Fried, S. D. — Page 2 | Search bioRxiv

bioRxiv2020

Non-Refoldability is Pervasive Across the E. coli Proteome

The foundational paradigm of protein folding is that the primary sequence of a protein contains all the information needed for it to adopt a specific native structure1,2. This remarkable property is explained by Anfinsens thermodynamic hypothesis3,4, which asserts that because native states minimize the Gibbs free energy of a protein molecule under physiological conditions, proteins can reliably navigate to their native structures and remain in those states by ergodically sampling their free energy landscapes5. Most experiments of protein folding - conducted on purified, small, single-domain soluble proteins - follow the proportion of protein molecules that are folded as a function of time, temperature, denaturant concentration, or sequence6-8, and have yielded immense insight into the molecular determinants that underpin stable globular folds9,10. However, our reliance on the thermodynamic hypothesis as a ground truth to interpret these experiments has limited our ability to study the folding of complex proteins or to consider alternative non-thermodynamic scenarios11,12. Here, we introduce an experimental approach to probe protein refolding for whole proteomes. We accomplish this by first unfolding and refolding E. coli lysates, and then interrogating the resulting protein structures using a permissive protease that preferentially cleaves at flexible regions. Using mass spectrometry, we analyze the digestion patterns to globally assess structural differences between native and refolded proteins. These studies reveal that following denaturation, many proteins are incapable of navigating back to their native structures. Our results signal a pervasive role for co-translational folding in shaping protein biogenesis, and suggest that the apparent stability of many native states derive from kinetic persistence rather than thermodynamic stability.

biophysics↗

bioRxiv2019

SurA is a "Groove-y" Chaperone That Expands Unfolded Outer Membrane Proteins

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 {beta}-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. Significance StatementOuter membrane proteins play critical roles in bacterial physiology and increasingly are exploited as antibiotic targets. SurA is the most important chaperone in the OMP biogenesis network and is thought to initiate their folding through an interaction with the BAM complex. We observe an unprecedented expansion of unfolded outer membrane proteins when bound to SurA. This expansion suggests a potential mechanism by which SurA can deliver uOMPs to the BAM complex. In addition, this study highlights the use of an integrative/hybrid structural biology approach and emerging methods to map highly heterogeneous structural ensembles such as that of an unfolded protein bound to a chaperone.

biophysics↗