We have used native mass spectrometry to analyze macromolecular complexes involved in the chaperonin-assisted refolding of gp23, the major capsid protein of bacteriophage T4. Adapting the instrumental methods allowed us to monitor all intermediate complexes involved in the chaperonin folding cycle. We found that GroEL can bind up to two unfolded gp23 substrate molecules. Notably, when GroEL is in complex with the cochaperonin gp31, it binds exclusively one gp23. We also demonstrated that the folding and assembly of gp23 into 336-kDa hexamers by GroEL-gp31 can be monitored directly by electrospray ionization mass spectrometry (ESI-MS). These data reinforce the great potential of ESI-MS as a technique to investigate structure-function relationships of protein assemblies in general and the chaperonin-protein folding machinery in particular. A major advantage of native mass spectrometry is that, given sufficient resolution, it allows the analysis at the picomole level of sensitivity of heterogeneous protein complexes with molecular masses up to several million daltons.

Secretion of the Escherichia coli toxin hemolysin A (HlyA) is catalyzed by the membrane protein complex HlyB-HlyD-TolC and requires a secretion sequence located within the last 60 amino acids of HlyA. The Hly translocator complex exports a variety of passenger proteins when fused N-terminal to this secretion sequence. However, not all fusions are secreted efficiently. Here, we demonstrate that the maltose binding protein (MalE) lacking its natural export signal and fused to the HlyA secretion signal is poorly secreted by the Hly system. We anticipated that folding kinetics might be limiting secretion, and we therefore introduced the "folding" mutation Y283D. Indeed this mutant fusion protein was secreted at a much higher level. This level was further enhanced by the introduction of a second MalE folding mutation (V8G or A276G). Secretion did not require the molecular chaperone SecB. Folding analysis revealed that all mutations reduced the refolding rate of the substrate, whereas the unfolding rate was unaffected. Thus, the efficiency of secretion by the Hly system is dictated by the folding rate of the substrate. Moreover, we demonstrate that fusion proteins defective in export can be engineered for secretion while still retaining function.

Nisin is a posttranslationally modified antimicrobial peptide containing the cyclic thioether amino acids lanthionine and methyllanthionine. Although much is known about its antimicrobial activity and mode of action, knowledge about the nisin modification process is still rather limited. The dehydratase NisB is believed to be the initial interaction partner in modification. NisB dehydrates specific serine and threonine residues in prenisin, whereas the cyclase NisC catalyzes the (methyl)lanthionine formation. The fully modified prenisin is exported and the leader peptide is cleaved off by the extracellular protease NisP. Light scattering analysis demonstrated that purified NisB is a dimer in solution. Using size exclusion chromatography and surface plasmon resonance, the interaction of NisB and prenisin, including several of its modified derivatives, was studied. Unmodified prenisin binds to NisB with an affinity of 1.05 ؎ 0.25 M, whereas the dehydrated and the fully modified derivatives bind with respective affinities of 0.31 ؎ 0.07 and 10.5 ؎ 1.7 M. The much lower affinity for the fully modified prenisin was related to a >20-fold higher off-rate. For all three peptides the stoichiometry of binding was 1:1. Active nisin, which is the equivalent of fully modified prenisin lacking the leader peptide did not bind to NisB, nor did prenisin in which the highly conserved FNLD box within the leader peptide was mutated to AAAA. Taken together our data indicate that the leader peptide is essential for initial recognition and binding of prenisin to NisB.

It has been suggested that the bacterial GroEL chaperonin accommodates only one substrate at any given time, due to conformational changes to both the cis and trans ring that are induced upon substrate binding. Using electrospray ionization mass spectrometry, we show that indeed GroEL binds only one molecule of the model substrate Rubisco. In contrast, the capsid protein of bacteriophage T4, a natural GroEL substrate, can occupy both rings simultaneously. As these substrates are of similar size, the data indicate that each substrate induces distinct conformational changes in the GroEL chaperonin. The distinctive binding behavior of Rubisco and the capsid protein was further investigated using tandem mass spectrometry on the intact 800-914 kDa GroEL-substrate complexes. Our data suggest that even in the gas phase the substrates remain bound inside the GroEL cavity. The analysis revealed further that binding of Rubisco to the GroEL oligomer stabilizes the chaperonin complex significantly, whereas binding of one capsid protein did not have the same effect. However, addition of a second capsid protein molecule to GroEL resulted in a similar stabilizing effect to that obtained after the binding of a single Rubisco. On the basis of the stoichiometry of the GroEL chaperonin-substrate complex and the dissociation behavior of the two different substrates, we hypothesize that the binding of a single capsid polypeptide does not induce significant conformational changes in the GroEL trans ring, and hence the unoccupied GroEL ring remains accessible for a second capsid molecule.

A subset of essential cellular proteins requires the assistance of chaperonins (in E. coli, GroEL and GroES), double-ring complexes in which the two rings act alternately to bind, encapsulate and fold nascent or stress-denatured proteins1,2,3,4,5. This process starts by the trapping of a substrate protein on hydrophobic surfaces in the central cavity of a GroEL ring6,7,8,9,10. Then, binding of ATP and co-chaperonin GroES to that ring ejects the non-native protein from its binding sites, through forced unfolding or other major conformational changes, and encloses it in a hydrophilic chamber for folding11,12,13,14. ATP hydrolysis and subsequent ATP binding to the opposite ring trigger dissociation of the chamber and release of the substrate protein15,2. The bacteriophage T4 requires its own version of GroES, gp31, that forms a taller folding chamber, to fold the major viral capsid protein gp2316, 17,18,19,20. Polypeptides are known to fold inside the chaperonin complex, but the conformation of an encapsulated protein has not previously been visualized. Here we present structures of gp23-chaperonin complexes, showing both the initial captured state and the final, close-to-native state with gp23 encapsulated in the folding chamber. Although the chamber is expanded, it is still barely large enough to contain the elongated gp23 monomer, explaining why the GroEL-GroES complex is not able to fold gp23 and showing how the chaperonin structure distorts to enclose a large, physiological substrate protein.Chaperonin-substrate (binary) complexes were formed by rapidly mixing urea-denatured gp23 with GroEL in 2.5-fold molar excess over GroEL oligomer. Ternary complexes were generated by adding gp31 (bacteriophage T4 GroES homologue) and the ATP transition state analogue ADP•AlF 3 . Cryo electron microscopy (EM) data sets of 30-35,000 particles were collected of both preparations and initial 3D maps were obtained by treating each data set as a single structure with 7-fold symmetry. The resulting maps showed GroEL and GroEL-gp31 complexes with some additional densities in the binding cavities (Supplementary figure 1). As in our earlier study on malate dehydrogenase (MDH) folding, we expected the non-native substrate to form heterogeneous and asymmetric complexes with the chaperonins. Therefore we used a combination of multivariate statistical analysis (MSA) The binary complexes were resolved into 5 classes (Supplementary Figure 2 and Supplementary Table 1). In agreement with our mass spectrometry results22, we observed empty GroEL (20% of the images) and GroEL with extra density in either one (40%) or both rings (40%). The classes displaying the largest fraction of the substrate density are shown in Figure 1, with the GroEL subunit domains fitted into the maps as separate rigid bodies. The GroEL rings deviate little from 7-fold symmetry ( Figure 1c, d, g, h and rotational correlation analysis, not shown). This is unlike GroEL-MDH complexes in which the GroEL apical domains are bunched together on the side of the ring w...