SUMMARY Secretion systems require high fidelity mechanisms to discriminate substrates amongst the vast cytoplasmic pool of proteins. Factors mediating substrate recognition by the type VI secretion system (T6SS) of Gram-negative bacteria, a widespread pathway that translocates effector proteins into target bacterial cells, have not been defined. We report that haemolysin co-regulated protein (Hcp), a ring-shaped hexamer secreted by all characterized T6SSs, binds specifically to cognate effector molecules. Electron microscopy analysis of an Hcp–effector complex from Pseudomonas aeruginosa revealed the effector bound to the inner surface of Hcp. Further studies demonstrated that interaction with the Hcp pore is a general requirement for secretion of diverse effectors encompassing several enzymatic classes. Though previous models depict Hcp as a static conduit, our data indicate it is a chaperone and receptor of substrates. These unique functions of a secreted protein highlight fundamental differences between the export mechanism of T6 and other characterized secretory pathways.

Many GFP variants have been developed for use as fluorescent tags, and recently a superfolder GFP (sfGFP) has been developed as a robust folding reporter. This new variant shows increased stability and improved folding kinetics, as well as 100% recovery of native protein after denaturation. Here, we characterize sfGFP, and find that this variant exhibits hysteresis as unfolding and refolding equilibrium titration curves are non-coincident even after equilibration for more than eight half-lives as estimated from kinetic unfolding and refolding studies. This hysteresis is attributed to trapping in a native-like intermediate state. Mutational studies directed towards inhibiting chromophore formation indicate that the novel backbone cyclization is responsible for the hysteresis observed in equilibrium titrations of sfGFP. Slow equilibration and the presence of intermediates imply a rough landscape. However, de novo folding in the absence of the chromophore is dominated by a smoother energy landscape than that sampled during unfolding and refolding of the post-translationally modified polypeptide.

Recent experimental studies suggest that the mature GFP has an unconventional landscape composed of an early folding event with a typical funneled landscape, followed by a very slow search and rearrangement step into the locked, active chromophore-containing structure. As we have shown previously, the substantial difference in time scales is what generates the observed hysteresis in thermodynamic folding. The interconversion between locked and the soft folding structures at intermediate denaturant concentrations is so slow that it is not observed under the typical experimental observation time. Simulations of a coarse-grained model were used to describe the fast folding event as well as identify native-like intermediates on energy landscapes enroute to the fluorescent native fold. Interestingly, these simulations reveal structural features of the slow dynamic transition to chromophore activation. Experimental evidence presented here shows that the trapped, native-like intermediate has structural heterogeneity in residues previously linked to chromophore formation. We propose that the final step of GFP folding is a ''locking'' mechanism leading to chromophore formation and high stability. The combination of previous experimental work and current simulation work is explained in the context of a dual-basin folding mechanism described above. molecular dynamics ͉ multicanonical method ͉ protein folding ͉ topological frustration ͉ folding funnel G FP has become a common reagent in biochemistry laboratories because of its ability to fold and form a visually fluorescent chromophore through autocatalytic cyclization and dehydration/oxidation reactions (1). Recent studies on the folding of GFP have shown multiple kinetic phases (2, 3) and high barriers to folding that lead to a slow ''drift'' of the unfolding equilibrium transition curve (4). In fact, a ''superfolder'' variant of GFP (sfGFP) (5) has an apparent hysteresis in equilibrium folding that is observed in the experiment (6, 7) shown in Fig. 1A. Considering the apparent nonthermodynamic behavior observed in experiment, can we use simulation to probe the free-energy landscape and explain these results?Proteins whose folding has been optimized by evolution have a sufficiently smooth, funneled energy landscape, with an ensemble of folding routes directed toward the native structural basin (8-10). Coarse-grained models with potentials designed to bias the protein toward the native basin, structure-based (Go ) models (11), have been able to capture the main structural features of the folding mechanism observed in experiments. Because these models are energetically unfrustrated, the agreement to experiments demonstrate that typical well folding proteins have such small amounts of residual energetic frustration that the observed structural heterogeneity observed in folding is mostly determined by geometrical properties of the native structure.Although it is a good folder, GFP has a folding landscape that differs from most proteins because of a structural rearrangement ne...

Topologically complex proteins fold by multiple routes as a result of hard-to-fold regions of the proteins. Oftentimes these regions are introduced into the protein scaffold for function and increase frustration in the otherwise smooth-funneled landscape. Interestingly, while functional regions add complexity to folding landscapes, they may also contribute to a unique behavior referred to as hysteresis. While hysteresis is predicted to be rare, it is observed in various proteins, including proteins containing a unique peptide cyclization to form a fluorescent chromophore as well as proteins containing a knotted topology in their native fold. Here, hysteresis is demonstrated to be a consequence of the decoupling of unfolding events from the isomerization or hula-twist of a chromophore in one protein and the untying of the knot in a second protein system. The question now is- can hysteresis be a marker for the interplay of landscapes where complex folding and functional regions overlap?

Green fluorescent protein (GFP) possesses a unique folding landscape with a dual basin, leading to the hysteretic folding behavior observed in experiment. While theoretical data do not have the resolution necessary to observe details of the chromophore during refolding, experimental results point to the chromophore as the cause of the observed hysteresis. Using NMR spectroscopy, which probes at the level of the individual residue, the hysteretic intermediate state is further characterized in the context of the loosely-folded native-like state {Niso} predicted in simulation. In the present study, several residues located in the lid of GFP indicate heterogeneity of the native states. Some of these residues show chemical shifts when the native-like intermediate {Niso} responsible for GFP's hysteretic folding behavior is trapped. Observed changes in the chromophore are consistent with increased flexibility or isomerization in {Niso} as predicted in recent theoretical work. Here we observe multiple chromophore environments within the native state are averaged in the trapped intermediate, linking chromophore flexibility to mispacking in the trapped intermediate. The present work is experimental evidence for the proposed final “locking” mechanism in GFP folding forming an incorrectly or loosely packed barrel under intermediate (hysteretic) folding conditions.