Effect of macromolecular crowding on protein folding dynamics at the secondary structure level

Abstract

Macromolecular crowding is one of the key characteristics of the cellular environment and is therefore intimately coupled to the process of protein folding in vivo. While previous studies have provided invaluable insight into the effect of crowding on the stability and folding rate of protein tertiary structures, very little is known about how crowding affects protein folding dynamics at the secondary structure level. In this study, we examined the thermal stability and folding-unfolding kinetics of three small folding motifs (i.e., a 34-residue alpha-helix, a 34-residue cross-linked helix-turn-helix, and a 16-residue beta-hairpin) in the presence of two commonly used crowding agents, Dextran 70 (200 g/L) and Ficoll 70 (200 g/L). We found that these polymers do not induce any appreciable changes in the folding kinetics of the two helical peptides, which is somewhat surprising as the helix-coil transition kinetics have been shown to depend on viscosity. Also to our surprise and in contrast to what has been observed for larger proteins, we found that crowding leads to an appreciable decrease in the folding rate of the shortest beta-hairpin peptide, indicating that besides the excluded volume effect, other factors also need to be considered when evaluating the net effect of crowding on protein folding kinetics. A model considering both the static and the dynamic effects arising from the presence of the crowding agent is proposed to rationalize these results.

Figure 1

(A) CD thermal melting curves of L9:41-74 in 200 g/L Dextran 70 (red) and in 200 g/L Ficoll 70 (blue), respectively. (B) Arrhenius plot of the T-jump induced conformational relaxation rates of L9:41-74 in 200 g/L Dextran 70 (red) and in 200 g/L Ficoll 70 (blue), respectively. Also shown for comparison are the CD thermal melting curve (black crosses in A) and relaxation rates (black dashed line in B) of the same peptide in 20 mM phosphate D₂O buffer (derived from reference 22).

Figure 2

(A) CD thermal melting curves of Z34C-m1 in 200 g/L Dextran 70 (red) and in 200 g/L Ficoll 70 (blue), respectively. Lines are global fits of these data to the two-state model described in the text. (B) Arrhenius plot of the T-jump induced conformational relaxation rates (open circles) of Z34C-m1 in 200 g/L Dextran 70 (red) and in 200 g/L Ficoll 70 (blue), respectively. Open triangles and squares correspond to the two-state folding and unfolding rates of Z34C-m1 in 200 g/L Dextran 70. Also shown are the CD thermal melting curve (black crosses in A) and folding (black dashed line in B) and unfolding (black solid line in B) rate constants of Z34C-m1 in 20 mM phosphate D₂O buffer (derived from reference 23).

Figure 3

CD thermal melting curves of trpzip4-m1 in 200 g/L Dextran 70 (red) and in 200 g/L Ficoll 70 (blue), respectively. Lines are global fits of these data to the two-state model described in the text. Also shown is the CD thermal melting curve (black) of the same peptide in 20 mM phosphate D₂O buffer (derived from reference 24).

Figure 4

Arrhenius plots of the T-jump induced conformational relaxation rates (open circles) of trpzip4-m1 in 200 g/L Dextran 70 (A) and in 200 g/L Ficoll 70 (B), respectively. Open triangles and squares correspond to their respective two-state folding and unfolding rates. Also shown in each case are the folding (blue dashed line) and unfolding (red dashed line) rate constants of trpzip4-m1 in 20 mM phosphate D₂O buffer (derived from reference 24).