Hydrodynamic properties are useful parameters for estimating the size and shape of proteins and nucleic acids in solution. The calculation of such properties from structural models informs on the ...solution properties of these molecules and complements corresponding structural studies. Here we report, to our knowledge, a new method to accurately predict the hydrodynamic properties of molecular structures. This method uses a convex hull model to estimate the hydrodynamic volume of the molecule and is orders of magnitude faster than common methods. It works well for both folded proteins and ensembles of conformationally heterogeneous proteins and for nucleic acids. Because of its simplicity and speed, the method should be useful for the modification of computer-generated, intrinsically disordered protein ensembles and ensembles of flexible, but folded, molecules in which rapid calculation of experimental parameters is needed. The convex hull method is implemented in a Python script called HullRad. The use of the method is facilitated by a web server and the code is freely available for batch applications.
Protein design is a powerful tool for elucidating mechanisms of function and engineering new therapeutics and nanotechnologies. Although soluble protein design has advanced, membrane protein design ...remains challenging because of difficulties in modeling the lipid bilayer. In this work, we developed an implicit approach that captures the anisotropic structure, shape of water-filled pores, and nanoscale dimensions of membranes with different lipid compositions. The model improves performance in computational benchmarks against experimental targets, including prediction of protein orientations in the bilayer, ΔΔG calculations, native structure discrimination, and native sequence recovery. When applied to de novo protein design, this approach designs sequences with an amino acid distribution near the native amino acid distribution in membrane proteins, overcoming a critical flaw in previous membrane models that were prone to generating leucine-rich designs. Furthermore, the proteins designed in the new membrane model exhibit native-like features including interfacial aromatic side chains, hydrophobic lengths compatible with bilayer thickness, and polar pores. Our method advances high-resolution membrane protein structure prediction and design toward tackling key biological questions and engineering challenges.
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 outer membrane of Gram-negative bacteria is a unique asymmetric lipid bilayer composed of phospholipids (PLs) in the inner leaflet and lipopolysaccharides (LPSs) in the outer leaflet. Its ...function as a selective barrier is crucial for the survival of bacteria in many distinct environments, and it also renders Gram-negative bacteria more resistant to antibiotics than their Gram-positive counterparts. Here, we report the structural properties of a model of the Escherichia coli outer membrane and its interaction with outer membrane phospholipase A (OmpLA) utilizing molecular dynamics simulations. Our results reveal that given the lipid composition used here, the hydrophobic thickness of the outer membrane is ∼3 Å thinner than the corresponding PL bilayer, mainly because of the thinner LPS leaflet. Further thinning in the vicinity of OmpLA is observed due to hydrophobic matching. The particular shape of the OmpLA barrel induces various interactions between LPS and PL leaflets, resulting in asymmetric thinning around the protein. The interaction between OmpLA extracellular loops and LPS (headgroups and core oligosaccharides) stabilizes the loop conformation with reduced dynamics, which leads to secondary structure variation and loop displacement compared to that in a DLPC bilayer. In addition, we demonstrate that the LPS/PL ratios in asymmetric bilayers can be reliably estimated by the per-lipid surface area of each lipid type, and there is no statistical difference in the overall membrane structure for the outer membranes with one more or less LPS in the outer leaflet, although individual lipid properties vary slightly.
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 addition, 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.
Thermodynamic stabilities are pivotal for understanding structure–function relationships of proteins, and yet such determinations are rare for membrane proteins. Moreover, the few measurements that ...are available have been conducted under very different experimental conditions, which compromises a straightforward extraction of physical principles underlying stability differences. Here, we have overcome this obstacle and provided structure–stability comparisons for multiple membrane proteins. This was enabled by measurements of the free energies of folding and the m values for the transmembrane proteins PhoP/PhoQ-activated gene product (PagP) and outer membrane protein W (OmpW) from Escherichia coli . Our data were collected in the same lipid bilayer and buffer system we previously used to determine those parameters for E. coli outer membrane phospholipase A (OmpLA). Biophysically, our results suggest that the stabilities of these proteins are strongly correlated to the water-to-bilayer transfer free energy of the lipid-facing residues in their transmembrane regions. We further discovered that the sensitivities of these membrane proteins to chemical denaturation, as judged by their m values, was consistent with that previously observed for water-soluble proteins having comparable differences in solvent exposure between their folded and unfolded states. From a biological perspective, our findings suggest that the folding free energies for these membrane proteins may be the thermodynamic sink that establishes an energy gradient across the periplasm, thus driving their sorting by chaperones to the outer membranes in living bacteria. Binding free energies of these outer membrane proteins with periplasmic chaperones support this energy sink hypothesis.
The outer membrane of Gram-negative bacteria is an asymmetric membrane with lipopolysaccharides on the external leaflet and phospholipids on the periplasmic leaflet. This outer membrane contains ...mainly β-barrel transmembrane proteins and lipidated periplasmic proteins (lipoproteins). The multisubunit protein β-barrel assembly machine (BAM) catalyzes the insertion and folding of the β-barrel proteins into this membrane. In Escherichia coli, the BAM complex consists of five subunits, a core transmembrane β-barrel with a long periplasmic domain (BamA) and four lipoproteins (BamB/C/D/E). The BamA periplasmic domain is composed of five globular subdomains in tandem called POTRA motifs that are key to BAM complex formation and interaction with the substrate β-barrel proteins. The BAM complex is believed to undergo conformational cycling while facilitating insertion of client proteins into the outer membrane. Reports describing variable conformations and dynamics of the periplasmic POTRA domain have been published. Therefore, elucidation of the conformational dynamics of the POTRA domain in full-length BamA is important to understand the function of this molecular complex. Using molecular dynamics simulations, we present evidence that the conformational flexibility of the POTRA domain is modulated by binding to the periplasmic surface of a native lipid membrane. Furthermore, membrane binding of the POTRA domain is compatible with both BamB and BamD binding, suggesting that conformational selection of different POTRA domain conformations may be involved in the mechanism of BAM-facilitated insertion of outer membrane β-barrel proteins.
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.
Outer membrane protein (OMP) biogenesis is critical to bacterial physiology because the cellular envelope is vital to bacterial pathogenesis and antibiotic resistance. The process of OMP biogenesis ...has been studied in vivo, and each of its components has been studied in isolation in vitro. This work integrates parameters and observations from both in vivo and in vitro experiments into a holistic computational model termed “Outer Membrane Protein Biogenesis Model” (OMPBioM). We use OMPBioM to assess OMP biogenesis mathematically in a global manner. Using deterministic and stochastic methods, we are able to simulate OMP biogenesis under varying genetic conditions, each of which successfully replicates experimental observations. We observe that OMPs have a prolonged lifetime in the periplasm where an unfolded OMP makes, on average, hundreds of short-lived interactions with chaperones before folding into its native state. We find that some periplasmic chaperones function primarily as quality-control factors; this function complements the folding catalysis function of other chaperones. Additionally, the effective rate for the β-barrel assembly machinery complex necessary for physiological folding was found to be higher than has currently been observed in vitro. Overall, we find a finely tuned balance between thermodynamic and kinetic parameters maximizes OMP folding flux and minimizes aggregation and unnecessary degradation. In sum, OMPBioM provides a global view of OMP biogenesis that yields unique insights into this essential pathway.
A Backbone-Based Theory of Protein Folding Rose, George D.; Fleming, Patrick J.; Banavar, Jayanth R. ...
Proceedings of the National Academy of Sciences - PNAS,
11/2006, Volume:
103, Issue:
45
Journal Article
Peer reviewed
Open access
Under physiological conditions, a protein undergoes a spontaneous disorder$\rightlefthapoon$order transition called "folding." The protein polymer is highly flexible when unfolded but adopts its ...unique native, three-dimensional structure when folded. Current experimental knowledge comes primarily from thermodynamic measurements in solution or the structures of individual molecules, elucidated by either x-ray crystallography or NMR spectroscopy. From the former, we know the enthalpy, entropy, and free energy differences between the folded and unfolded forms of hundreds of proteins under a variety of solvent/cosolvent conditions. From the latter, we know the structures of ≈35,000 proteins, which are built on scaffolds of hydrogen-bonded structural elements, α-helix and β-sheet. Anfinsen showed that the amino acid sequence alone is sufficient to determine a protein's structure, but the molecular mechanism responsible for self-assembly remains an open question, probably the most fundamental open question in biochemistry. This perspective is a hybrid: partly review, partly proposal. First, we summarize key ideas regarding protein folding developed over the past half-century and culminating in the current mindset. In this view, the energetics of side-chain interactions dominate the folding process, driving the chain to self-organize under folding conditions. Next, having taken stock, we propose an alternative model that inverts the prevailing side-chain/backbone paradigm. Here, the energetics of backbone hydrogen bonds dominate the folding process, with preorganization in the unfolded state. Then, under folding conditions, the resultant fold is selected from a limited repertoire of structural possibilities, each corresponding to a distinct hydrogen-bonded arrangement of α-helices and/or strands of β-sheet.
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