alphaB-Crystallin: Einblicke in ein kleines Hitzeschockprotein mit NMR-Spektroskopie

The ubiquitous alphaB-crystallin belongs to the small heat shock proteins, a protein class involved in the maintenance of the homeostasis in cells. As a chaperone it prevents the aggregation of misfolded proteins by keeping them in the dissolved state. alphaB-crystallin is found upregulated among others in neurodegenerative disease states like Alzheimer’s and Parkinson and cardiomyopathies as well as certain cancer types. The remarkable palette of substrate proteins, the number of disease-states in which upregulation occurs and the variation in substrate affinity in dependency of posttranslational modifications, all indicate an important role of alphaB-crystallin in the homeostasis of living cells. Although the description of structural features of the protein is constantly being refined, so far, there are only models available to describe its quaternary structure of the oligomeric ensemble and an atomic structure for the full-length protein stays elusive. Being essential for both, substrate recognition and formation of higher order oligomers, the role and structural features of the N-terminus remains enigmatic. In this work we aimed to gain a deeper understanding of the role of the N-terminus and the two terminal IXI-motifs, 3 – IAI – 5 and 159 – IPI – 161, by making use of different NMR-techniques for their investigation. The C-terminal motif is known for binding to a hydrophobic groove formed by the strands beta4 and beta8. We use both IXI-motifs as anchor points in our investigations by comparing the wild-type protein and a set of valine-mutants of the two IXI-motifs, namely alphaB I3/5V, alphaB I159/161V and alphaB I3/5/159/161V via MAS solid-state and solution NMR-spectroscopy methods. It was possible to identify and assign the N-terminal motif-related isoleucines for the first time. Carbon correlation spectra revealed very similar chemical shifts for the isoleucine sidechain atoms for both motifs, which is interpreted as both motifs being located in a very similar chemical environment within the complex oligomer. This strongly implicates that the N-terminal IXI-motif can also bind to the hydrophobic groove. Furthermore, through carbon direct-excitation experiments in solution NMR and subsequent subtraction of the resulting spectra of the different samples, the amount of bound C-terminal motif could be determined via integration of the signals, as well as the ratio of bound N- and C-termini. We found that the C-terminus is being bound to an extend of approximately 50% in the temperature range of 290 – 310 K. The binding of the N-terminus is more temperature dependent, the lower the temperature, the more of the N-terminal IXI-motif is bound to the groove. The binding of the N-terminus serves as an additional way for self-regulation and oligomerization. This provides a rational explanation for the broad distribution of oligomers. To probe the proximity of the IXI-motifs to the alpha-crystallin domain we mutated the residue between the isoleucines to cysteines (alphaB A4C and alphaB P160C). These mutants were spin-labelled with MTSL and in case of the C-terminal variant also with a TOTAPOL derivative. It turned out that the label is cleaved off very quickly in case of the N-terminal mutant, implying that the cysteines are very close to each other. In case of the C-terminal spin labelled variants, the paramagnetic relaxation enhancement-effect on the signals in the PDSD spectra was analyzed, the effect evaluated and discussed on the two existing 24mer models by Jehle and Weinkauf