Structural and spectroscopic studies of proteins in stress conditions

Following Albumin changes in solution with small-angle X ray scattering, UV-vis and fluorescence

The study of molecular structure under varying conditions can help elucidating the underlying  mechanism of complex processes and linking structure to function. In the case of biological macromolecules the interesting length-scales of phenomena like conformational changes or formation of supramolecular complexes can be monitored with the technique of small angle X-ray scattering, which offers the advantage of probing the structure directly in solution conditions close to the natural functioning state, or modified in a controlled way to discover structural responses upon “stress”-inducing environments. In addition, the power to resolve the mechanism of convoluted effects can be enhanced by the combination of techniques that can report on both the structure and chemistry of the samples. As an example investigation methods based on SAXS analysis aided by optical spectroscopy can give a useful structural insight into complex processes involving proteins under changing solution environment or application of external perturbations [1].

This idea was applied to the case of Human Serum Albumin, the most  abundant protein of body fluids which has a “modular” three-domain structure potentially responding to stress by means of changes of conformation [2].

Besides acting as the main carrier protein of the circulation, HSA is also an obvious target of extracellular reactive oxidant species due to its high abundance in plasma. It is for this reason considered the main anti-oxidant defense in blood [3]. It is foreseeable that repeated exposure of HSA to oxidative environments could significantly affect its biological activity and a correlation between the structure and the oxidation state of HSA is expected to be crucial for rationalizing the effect of oxidative modifications on the functional ability of this protein.

In the first part of the seminar a study addressed at probing the structural changes of albumin induced by the chemical damage caused by the oxidant hypochlorite will be presented [4].  The experiments benefited from the use of a multi-technique instrumental platform which combined the simultaneous collection of SAXS, UV-vis absorbance spectra and fluorescence emission on the same sample volume [1]. Despite the chemical modification, the native shape was preserved up to oxidant/HSA molar ratio < 80, above which a structural transition occurred in the critical oxidant/HSA molar ratio range between 80-120. This conformational variation involved the drifting of one of the end-domains from the rest of the protein and corresponded to the loss of one third of the alpha-helix and a net increase of the protein negative charge. The high reproducibility and well-defined nature of this transition suggested that it represents a structural response characteristic of this multi-domain protein that was never observed before.

HSA is also known to undergo conformational variations as a function of pH and in particular the rearrangements observed when going from neutral to acid pH are defined as the the N-F transition (pH 4.3) and the acid expansion (below pH 3.5) [5]. In the second part of the seminar the results of a SAXS investigation combined with the monitoring of the protein intrinsic fluorescence are  described. We managed to implement an experimental setup in which the process of acid unfolding could be followed in-situ as a function of a continuous decrease of pH in time [6]. This allowed for the correlation between the structural and spectroscopic data as a function of pH together with an attempt to resolve the structural conformers involved in the transition. In addition, to test the stabilizing effect of physiological ligands of albumin as fatty acids, we compared the acid unfolding process of albumin with and without palmitic acid. We found that when binding the ligand the native conformation was favored up to lower pH values.

[1]         S. Haas, T.S. Plivelic, C. Dicko, Combined SAXS/UV-vis/Raman as a diagnostic and structure resolving tool in materials and life sciences applications, J. Phys. Chem. B, 118 (2014) 2264–2273.

[2]         D.C. Carter, J.X. Ho, Structure of serum albumins, Adv. Protein Chem., 45 (1994) 153–203.

[3]         M. Roche, P. Rondeau, N.R. Singh, E. Tarnus, E. Bourdon, The antioxidant properties of serum albumin, FEBS Lett., 582 (2008) 1783–1787.

[4]         A. Del Giudice, C. Dicko, L. Galantini, N. V. Pavel, Structural Response of Human Serum Albumin to Oxidation: Biological Buffer to Local Formation of Hypochlorite, J. Phys. Chem. B, 120 (2016) 12261–12271.

[5]         T.J. Peters, All About Albumin, Elsevier, 1995.

[6]         A. Del Giudice, C. Dicko, L. Galantini, N.V. Pavel, Time-dependent pH Scanning of the Acid-Induced Unfolding of Human Serum Albumin Reveals Stabilization of the Native Form by Palmitic Acid Binding, J. Phys. Chem. B, Under Revision (2017).