To import large metabolites across the outer membrane of Gram-negative bacteria, TonB dependent transporters (TBDTs) undergo significant conformational change. After substrate binding in BtuB, the E. coli vitamin B12 TBDT, TonB binds and couples BtuB to the inner membrane proton motive force that powers transport (1). But, the role of TonB in rearranging the plug domain to form a putative pore remains enigmatic. Some studies focus on force-mediated unfolding (2) while others propose force-independent pore formation (3) by TonB binding leading to breakage of a salt bridge termed the "Ionic Lock". Our hydrogen exchange/mass spectrometry measurements in E. coli outer membranes find that the region surrounding the Ionic Lock, far from the B12 site, is fully destabilized upon substrate binding. A comparison of the exchange between the B12 bound and the B12&TonB bound complexes indicates that B12 binding is sufficient to unfold the Ionic Lock region with the subsequent binding of a TonB fragment having much weaker effects. TonB binding accelerates exchange in the third substrate binding loop, but pore formation does not obviously occur in this or any region. This study provides a detailed structural and energetic description of the early stages of B12 passage that provides support both for and against current models of the transport process.

The denaturant dependence of hydrogen−deuterium exchange (HDX) is a powerful measurement to identify the breaking of individual Hbonds and map the free energy surface (FES) of a protein including the very rare states. Molecular dynamics (MD) can identify each partial unfolding event with atomic-level resolution. Hence, their combination provides a great opportunity to test the accuracy of simulations and to verify the interpretation of HDX data. For this comparison, we use Upside, our new and extremely fast MD package that is capable of folding proteins with an accuracy comparable to that of all-atom methods. The FESs of two naturally occurring and two designed proteins are so generated and compared to our NMR/HDX data. We find that Upside's accuracy is considerably improved upon modifying the energy function using a new machine-learning procedure that trains for proper protein behavior including realistic denatured states in addition to stable native states. The resulting increase in cooperativity is critical for replicating the HDX data and protein stability, indicating that we have properly encoded the underlying physiochemical interactions into an MD package. We did observe some mismatch, however, underscoring the ongoing challenges faced by simulations in calculating accurate FESs. Nevertheless, our ensembles can identify the properties of the fluctuations that lead to HDX, whether they be small-, medium-, or large-scale openings, and can speak to the breadth of the native ensemble that has been a matter of debate.

Significance TonB - dependent transporters such as BtuB are found in the outer membranes of gram-negative bacteria. They import scarce nutrients essential for growth, such as B12, the substrate of BtuB. Many transport steps remain enigmatic. Recent studies have emphasized force-mediated unfolding or the breakage of the “Ionic Lock,” a moiety far from the B12 binding site. A strong dependence on the membrane environment has been noted. Accordingly, we measured hydrogen exchange on BtuB still embedded in native outer membranes and found that B12 binding is sufficient to break the Ionic Lock. The amino terminus then extends into the periplasm to bind TonB, but we find no evidence of pore formation, which likely requires energy transduction from the inner membrane by TonB.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful tool that monitors protein dynamics in solution. However, the reversible nature of HDX labels has largely limited the application to in vitro systems. Here, we describe a protocol for measuring HDX-MS in living Escherichia coli cells applied to BtuB, a TonB-dependent transporter found in outer membranes (OMs). BtuB is a convenient and biologically interesting system for testing in vivo HDX-MS due to its controllable HDX behavior and large structural rearrangements that occur during the B12 transport cycle. Our previous HDX-MS study in native OMs provided evidence for B12 binding and breaking of a salt bridge termed the Ionic Lock, an event that leads to the unfolding of the amino terminus. Although purified OMs provide a more native-like environment than reconstituted systems, disruption of the cell envelope during lysis perturbs the linkage between BtuB and the TonB complex that drives B12 transport. The in vivo HDX response of BtuB's plug domain (BtuBp) to B12 binding corroborates our previous in vitro findings that B12 alone is sufficient to break the Ionic Lock. In addition, we still find no evidence of B12 bindinginduced unfolding in other regions of BtuBp that could enable B12 passage. Our protocol was successful in reporting on the HDX of several endogenous E. coli proteins measured in the same measurement. Our success in performing HDX in live cells opens the possibility for future HDX-MS studies in a native cellular environment. IMPORTANCEWe present a protocol for performing in vivo HDX-MS, focusing on BtuB, a protein whose native membrane environment is believed to be mechanistically

The relationship between folding cooperativity and downhill, or barrier-free, folding of proteins under highly stabilizing conditions remains an unresolved topic, especially for proteins such as λ-repressor that fold on the microsecond timescale. Under aqueous conditions where downhill folding is most likely to occur, we measure the stability of multiple H bonds, using hydrogen exchange (HX) in a λ YA variant that is suggested to be an incipient downhill folder having an extrapolated folding rate constant of 2 × 10 5 s −1 and a stability of 7.4 kcal·mol −1 at 298 K. At least one H bond on each of the three largest helices (α1, α3, and α4) breaks during a common unfolding event that reflects global denaturation. The use of HX enables us to both examine folding under highly stabilizing, native-like conditions and probe the pretransition state region for stable species without the need to initiate the folding reaction. The equivalence of the stability determined at zero and high denaturant indicates that any residual denatured state structure minimally affects the stability even under native conditions. Using our ψ analysis method along with mutational ϕ analysis, we find that the three aforementioned helices are all present in the folding transition state. Hence, the free energy surface has a sufficiently high barrier separating the denatured and native states that folding appears cooperative even under extremely stable and fast folding conditions. T he nature of the energy landscape when folding occurs on the microsecond timescale continues to be investigated using both experimental and computational approaches. The 80-residue subdomain of the λ-repressor transcription factor, λ 6-85 (1-11), is an appealing system as it contains five α-helices arranged in a nonsymmetric pattern, folds in microseconds, and is nearly a downhill folder (12), a condition where a continuous series of folding events may be characterized. These characteristics along with a folding time that nears the upper limit for current all-atom simulations (13-19) make this protein appropriate for detailed comparisons between experiment and simulations.The energy landscape of λ 6-85 is significantly influenced by its sequence. Oas and coworkers found that the wild type and a fasterfolding mutant with two helix-promoting substitutions (λ AA with G46A/G48A, Fig. S1A) fold cooperatively (1-4). The transition state ensemble (TSE) characterized using ϕ analysis minimally contains α1 and α4 (3). Kinetic H/D isotope effect measurements in the Sosnick laboratory found that both λ 6-85 and λ AA fold cooperatively with ∼70% of the H bonds being formed in the TSE, matching the degree of surface buried in the TSE [beta Tanford value (β Tanford )] (5).Using nanosecond T-jump and other methods, Gruebele and coworkers proposed that faster-folding and stabilized variants, such as λ YA (λ AA + Q33Y) and λ D14A (λ YA + D14A), fold in an incipient or fully downhill manner, especially under highly stable conditions at lower temperatures (6-9). Key signatures of downhi...