detect and analyse structural changes of proteins in the low nanomolar concentration range when forming stable non-covalent protein-metallic nanoparticle complexes. In particular, we measured the secondary structure of human serum albumin (HSA) in a well-defined stoichiometric complex with silver (AgNP) and gold (AuNP) nanoparticles at nanomolar concentrations. An example of these studies is illustrated in Fig.1, where the SRCD data of the HSA/AgNP system are reported.
|Figure 2: Thermal unfolding of free HSA 80nM. (a) CD spectra at selected temperatures. (b) Complete temperature unfolding profile at 222nm with sigmoidal fit (red line) to calculate melting temperature.|
In addition we have been also able to follow the changes in the thermal stability of human serum albumin when complexed to metallic nanoparticles. Fig. 2 shows the thermal unfolding of free HSA and the fitting of the data used to calculate the melting temperature (Tm) of the protein. By collecting the CD spectra of the protein at variable temperatures between 20°C and 90°C and fitting the data of the protein unfolding with a sigmoid equation (indicative of a cooperative unfolding process) we calculated a melting temperature of 75.1°C for the free HSA protein.
|Figure 3: Thermal unfolding of free HSA and HSA-AgNP complex.|
By repeating this experiment with HSA-AuNP and HSA-AgNP we could calculate the melting temperature of the human serum albumin protein when interacting with AuNP (Tm = 74.8° C) and with AgNP (Tm = 69.1° C). The comparison of the unfolding of HSA in the three conditions (alone, -AgNP, -AuNP ), partially shown in Fig.3, indicates that upon interaction with silver nanoparticles the protein changes its melting temperature from 75.1°C to 69.1°C. This decrease of 6°C in the melting temperature indicates that upon interaction with AgNP the serum albumin significantly reduces its thermal stability. On the other hand a similar destabilisation is not observed in the case of HSA-AuNP complexes, which have a melting temperature of 74.8°C, experimentally indistinguishable from the Tm value for the protein alone.
Our work shows that by using synchrotron radiation circular dichroism it is possible to analyse the secondary structure and stability of proteins in the low nanomolar concentration range, thus providing a unique method for detecting the relative stability of key biological proteins interacting with nanoparticles. In particular, this extreme sensitivity has allowed us to show B23that human serum albumin is significantly destabilised when interacting with silver nanoparticles, while its stability is not affected when interacting with gold nanoparticles. The high sensitivity provides structural information on protein-nanoparticles complexes at near equimolar ratios and allows access to detailed information that has been very difficult to obtain until now.
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