Abstract
Folding of proteins usually involves intermediates, of which an important type is the molten globule (MG). MGs are ensembles of interconverting conformers that contain (non-)native secondary structure and lack the tightly packed tertiary structure of natively folded globular proteins. Whereas MGs of various purified proteins have been probed to date, no data are available on their presence and/or effect during protein synthesis. To study whether MGs arise during translation, we use ribosome-nascent chain (RNC) complexes of the electron transfer protein flavodoxin. Full-length isolated flavodoxin, which contains a non-covalently bound flavin mononucleotide (FMN) as cofactor, acquires its native α/β parallel topology via a folding mechanism that contains an off-pathway intermediate with molten globular characteristics. Extensive population of this MG state occurs at physiological ionic strength for apoflavodoxin variant F44Y, in which a phenylalanine at position 44 is changed to a tyrosine. Here, we show for the first time that ascertaining the binding rate of FMN as a function of ionic strength can be used as a tool to determine the presence of the off-pathway MG on the ribosome. Application of this methodology to F44Y apoflavodoxin RNCs shows that at physiological ionic strength the ribosome influences formation of the off-pathway MG and forces the nascent chain toward the native state.
Highlights
Proteins need to fold into their correct native conformations to perform their functions
The molten globule (MG) state is characterized by fluorescence anisotropy that is higher than native apoflavodoxin
All Purified F44YP Contains flavin mononucleotide (FMN)—In a previous study, we showed that when FMN binds to a released C-terminally shortened C69A flavodoxin construct, this construct is protected against intracellular proteolysis, due to the increased thermodynamic stability conferred by incorporated FMN [44]
Summary
Dependence of Apoflavodoxin’s Off-pathway MG on Ionic Strength and Temperature—Decreasing salt concentration is a well known method to destabilize folded proteins [48]. Observation of an altering slope in FMN fluorescence titration data shows that C69ARNC binds FMN (Fig. 5A) This difference in FMN occupation of F44YRNC and C69ARNC, and the observation that F44YP is fully saturated with FMN, suggests that when the apo-form of F44Y protein is present in E. coli, it is not as stable in the cellular environment of E. coli as C69A apoprotein is. Increasing potassium PPi concentration from 10 to 100 mM, or addition of 290 mM NaCl to buffer I, leads to an increase in FMN binding rate This observation is due to increased population of the native state of apoflavodoxin. Upon addition of salt (buffer I with 290 mM NaCl), the FMN binding rate of apoF44YP is similar to the rate observed for apo-F44YP in 100 mM potassium PPi, as both proteins are natively folded. The presence of the triple Strep tag, the Smt domain, the linker, and the SecM sequence of the construct used for RNC produc-
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