Modular Assembly Revealed by Tryptophan and Other Optical Probes in Staphylococcal Nuclease Folding

This is the authors' abstract. We don't add key points for this paper.

How do different molecular events amalgamate to form kinetic funnels for protein folding? To probe local events and dissect folding of sub‐domains new tryptophan probes have been implanted in two locations of staphylococcal nuclease (SNase): the β‐barrel (W140F+L25W) and the C‐helix (W140F+L125W). 8‐Anilino‐l‐naphthalensulfonate (ANS) was also used to trace a transient appearance of hydrophobic clusters. By use of these fluorescence probes in addition to the CD222nm signal of protein, several molecular events for the folding of SNase and its mutants have been resolved and activation barriers for these reactions determined. Folding from a GdmHCl unfolded state begins a burst with the formation of 20% of the secondary structure (< 20 ms in CD mode). The next event (55 ms) is the clustering of hydrophobic side chains in the C‐ and the oligonucleotide binding (OB)‐domains. These hydrophobic clusters are capable of binding ANS and they continue to grow in 500–700 ms. ANS binding sites subsequently sequester and become inaccessible to ANS in a 1.7‐s reaction. Merging of the OB‐ and the C‐domains that follows is a complex tri‐phasic process (134 ms, 457 ms, and 31.3 s). Global folding measured by the CD222nm signal unveils yet another slow reaction (1,600 s) which was shown to originate from the trans/cis isomerization of Pro117. A modular assembly model depicting protein folding‐by‐parts or origami of folding is proposed and discussed in light of these observations.