Solid state fluorescence of proteins in high throughput mode and its applications [version 2; peer review: 2 approved]

Abstract

Direct comparison between fluorescence spectra of a sample in solution and solid state form is valuable to monitor the changes in protein structure when it is “dried” or immobilized on a solid surface (for biocatalysis or sensor applications). We describe here a simple method for recording fluorescence emission spectra of protein powders without using any dedicated accessory for solid samples in a high-throughput format. The 96-well plate used in our studies, was coated black from all the sides and the excitation and emission paths are identical and are from the top of the well. These two features minimize scatter and provide fairly noise free spectra. Even then the fluorescence intensity may be dependent upon many factors such as the extent of protein aggregation, morphology and sizes of the protein particles. Hence, (changes in) λmax emission may be a more reliable metric in the case of fluorescence spectra of proteins in the solid state. However, any large changes in the intensity could indicate changes in the microenvironment of the fluorophore. The fluorescence emission spectra were blue-shifted (4 to 9 nm), showed an increase in the intensity for different proteins studied upon lyophilization, and were similar to what has been reported by others using available commercial accessories for solid state samples. After validating that our method worked just as well as the dedicated accessories, we applied the method to compare the fluorescence emission spectra of α-chymotrypsin in solution, precipitated form, and the lyophilized powder form. We further examined the fluorescence emission spectra of green fluorescent protein (GFP) in solution and solid form. We also analyzed fluorescence resonance energy transfer (FRET) between tryptophan (Trp57) and the cyclic chromophore of GFP. These findings pointed towards the change in the microenvironment around the cyclic chromophore in GFP upon lyophilization.

Keywords

• Experimental Biophysical Methods
• Protein Chemistry & Proteomics