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The survival and virulence of Gram-negative bacteria require proper biogenesis and maintenance of the outer membrane (OM), which is densely packed with à-barrel OM proteins (OMPs). Before reaching the OM, precursor unfolded OMPs (uOMPs) must cross the whole cell envelope. A network of periplasmic chaperones and proteases maintains unfolded but folding-competent conformations of these membrane proteins in the aqueous periplasm while simultaneously preventing off-pathway aggregation. These periplasmic proteins utilize different strategies, including conformational heterogeneity, oligomerization, multivalency, and ki- netic partitioning, to perform and regulate their functions. Redundant and unique characteristics of the individual periplasmic players synergize to create a protein quality control team capable responding to changing environmental stresses. 

Polyethylene glycol (PEG) conjugation provides a protective modification that enhances the pharmacokinetics and solubility of proteins for therapeutic use. A knowledge of the structural ensemble of these PEGylated proteins is necessary to understand the molecular details that contribute to their hydrodynamic and colligative properties. Because of the large size and dynamic flexibility of pharmaceutically important PEGylated proteins, the determination of structure is challenging. In addi- tion, the hydration of these conjugates that contain large polymers is difficult to determine with traditional methods that identify only first shell hydration water, which does not account for the complete hydrodynamic volume of a macromolecule. Here, we demonstrate that structural ensembles, generated by coarse-grained simulations, can be analyzed with HullRad and used to predict sedimentation coefficients and concentration-dependent hydrodynamic and diffusion nonideality coefficients of PEGylated proteins. A knowledge of these concentration-dependent properties enhances the ability to design and analyze new modified protein therapeutics. HullRad accomplishes this analysis by effectively accounting for the complete hydration of a macromolecule, including that of flexible polymers. 

Perspective article commenting on another paper published in PNAS 

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 the experimental properties of two well-studied OMPs. The overall sizes and shapes of the unfolded ensembles in the absence of a denaturant 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 have polymer properties different from unfolded soluble and intrinsically disordered proteins and reveal inherent differences in the unfolded states that necessitate further investigation. Building these uOMP ensembles advances the understanding of OMP biogenesis and provides essential information for interpreting structures of uOMP-chaperone complexes. 

Hydration of biological macromolecules is important for their stability and function. Historically, attempts have been made to describe the degree of macromolecular hydration using a single parameter over a narrow range of values. Here, we describe a method to calculate two types of hydration: surface shell water and entrained water. A consideration of these two types of hydration helps to explain the “hydration problem” in hydrodynamics. The combination of these two types of hydration allows accurate calculation of hydrodynamic volume and related macromolecular properties such as sedimentation and diffusion coefficients, intrinsic viscosities, and the concentration-dependent non-ideality identified with sedimentation velocity experiments. 

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.