Infrared spectroscopy of biomolecules: fibril assembly and protein-membrane interaction


Study on Cytochrome C fibril assembly


Amyloidogenic proteins have the capability to self-assembly into beta-sheet organized aggregates (fibrils). First associated to a wide range of degenerative diseases, amyloid fibrils were then recognized to have functional roles in bacterial coatings, branching hyphae in fungi, insect egg envelop protection, catalytic scaffolds and epigenetic transformations [1]. Recently, the study of Cytochrome C aggregation, an evolutionarily conserved heme protein involved in many cellular processes, attracted attention within the scientific community: indeed, the aggregation and brillation dynamics of his protein, as well as the mechanisms leading to organized beta-rich conformations, are partially known. Experimental studies  were performed on Cytochrome C aggregates by Scanning Electron Microscopy, fluorescence microscopy, micro-Raman and Infrared spectroscopy, aimed at elucidating the morphology, the kinetic of formation and the secondary structure of the aggregates. It was observed that beta-rich aggregates with different morphologies (mature fibrils, spherules and platelets) are built up, after destabilization of the protein from the native fold, through continuous provision of oligomers and/or amino-acids, over timescales varying from tenths to hundreds of minutes [2].

Fig. 1  The infrared absorption spectrum of Cytochrome C at pH 9 after thermal incubation at 70 C . The green filled Gaussian at 1617 cm-1 is the amyloid fibrils contribution. In the inset, a  fluorescence image of fibrils in the same sample is shown.


Study on Cardiolipin-Cytochrome C interaction as a model for mitochondrial membrane functioning.


The interaction lipid membrane-protein is an intermediate but crucial step towards the understanding of a class of degenerative diseases. Indeed, at the level of cell cytoplasm, nanofibrils and protein aggregates might be signature of organelles misfunctioning and could themselves induce cytotoxicity [3]. The adhesion of native or mutated proteins to lipidic multilayers provides disordered segments of the CH2 groups with formation of complexes that remarkably affect the number and the intensities of the infrared vibrational modes of the membrane. Large Unilamellar Vesicles (LUV) of cardiolipin, mimic of the mitochondrial membrane, and Cytochrome C in native and mutated forms, both highly interactive with the lipidic membranes, were used as models. The infrared absorption spectra revealed local distorsions or breakage of the lipid framework and provided insight on the protein-membrane binding mechanism, on the membrane stiffness and on the polarity of the environment as well [2].



Fig. 2  CH2 symmetric stretching energy in LUV-Cytochrome C solutions as a function of the protein concentration. Dashed line is a guide to the eyes. ATR spectra were
taken at the infrared beamline SINBAD.


[1]  M.Carbonaro et al., International Journal of Biological Macromolecules 115 1157 (2018) DOI:10.1016/j.ijbiomac.2018.04.134

[2] A. Nucara et al.,International Journal of Biological Macromolecules 138 106 (2019) DOI: 10.1016/j.ijbiomac.2019.07.060.

[3]  D. H. J. Lopes et al., Biophysical Journal 93 3132 (2007)



Prof. Alessandro Nucara
Dipartimento di Fisica, Università La Sapienza di Roma