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Abstract Ultrafast folding proteins have become an important paradigm in the study of protein folding dynamics. Due to their low energetic barriers and fast kinetics, they are amenable for study by both experiment and simulation. However, single molecule force spectroscopy experiments on these systems are challenging as these proteins do not provide the mechanical fingerprints characteristic of more mechanically stable proteins, which makes it difficult to extract information about the folding dynamics of the molecule. Here, we investigate the unfolding of the ultrafast protein Engrailed Homeodomain (EnHD) by single-molecule atomic force microscopy experiments. Constant speed experiments on EnHD result in featureless transitions typical of compliant proteins. However, in the force-ramp mode we recover sigmoidal curves that we interpret as a very compliant protein that folds and unfolds many times over a marginal barrier. This is supported by a simple theoretical model and coarse-grained molecular simulations. Our results show the ability of force to modulate the unfolding dynamics of ultrafast folding proteins. 

Intrinsically disordered proteins (IDPs) fold upon binding to select/recruit multiple partners, morph around the partner's structure, and exhibit allostery. However, we do not know whether these properties emerge passively from disorder, or rather are encoded into the IDP's folding mechanisms. A main reason for this gap is the lack of suitable methods to dissect the energetics of IDP conformational landscapes without partners. Here we introduce such an approach that we term molecular LEGO, and apply it to NCBD, a helical, molten globule–like IDP, as proof of concept. The approach entails the experimental and computational characterization of the protein, its separate secondary structure elements (LEGO building blocks), and their supersecondary combinations. Comparative analysis uncovers specific, yet inconspicuous, energetic biases in the conformational/folding landscape of NCBD, including 1) strong local signals that define the three native helices, 2) stabilization of helix–helix interfaces via soft pairwise tertiary interactions, 3) cooperative stabilization of a heterogeneous three-helix bundle fold, and 4) a dynamic exchange between sets of tertiary interactions (native and nonnative) that recapitulate the different structures NCBD adopts in complex with various partners. Crucially, a tug of war between sets of interactions makes NCBD gradually shift between structural subensembles as a conformational rheostat. Such conformational rheostatic behavior provides a built-in mechanism to modulate binding and switch/recruit partners that is likely at the core of NCBD's function as transcriptional coactivator. Hence, the molecular LEGO approach emerges as a powerful tool to dissect the conformational landscapes of unbound IDPs and rationalize their functional mechanisms. 

“I can’t believe another year has passed already” is what most of us think when another birthday is upon us or when we see our children grow [...] 

Transcription factors must scan genomic DNA, recognize the cognate sequence of their control element(s), and bind tightly to them. The DNA recognition process is primarily carried out by their DNA binding domains (DBD), which interact with the cognate site with high affinity and more weakly with any other DNA sequence. DBDs are generally thought to bind to their cognate DNA without changing conformation (lock-and-key). Here, we used nuclear magnetic resonance and circular dichroism to investigate the interplay between DNA recognition and DBD conformation in the engrailed homeodomain (enHD), as a model case for the homeodomain family of eukaryotic DBDs. We found that the conformational ensemble of enHD is rather flexible and becomes gradually more disordered as ionic strength decreases following a Debye–Hückel’s dependence. Our analysis indicates that enHD’s response to ionic strength is mediated by a built-in electrostatic spring-loaded latch that operates as a conformational transducer. We also found that, at moderate ionic strengths, enHD changes conformation upon binding to cognate DNA. This change is of larger amplitude and somewhat orthogonal to the response to ionic strength. As a consequence, very high ionic strengths (e.g., 700 mM) block the electrostatic-spring-loaded latch and binding to cognate DNA becomes lock-and-key. However, the interplay between enHD conformation and cognate DNA binding is robust across a range of ionic strengths (i.e., 45 to 300 mM) that covers the physiologically-relevant conditions. Therefore, our results demonstrate the presence of a mechanism for the conformational control of cognate DNA recognition on a eukaryotic DBD. This mechanism can function as a signal transducer that locks the DBD in place upon encountering the cognate site during active DNA scanning. The electrostatic-spring-loaded latch of enHD can also enable the fine control of DNA recognition in response to transient changes in local ionic strength induced by variate physiological processes.