Role of cis- and trans-interactions in manifestations of amyloidogenic properties of variable domains of Bence-Jones proteins TIM and LUS

Abstract

Intact Bence-Jones proteins TIM and LUS under simulated physiological conditions (10 mM phosphate buffer, pH 7.0, 100 mM NaCl, 37°C) did not display amyloidogenic properties. However, their isolated variable domains exhibit these qualities in full measure. Therefore, both intact proteins and their variable domains were studied using a complex of physical methods (scanning microcalorimetry, analytical centrifugation, optics) that allowed us to assess the stability of their tertiary and quaternary structures. The experimentally obtained thermodynamic functions indicated that the stability of isolated variable domains of TIM and LUS was comparable to the stability of similar domains in amyloidogenic proteins described earlier. However, inside the whole protein their stability was comparable to the stability of VL domains of ordinary Bence-Jones proteins. The decreased stability of the isolated variable domains of TIM and LUS was shown to be due both to weak interactions between a pair of variable domains (trans-interaction) and to a natural lack of interaction with the constant domains (cis-interaction).

The Folding Pathway of 6aJL2

“…While no such intermediate was observed at 25 °C, other studies on similar proteins have presented evidence from kinetic data for equilibrium intermediates under different conditions. 4,10,21,39 The estimated ΔG H 2 O value presented here is consistent with those reported for 6aJL2 5,[7][8][9]34 and for similar proteins, particularly among the V L proteins where it has been recognized that V L κ-type are more stable (∼5.975−8.365 kcal/mol) than λ-type (∼3.585−6.7 kcal/mol) proteins for the same denaturant agent. 10,40 The m-value is consistent with those of proteins with the same number of residues.…”

“…The estimated Δ G H 2 O value presented here is consistent with those reported for 6aJL2 ,− , and for similar proteins, particularly among the V L proteins where it has been recognized that V L κ-type are more stable (∼5.975–8.365 kcal/mol) than λ-type (∼3.585–6.7 kcal/mol) proteins for the same denaturant agent. , The m -value is consistent with those of proteins with the same number of residues . Detection of folding intermediates in equilibrium data may be difficult due to several factors including low population, indistinct signal differences between species, similar unfolding transitions, etc.…”

“…The SVD data analysis presented here suggests the existence of an intermediate state in equilibrium conditions, and as shown in the results section, the accompanying folding mechanism is complex. Regarding higher order processes, Tishchenko suggests the presence/formation of dimers in 6aJL2 based on Bence–Jones proteins evidence, and indeed Blancas et al observed higher order structure at 37 °C but not at 25 °C. , In the present study, no protein concentration dependent changes are observed suggesting the absence of dimer formation.…”

“…Regarding higher order processes, Tishchenko suggests the presence/ formation of dimers in 6aJL2 based on Bence−Jones proteins evidence, and indeed Blancas et al observed higher order structure at 37 °C but not at 25 °C. 7,34 In the present study, no protein concentration dependent changes are observed suggesting the absence of dimer formation.…”

“…V L chains can be isolated and studied as single domains, but they are found as part of bigger complexes in a natural state (as antibodies); i.e., they show clear tendencies toward quaternary association and also toward nonspecific aggregation. ,,, Therefore, another possible source of heterogeneity in the unfolded state of a single V L domain could be quaternary association, provided nonspecific aggregation is been avoided in the experimental conditions. , Refolding experiments from 7.00 to 1.50 M urea, a final urea concentration in the native region for 6aJL2, results in three refolding time constants, ≈6, 10, and 32 s, at all tested 6aJL2 concentrations (Figure a). These three time constants are in good agreement with those reported in the chevron plot for 1.50 M urea.…”