Biophysics

We have three main research lines in biophysics:

1. Infrared (IR) nanospectroscopy of rhodopsins – Monitoring, for the first time at the nanoscale, the conformational changes of transmembrane proteins triggered by visible light.

2. IR nanospectroscopy of protein aggregates – Investigating, at the nanoscale, the structural modifications of proteins during their aggregation process, which is relevant to neurodegenerative diseases.

3. Functional studies of proteins – Evaluating the relationship between the structural properties of proteins and their nutrient utilization.

Spectroscopic identification at the nanoscale is essential for advancing our understanding of the local properties of inhomogeneous materials and biological matter. Mid-infrared (mid-IR) spectroscopy is a powerful tool for studying the vibrational structure of molecules and the low-energy electrodynamics of solid-state samples.

In our lab, we employ an alternative approach to near-field scattering by detecting the force exerted on the atomic force microscope (AFM) tip by the sample when its molecules are selectively excited by mid-IR radiation. This method enables the acquisition of high-sensitivity infrared chemical maps with a lateral resolution better than 100 × 100 nm².

For biological samples, this approach allows us to investigate chemical composition and functional secondary structure modifications at the scale of a single membrane or even a single protein aggregate.

1. IR Nanospectroscopy of Rhodopsin Triggered by Visible Light

Rhodopsins are fundamental cell receptors that function as ion pumps and channels through the cell membrane. They are extensively studied for applications in neuroscience and bioelectronics.

We have applied IR near-field nanospectroscopy in difference mode—acquiring absorption spectra under visible light ON and OFF conditions—to investigate, for the first time, the conformational changes of both Bacteriorhodopsin and, more recently, Channelrhodopsin in their native environment: a 5 nm-thick, cell-free membrane patch.

Our findings demonstrate that these proteins retain their ion channel function even in contact with gold surfaces, apart from minor modifications in their kinetics (1-3).

[1] V. Giliberti et al., Nano Lett., 19(5), 3104-3114 (2019)

[2] M. E. Temperini et al., OSA Continuum 3.9: 2564-2572 (2020)

[3] R. Polito et al., Phys. Rev. Applied 16, 014048 (2021)

2. IR Nanospectroscopy of Proteins During Their Aggregation Process

Protein aggregation into amyloid fibrils is a ubiquitous phenomenon in nature, occurring in both physiological and pathological conditions. When happening within cerebral tissues, aggregation of proteins is of increasing interest due to the identified connection between macroscopic insoluble protein aggregates and neurodegenerative diseases. 

In our lab, we are investigating the in-vitro aggregation process of two proteins relevant to neurodegenerative diseases: TAR DNA-binding protein of 43 kDa (TDP-43) and α-synuclein. We are also studying the role of RNA in the aggregation process of α-synuclein, specifically on protein aggregation kinetics and structural properties of fibrils (1,2). For α-synuclein, we have demonstrated that RNA alters the α-synuclein fibril architecture and modifies the morphological properties of fibrils.

Research in this area, could shed light on the pathological implications of neurodegenerative diseases, where intracellular amyloid formation may sequester RNA. This interaction could potentially influence disease progression and aid the development of RNA-based therapeutics and diagnostics.

[1] A. Intze. PhD Thesis, Sapienza University of Rome (2025).

[2] J. Rupert et al., Nucleic Acids Res., 51(21), 11466-11478 (2023).

3. Functional Studies of Proteins: Structural Properties and Nutrient Utilization

We have studied the secondary structure of plant and animal proteins of nutritional relevance using Diffuse Reflectance FT-IR Spectroscopy (DRIFTS). Our results, obtained from various proteins with different structures, have been validated through comparisons with X-ray crystallographic and IR/Raman semiquantitative data available in the literature. The same methodology has been applied to proteins within whole food matrices, revealing structural differences between untreated and processed foods (1).

In addition to the major amide I contributions—β-sheets (1633–1638 cm⁻¹), random coil (1649 cm⁻¹), α-helix (1654–1658 cm⁻¹), and β-turns (1671–1678 cm⁻¹)—we have quantified minor contributions associated with antiparallel β-sheet structures at 1606–1620 cm⁻¹ and 1690–1696 cm⁻¹.

Application of this method to protein-rich legume seed flour has allowed us to monitor changes in protein secondary structure upon heat processing of increasing intensity. Our findings indicate that multimeric complexes form from food proteins of plant origin through different mechanisms, depending on the initial β-sheet content. Notably, proteins with a higher β-sheet content exhibit greater stability in these complexes, which may adversely impact protein utilization and overall nutritional quality (2).

Furthermore, we have investigated the interaction between β-lactoglobulin B, a whey carrier protein, and (-)-epicatechin, a major dietary flavonoid with health-promoting biological activities, using FT-IR under physiological conditions. Our results suggest that epicatechin binds to the β-lactoglobulin B surface, leading to protein dissociation at molar ratios ≤2, with minimal changes in secondary structure. This finding supports the potential use of β-lactoglobulin B as a carrier for flavonoids, including epicatechin (3).

[1] M. Carbonaro et al., Food Chem. 108, 361-368 (2008).

[2] M. Carbonaro et al., Amino Acids 43, 911-921 (2012).

[3] A.  Nucara et al. SpringerPlus, 2, 661 (2013).