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The journey by which proteins navigate their energy landscapes to their native structures is complex, involving (and sometimes requiring) many cellular factors and processes operating in partnership with a given polypeptide chain’s intrinsic energy landscape. The cytosolic environment and its complement of chaperones play critical roles in granting many proteins safe passage to their native states; however, it is challenging to interrogate the folding process for large numbers of proteins in a complex background with most biophysical techniques. Hence, most chaperone-assisted protein refolding studies are conducted in defined buffers on single purified clients. Here, we develop a limited proteolysis–mass spectrometry approach paired with an isotope-labeling strategy to globally monitor the structures of refolding Escherichia coli proteins in the cytosolic medium and with the chaperones, GroEL/ES (Hsp60) and DnaK/DnaJ/GrpE (Hsp70/40). GroEL can refold the majority (85%) of the E. coli proteins for which we have data and is particularly important for restoring acidic proteins and proteins with high molecular weight, trends that come to light because our assay measures the structural outcome of the refolding process itself, rather than binding or aggregation. For the most part, DnaK and GroEL refold a similar set of proteins, supporting the view that despite their vastly different structures, these two chaperones unfold misfolded states, as one mechanism in common. Finally, we identify a cohort of proteins that are intransigent to being refolded with either chaperone. We suggest that these proteins may fold most efficiently cotranslationally, and then remain kinetically trapped in their native conformations.

Outer membrane proteins (OMPs) must exist as an unfolded ensemble while interacting with a chaperone network in the periplasm of Gram-negative bacteria. Here, we developed a method to model unfolded OMP (uOMP) conformational ensembles using experimental properties of two well-studied OMPs. The overall size and shape of the unfolded ensembles in water were experimentally defined by measuring the sedimentation coefficient as a function of urea concentration. We used these data to model a full range of unfolded conformations by parameterizing a targeted coarse-grained simulation protocol. The ensemble members were further refined by short molecular dynamics simulations to reflect proper torsion angles. The final conformational ensembles reveal inherent differences in the unfolded states that necessitate further investigation. Building these uOMP ensembles advances the understanding of OMP biogenesis and produces essential information for interpreting structures of uOMP-chaperone complexes.

The periplasmic chaperone network ensures the biogenesis of bacterial outer membrane proteins (OMPs) and has recently been identified as a promising target for antibiotics. SurA is the most important member of this network, both due to its genetic interaction with the β-barrel assembly machinery complex as well as its ability to prevent unfolded OMP (uOMP) aggregation. Using only binding energy, the mechanism by which SurA carries out these two functions is not well-understood. Here, we use a combination of photo-crosslinking, mass spectrometry, solution scattering, and molecular modeling techniques to elucidate the key structural features that define how SurA solubilizes uOMPs. Our experimental data support a model in which SurA binds uOMPs in a groove formed between the core and P1 domains. This binding event results in a drastic expansion of the rest of the uOMP, which has many biological implications. Using these experimental data as restraints, we adopted an integrative modeling approach to create a sparse ensemble of models of a SurA•uOMP complex. We validated key structural features of the SurA•uOMP ensemble using independent scattering and chemical crosslinking data. Our data suggest that SurA utilizes three distinct binding modes to interact with uOMPs and that more than one SurA can bind a uOMP at a time. This work demonstrates that SurA operates in a distinct fashion compared to other chaperones in the OMP biogenesis network.

The journey by which proteins navigate their energy landscapes to their native structures is complex, involving (and sometimes requiring) many cellular factors and processes operating in partnership with a given polypeptide chains intrinsic energy landscape. The cytosolic environment and its complement of chaperones play critical roles in granting proteins safe passage to their native states; however, the complexity of this medium has generally precluded biophysical techniques from interrogating protein folding under cellular-like conditions for single proteins, let alone entire proteomes. Here, we develop a limited-proteolysis mass spectrometry approach paired within an isotope-labeling strategy to globally monitor the structures of refolding E. coli proteins in the cytosolic medium and with the chaperones, GroEL/ES (Hsp60) and DnaK/DnaJ/GrpE (Hsp70/40). GroEL can refold the majority (85%) of the E. coli proteins for which we have data, and is particularly important for restoring acidic proteins and proteins with high molecular weight, trends that come to light because our assay measures the structural outcome of the refolding process itself, rather than indirect measures like binding or aggregation. For the most part, DnaK and GroEL refold a similar set of proteins, supporting the view that despite their vastly different structures, these two chaperones both unfold misfolded states, as one mechanism in common. Finally, we identify a cohort of proteins that are intransigent to being refolded with either chaperone. The data support a model in which chaperone-nonrefolders have evolved to fold efficiently once and only once, co-translationally, and remain kinetically trapped in their native conformations. HIGHLIGHTS- New proteomic methods are developed to probe protein refolding globally and sensitively in a complex background - The results revise the consensus model of which E. coli proteins require the GroEL chaperonin for efficient refolding - DnaK (Hsp70) and GroEL refold largely the same clientele, suggesting that these distinct chaperones share a common unifying mechanism - A small cohort of proteins cannot be fully restored by chaperones. These chaperone- nonrefolders tend to be involved in tRNA aminoacylation and glycolysis, and may have kinetically-trapped native states

The CuA center is the initial electron acceptor in cytochrome c oxidase, and it consists of two copper ions bridged by two cysteines and ligated by two histidines, a methionine, and a carbonyl in the peptide backbone of a nearby glutamine. The two ligating histidines are of particular interest as they may influence the electronic and redox properties of the metal center. To test for the presence of reactive ligating histidines, a portion of cytochrome c oxidase from the bacteria Thermus thermophilus that contains the CuA site (the TtCuA protein) was treated with the chemical modifier diethyl pyrocarbonate (DEPC) and the reaction followed through UV–visible, circular dichroism, and electron paramagnetic resonance spectroscopies at pH 5.0–9.0. A mutant protein (H40A/H117A) with the non-ligating histidines removed was similarly tested. Introduction of an electron-withdrawing DEPC-modification onto the ligating histidine 157 of TtCuA increased the reduction potential by over 70 mV, as assessed by cyclic voltammetry. Results from both proteins indicate that DEPC reacts with one of the two ligating histidines, modification of a ligating histidine raises the reduction potential of the CuA site, and formation of the DEPC adduct is reversible at room temperature. The existence of the reactive ligating histidine suggests that this residue may play a role in modulating the electronic and redox properties of TtCuA through kinetically-controlled proton exchange with the solvent. Lack of reactivity by the metalloproteins Sco and azurin, both of which contain a mononuclear copper center, indicate that reactivity toward DEPC is not a characteristic of all ligating histidines.

Chaperone proteins play a critical role in the biogenesis of many nascent polypeptides in vivo. In the periplasm of E. coli this role is partially fulfilled by SurA, which promotes the efficient assembly of unfolded outer membrane proteins (uOMPs) into the bacterial outer membrane, though the mechanism by which SurA interacts with uOMPs is not well understood. Here we identify multiple conformations of SurA in solution, one of which contains a cradle-like groove in which client uOMPs bind. Access to this binding groove by clients is gated by the intrinsic conformational dynamics of SurA. Crosslinking mass spectrometry experiments identify multiple regions of native client uOMPs that bind to SurA, providing insight into the molecular determinants of SurA-uOMP interactions. In contrast to other periplasmic chaperones that encapsulate uOMPs, small angle neutron scattering data demonstrate that SurA binding greatly expands client uOMPs. These data can explain the dual roles of SurA as both a holdase and a foldase. Using an integrative modeling approach that combines crosslinking, mass spectrometry, small angle neutron scattering, and simulation, we propose structural models of SurA in complex with an unfolded protein client. We further find that multiple SurA monomers are able to bind discrete sites on a single uOMP. The structural arrangement of SurA and uOMPs provides the basis for a possible mechanism by which SurA binds and expands clients in a manner that facilitates their folding into the outer membrane. Significance Statement Outer membrane proteins play critical roles in bacterial physiology and increasingly are being exploited as antibiotic targets. Their biogenesis requires chaperones in the bacterial periplasm to safely ferry them to their destination membrane. We used crosslinking, mass spectrometry, and small angle neutron scattering to propose an ensemble of structural models that explain how one chaperone, SurA, stabilizes client outer membrane proteins through expansion of their overall size, which positions them for delivery to the BAM complex. This study highlights the use of an hybrid integrative approach and emerging methods in structural biology to map highly heterogeneous structural ensembles like that of an unfolded protein bound to a chaperone.

The Cu(A) center is the initial electron acceptor in cytochrome c oxidase, and it consists of two copper ions bridged by two cysteines and ligated by two histidines, a methionine, and a carbonyl in the peptide backbone of a nearby glutamine. The two ligating histidines are of particular interest as they may influence the electronic and redox properties of the metal center. To test for the presence of reactive ligating histidines, a portion of cytochrome c oxidase from the bacteria Thermus thermophilus that contains the Cu(A) site (the TtCu(A) protein) was treated with the chemical modifier diethyl pyrocarbonate (DEPC) and the reaction followed through UV–visible, circular dichroism, and electron paramagnetic resonance spectroscopies at pH 5.0–9.0. A mutant protein (H40A/H117A) with the non-ligating histidines removed was similarly tested. Introduction of an electron-withdrawing DEPC-modification onto the ligating histidine 157 of TtCu(A) increased the reduction potential by over 70 mV, as assessed by cyclic voltammetry. Results from both proteins indicate that DEPC reacts with one of the two ligating histidines, modification of a ligating histidine raises the reduction potential of the Cu(A) site, and formation of the DEPC adduct is reversible at room temperature. The existence of the reactive ligating histidine suggests that this residue may play a role in modulating the electronic and redox properties of TtCu(A) through kinetically-controlled proton exchange with the solvent. Lack of reactivity by the metalloproteins Sco and azurin, both of which contain a mononuclear copper center, indicate that reactivity toward DEPC is not a characteristic of all ligating histidines.

A method for fitting sedimentation velocity experiments using whole boundary Lamm equation solutions is presented. The method, termed parametrically constrained spectrum analysis (PCSA), provides an optimized approach for simultaneously modeling heterogeneity in size and anisotropy of macromolecular mixtures. The solutions produced by PCSA are particularly useful for modeling polymerizing systems, where a single-valued relationship exists between the molar mass of the growing polymer chain and its corresponding anisotropy. The PCSA uses functional constraints to identify this relationship, and unlike other multidimensional grid methods, assures that only a single molar mass can be associated with a given anisotropy measurement. A description of the PCSA algorithm is presented, as well as several experimental and simulated examples that illustrate its utility and capabilities. The performance advantages of the PCSA method in comparison to other methods are documented. The method has been added to the UltraScan-III software suite, which is available for free download from http://www.ultrascan.uthscsa.edu.