Amyloids are insoluble protein fibrillar aggregates. The importance of characterizing their aggregation has steadily increased because of their link to human diseases and material science applications. In particular, misfolding and aggregation of the Josephin domain of ataxin-3 is implicated in spinocerebellar ataxia-3. Infrared nanospectroscopy, simultaneously exploiting atomic force microscopy and infrared spectroscopy, can characterize at the nanoscale the conformational rearrangements of proteins during their aggregation. Here we demonstrate that we can individually characterize the oligomeric and fibrillar species formed along the amyloid aggregation. We describe their secondary structure, monitoring at the nanoscale an α-to-β transition, and couple these studies with an independent measurement of the evolution of their intrinsic stiffness. These results suggest that the aggregation of Josephin proceeds from the monomer state to the formation of spheroidal intermediates with a native structure. Only successively, these intermediates evolve into misfolded aggregates and into the final fibrils.

At least nine dominant neurodegenerative diseases are caused by expansion of CAG repeats in coding regions of specific genes that result in abnormal elongation of polyglutamine (polyQ) tracts in the corresponding gene products. When above a threshold that is specific for each disease the expanded polyQ repeats promote protein aggregation, misfolding and neuronal cell death. The length of the polyQ tract inversely correlates with the age at disease onset. It has been observed that interruption of the CAG tract by silent (CAA) or missense (CAT) mutations may strongly modulate the effect of the expansion and delay the onset age. We have carried out an extensive study in which we have complemented DNA sequence determination with cellular and biophysical models. By sequencing cloned normal and expanded SCA1 alleles taken from our cohort of ataxia patients we have determined sequence variations not detected by allele sizing and observed for the first time that repeat instability can occur even in the presence of CAG interruptions. We show that histidine interrupted pathogenic alleles occur with relatively high frequency (11%) and that the age at onset inversely correlates linearly with the longer uninterrupted CAG stretch. This could be reproduced in a cellular model to support the hypothesis of a linear behaviour of polyQ. We clarified by in vitro studies the mechanism by which polyQ interruption slows down aggregation. Our study contributes to the understanding of the role of polyQ interruption in the SCA1 phenotype with regards to age at disease onset, prognosis and transmission. Spinocerebellar ataxia type 1 (SCA1) is a progressive neurodegenerative disorder resulting in loss of coordination and balance. It is caused by an expanded repeated DNA sequence (CAG) in the gene ATXN1. The CAG repeat region is normally interrupted by the DNA sequence CAT. Loss of this interruption is believed to cause instability whereby the CAG repeat expands beyond a key threshold resulting, ultimately, in polyglutamine protein aggregation and cell death. Here we examine how interruptions influence pathology in patients and establish a cellular model to support our findings. We distinguish our patients into two sub-groups based on whether or not their expanded CAG repeat stretches contained an interruption. This is not possible with conventional diagnostic techniques. Differentiating the sub-group with no interruptions led to improved accuracy in predicting their age at onset. The other sub-group, with interruptions, reveals a delay in age at onset that shows greater alignment with the longest stretch of CAG repeats. These findings are significant for genetic counselling and prognosis. Our cellular model and in vitro studies confirmed the relationship between disease severity and uninterrupted repeat length and showed that interruptions do not significantly affect the polyglutamine protein aggregation, but do slow down the aggregation rate.

Haem proteins have long been the most studied proteins in biophysics, and have become paradigms for the characterization of fundamental biomolecular processes as ligand binding and regulatory conformational transitions. The presence of the haem prosthetic group, the absorbance spectrum of which has a ligation sensitive region conveniently located in the UV-visible range, has offered a powerful and sensitive tool for the investigation of molecular functions. The central Fe atom is capable of reversibly binding diatomic ligands, including O₂, CO, and NO. The Fe-ligand bond is photolabile, and a reactive unligated state can be transiently generated with a pulsed laser. The photodissociated ligands quickly rebind to the haem and the process can be monitored by transient absorbance methods. The ligand rebinding kinetics reflects protein dynamics and ligand migration within the protein inner cavities. The characterization of these processes was done in the past mainly by low temperature experiments. The use of silica gels to trap proteins allows the characterization of internal ligand dynamics at room temperature. In order to show the potential of the laser flash photolysis techniques, combined with modern numerical analysis methods, we report experiments conducted on two non-symbiotic haemoglobins from Arabidopsis thaliana. The comparison between time courses recorded on haemoglobins in solution and encapsulated in silica gels allows for the highlighting of different interplays between protein dynamics and ligand migration.

Neuroglobin (Ngb), a member of the globin superfamily, was found in the brain of vertebrates and is suggested to play a neuroprotective function under hypoxic conditions by scavenging nitrogen monoxide (NO) through a dioxygenase activity. In order for such a reaction to efficiently take place and to minimize the release of reactive intermediates in the cytosol, the cosubstrates O₂ and NO and other unstable reaction intermediates should bind sequentially to docking sites in the protein matrix. We have characterized the accessibility of these sites by analyzing the geminate CO rebinding kinetics to the heme moiety observed upon nanosecond flash photolysis of the Ngb-CO complex encapsulated in silica gels. The geminate rebinding phase showed a remarkable complexity, revealing the presence of a system of secondary docking sites where ligands are stored for hundreds of microseconds. Most kinetics steps display little temperature dependence, demonstrating that ligands can easily migrate through the cavities, except for the slowest reaction intermediate, possibly reflecting a structural conformational change reshaping the system of cavities. This conformational change is unrelated with distal His E7 binding to the heme, as it persists for the HE7L mutant. Overall, data are consistent with the presence of a discrete system of docking sites, possibly acting as reservoirs for the putative cosubstrates and for other reactive species involved in the physiologically relevant reaction.