The elusive role of the N-terminal extension of βA3- and βAl-crystallin

Abstract

beta-Crystallins are structural lens proteins with a conserved two-domain structure and variable N- and C-terminal extensions. These extensions are assumed to be involved in quaternary interactions within the beta-crystallin oligomers or with other lens proteins. Therefore, the production of beta A3- and beta A1-crystallin from the single beta A3/A1 mRNA by dual translation initiation is of interest. These crystallins are identical, except that beta A1 has a much shorter N-terminal extension that beta A3. This rare mechanism has been conserved for over 250 million years during the evolution of the beta A3/A1 gene, suggesting that the generation of different N-terminal extensions confers a selective advantage. We therefore compared the stability and association behaviour of recombinant beta A3- and beta A1-crystallin. Both proteins are equally stable in urea- and pH-induced denaturation experiments. Gel filtration and analytical ultracentrifugation established that beta A3 and beta A1 both form homodimers. In the water-soluble proteins of bovine lens, beta A3 and beta A1 are present in the same molecular weight fractions, indicating that they oligomerize equally with other beta-crystallins. 1H-NMR spectroscopy showed that residues Met1 to Asn22 of the N-terminal extension of beta A3 have great flexibility and are solvent exposed, excluding them from protein interactions in the homodimer. These results indicate that the different N-terminal extensions of beta A3 and beta A1 do not affect their homo- or heteromeric interactions.

The mammalian small heat‐shock protein Hsp20 forms dimers and is a poor chaperone

“…To determine the actual mass, and thus the number of subunits in the Hsp20 oligomers, the protein was subjected to analytical ultracentrifugation, at initial protein concentration of 400 µg/ml. Although protein aggregation occurred during the run, high-speed sedimentation equilibria under meniscus depletion conditions [26] indicated the presence of an oligomer with a calculated molecular mass of 43.9Ϯ 3.4 kDa (data not shown). Because this is again too high for a dimer, and too low for a trimer, we also performed crosslinking experiments.…”

“…1 H-NMR spectroscopy was performed on rat Hsp20 (1.8 mM subunit concentration in 20 mM sodium phosphate buffer, pH 7.2, 0.05% NaN 3 , 10% D 2 O/90% H 2 O) at 25°C on a Bruker DMX-600 spectrometer. Two-dimensional spectra were acquired and processed using parameters described in Werten et al [26]. TOCSY spectra were acquired with mixing times of 30 ms and 70 ms and a NOESY spectrum was acquired with a mixing time of 130 ms.…”

Age-related Changes in Human Lens Crystallins Identified by Two-dimensional Electrophoresis and Mass Spectrometry

“…However, the function of the Nterminal extensions of β-crystallins remains unknown. They may play a role in stabilization of β-crystallin oligomers, but this hypothesis is still under investigation (Cooper, Carver and Truscott, 1993 ;Kroone et al, 1994 ;Trinkl, Glockshuber and Jaenicke, 1994 ;Werten et al, 1996 ;Ajaz et al, 1997). The Nterminal extensions may also act as intermolecular spacers to keep crystallins in elongating lens fibers dispersed.…”

Chaperones and chaperone–substrate complexes: Dynamic playgrounds for NMR spectroscopists

“…This polydispersity and dynamics have prevented structure determination by crystallography. Initial investigations by solution NMR spectroscopy of related sHsps revealed that the C-termini of B-crystallin, mouse Hsp25, and rat Hsp20, as well as the C-terminus of A-crystallin, are highly flexible and devoid of secondary structure, as evidenced by nearly random coil C chemical shifts, and are not involved in oligomerization [294][295][296][297]. Subsequent analysis demonstrated that these flexible extensions are not directly involved in substrate binding, but play an important role in retaining the quaternary structure, as evidenced by increased flexibility observed by 1 H-spectroscopy upon C-terminal truncation of mouse Hsp25, and subsequently by altered chaperone activity upon C-terminal immobilization of αA-crystallin through the introduction of bulky amino-acids [298,299].…”