Organic Ligand Binding by a Hydrophobic Cavity in a Designed Tetrameric Coiled‐Coil Protein

The design and characterization of a hydrophobic cavity in de novo designed proteins provides a wide range of information about the functions of de novo proteins. We designed a de novo tetrameric coiled‐coil protein with a hydrophobic pocketlike cavity. Tetrameric coiled coils with hydrophobic cavities have previously been reported. By replacing one Leu residue at the a position with Ala, hydrophobic cavities that did not flatten out due to loose peptide chains were reliably created. To perform a detailed examination of the ligand‐binding characteristics of the cavities, we originally designed two other coiled‐coil proteins: AM2, with eight Ala substitutions at the adjacent a and d positions at the center of a bundled structure, and AM2W, with one Trp and seven Ala substitutions at the same positions. To increase the association of the helical peptides, each helical peptide was connected with flexible linkers, which resulted in a single peptide chain. These proteins exhibited CD spectra corresponding to superhelical structures, despite weakened hydrophobic packing. AM2W exhibited binding affinity for size‐complementary organic compounds. The dissociation constants, K_d, of AM2W were 220 nM for adamantane, 81 μM for 1‐adamantanol, and 294 μM for 1‐adamantaneacetic acid, as measured by fluorescence titration analyses. Although it was contrary to expectations, AM2 did not exhibit any binding affinity, probably due to structural defects around the designed hydrophobic cavity. Interestingly, AM2W exhibited incremental structure stability through ligand binding. Plugging of structural defects with organic ligands would be expected to facilitate protein folding.     

Cellular Synthesis and X-ray Crystal Structure of a Designed Protein Heterocatenane

Herein, we report the biosynthesis of protein heterocatenanes using a programmed sequence of multiple post-translational processing events including intramolecular chain entanglement, in situ backbone cleavage, and spontaneous cyclization. The approach is general, autonomous, and can obviate the need for any additional enzymes. The catenane topology was convincingly proven using a combination of SDS-PAGE, LC-MS, size exclusion chromatography, controlled proteolytic digestion, and protein crystallography. The X-ray crystal structure clearly shows two mechanically interlocked protein rings with intact folded domains. It opens new avenues in the nascent field of protein-topology engineering.     

SpyTag/SpyCatcher Cyclization Confers Resilience to Boiling on a Mesophilic Enzyme

SpyTag is a peptide that spontaneously forms an amide bond with its protein partner SpyCatcher. SpyTag was fused at the N terminus of β‐lactamase and SpyCatcher at the C terminus so that the partners could react to lock together the termini of the enzyme. The wild‐type enzyme aggregates above 37 °C, with irreversible loss of activity. Cyclized β‐lactamase was soluble even after heating at 100 °C; after cooling, the catalytic activity was restored. SpyTag/SpyCatcher cyclization led to a much larger increase in stability than that achieved through point mutation or alternative approaches to cyclization. Cyclized dihydrofolate reductase was similarly resilient. Analyzing unfolding through calorimetry indicated that cyclization did not increase the unfolding temperature but rather facilitated refolding after thermal stress. SpyTag/SpyCatcher sandwiching represents a simple and efficient route to enzyme cyclization, with potential to greatly enhance the robustness of biocatalysts.     

Cellular Synthesis and X‐ray Crystal Structure of a Designed Protein Heterocatenane

Herein, we report the biosynthesis of protein heterocatenanes using a programmed sequence of multiple post‐translational processing events including intramolecular chain entanglement, in situ backbone cleavage, and spontaneous cyclization. The approach is general, autonomous, and can obviate the need for any additional enzymes. The catenane topology was convincingly proven using a combination of SDS‐PAGE, LC‐MS, size exclusion chromatography, controlled proteolytic digestion, and protein crystallography. The X‐ray crystal structure clearly shows two mechanically interlocked protein rings with intact folded domains. It opens new avenues in the nascent field of protein‐topology engineering.     

On a geometry-based approach to protein sequence alignment

We consider a novel numerical representation of proteins obtained by assigning to individual amino acids the polar coordinate on a unit circle. As a result one can represent protein sequence as one-dimensional numerical sequence, the entries of which when subtracted facilitates search for alignment between pairs of proteins of interest. The alignment is sought by shifting one sequence relative to another by several sequence units to the left or to the right. The novel approach is illustrated on two yeast proteins having 174 and 171 amino acids. Visiting Emeritus from the Department of Mathematics & Computer Science Drake University, Des Moines, Iowa.     

Substitution of an Internal Disulfide Bridge with a Diselenide Enhances both Foldability and Stability of Human Insulin

Insulin analogues, mainstays in the modern treatment of diabetes mellitus, exemplify the utility of protein engineering in molecular pharmacology. Whereas chemical syntheses of the individual A and B chains were accomplished in the early 1960s, their combination to form native insulin remains inefficient because of competing disulfide pairing and aggregation. To overcome these limitations, we envisioned an alternative approach: pairwise substitution of cysteine residues with selenocysteine (Sec, U). To this end, Cys^A6 and Cys^A11 (which form the internal intrachain A6–A11 disulfide bridge) were each replaced with Sec. The A chain[C6U, C11U] variant was prepared by solid-phase peptide synthesis; while sulfitolysis of biosynthetic human insulin provided wild-type B chain-di-S-sulfonate. The presence of selenium atoms at these sites markedly enhanced the rate and fidelity of chain combination, thus solving a long-standing challenge in chemical insulin synthesis. The affinity of the Se-insulin analogue for the lectin-purified insulin receptor was indistinguishable from that of WT-insulin. Remarkably, the thermodynamic stability of the analogue at 25 °C, as inferred from guanidine denaturation studies, was augmented (ΔΔG_u ≈0.8 kcal mol⁻¹). In accordance with such enhanced stability, reductive unfolding of the Se-insulin analogue and resistance to enzymatic cleavage by Glu-C protease occurred four times more slowly than that of WT-insulin. 2D-NMR and X-ray crystallographic studies demonstrated a native-like three-dimensional structure in which the diselenide bridge was accommodated in the hydrophobic core without steric clash.     

Dual Binding Mode of Metallacarborane Produces a Robust Shield on Proteins

An inorganic sandwich molecule, Na[Co(C₂B₉H₁₁)₂], able to produce vesicles through self-assembly and known to produce strong dihydrogen-bond interactions with amine groups is capable of interacting with proteins. This dual non-bonding ability of Na[Co(C₂B₉H₁₁)₂] is what makes this molecule unique: it can be firmly anchored to a protein surface and is capable of extending over it. To prove this, the widely available bovine serum albumin (BSA), which has many pendant amino groups in its structure, has been taken as the model protein. It has been found that around 100 molecules of Na[Co(C₂B₉H₁₁)₂] preserve the native structure of BSA, while endorsing it with a significantly increased stability with respect to chemical- and thermal-induced denaturation due to efficient encapsulation. The advantages of this encapsulation technique are two-fold; the first is its simplicity as it relies on the anchoring capacity of Na[Co(C₂B₉H₁₁)₂] to the surface of the protein through the amine-containing residues and the second is its self-assembling capacity allowing it to spread across the surface. The dense shield of protection offered by Na[Co(C₂B₉H₁₁)₂] has been demonstrated by the inhibition of BSA pseudo-esterase activity, which indicates that the inorganic corset around BSA protects its reactive surface residues, thereby preventing their acetylation.     

Construction of a Triangle‐Shaped Trimer and a Tetrahedron Using an α‐Helix‐Inserted Circular Permutant of Cytochrome c555

Highly‐ordered protein structures have gained interest for future uses for biomaterials. Herein, we constructed a building block protein (BBP) by the circular permutation of the hyperthermostable Aquifex aeolicus cytochrome (cyt) c₅₅₅, and assembled BBP into a triangle‐shaped trimer and a tetrahedron. The angle of the intermolecular interactions of BBP was controlled by cleaving the domain‐swapping hinge loop of cyt c₅₅₅ and connecting the original N‐ and C‐terminal α‐helices with an α‐helical linker. We obtained BBP oligomers up to ≈40 mers, with a relatively large amount of trimers. According to the X‐ray crystallographic analysis of the BBP trimer, the N‐terminal region of one BBP molecule interacted intermolecularly with the C‐terminal region of another BBP molecule, resulting in a triangle‐shaped structure with an edge length of 68 Å. Additionally, four trimers assembled into a unique tetrahedron in the crystal. These results demonstrate that the circular permutation connecting the original N‐ and C‐terminal α‐helices with an α‐helical linker may be useful for constructing organized protein structures.     

Backbone building from quadrilaterals: a fast and accurate algorithm for protein backbone reconstruction from alpha carbon coordinates

In this contribution, we present an algorithm for protein backbone reconstruction that comprises very high computational efficiency with high accuracy. Reconstruction of the main chain atomic coordinates from the alpha carbon trace is a common task in protein modeling, including de novo structure prediction, comparative modeling, and processing experimental data. The method employed in this work follows the main idea of some earlier approaches to the problem. The details and careful design of the present approach are new and lead to the algorithm that outperforms all commonly used earlier applications. BBQ (Backbone Building from Quadrilaterals) program has been extensively tested both on native structures as well as on near-native decoy models and compared with the different available existing methods. Obtained results provide a comprehensive benchmark of existing tools and evaluate their applicability to a large scale modeling using a reduced representation of protein conformational space. The BBQ package is available for downloading from our website at http://biocomp.chem.uw.edu.pl/services/BBQ/. This webpage also provides a user manual that describes BBQ functions in detail.     

Structural Analysis of a Helical Peptide Unfolding Pathway

The analysis of the folding mechanism in peptides adopting well‐defined secondary structure is fundamental to understand protein folding. Herein, we describe the thermal unfolding of a 15‐mer vascular endothelial growth factor mimicking α‐helical peptide (QK_L10A) through the combination of spectroscopic and computational analyses. In particular, on the basis of the temperature dependencies of QK_L10A H_α chemical shifts we show that the first phase of the thermal helix unfolding, ending at around 320 K, involves mainly the terminal regions. A second phase of the transition, ending at around 333 K, comprises the central helical region of the peptide. The determination of high‐resolution QK_L10A conformational preferences in water at 313 K allowed us to identify, at atomic resolution, one intermediate of the folding–unfolding pathway. Molecular dynamics simulations corroborate experimental observations detecting a stable central helical turn, which represents the most probable site for the helix nucleation in the folding direction. The data presented herein allows us to draw a folding–unfolding picture for the small peptide QK_L10A compatible with the nucleation–propagation model. This study, besides contributing to the basic field of peptide helix folding, is useful to gain an insight into the design of stable helical peptides, which could find applications as molecular scaffolds to target protein–protein interactions.     

SDOCK: a global protein-protein docking program using stepwise force-field potentials

Fast Fourier transform (FFT) method limits the forms of scoring functions in global protein-protein docking. On the other hand, force field potentials can effectively describe the energy hyper surface of biological macromolecules. In this study, we developed a new protein-protein docking program, SDOCK, that incorporates van der Waals attractive potential, geometric collision, screened electrostatic potential, and Lazaridis-Karplus desolvation energy into the scoring function in the global searching process. Stepwise potentials were generated from the corresponding continuous forms to treat the structure flexibility. After optimization of the atom solvation parameters and the weights of different potential terms based on a new docking test set that contains 142 cases with small or moderate conformational changes upon binding, SDOCK slightly outperformed the well-known FFT based global docking program ZDOCK3.0. Among the 142 cases tested, 52.8% gave at least one near-native solutions in the top 100 solutions. SDOCK was also tested on six blind testing cases in Critical Assessment of Predicted Interactions rounds 13 to 18. In all six cases, the near-native solutions could be found within the top 350 solutions. Because the SDOCK approach performs global docking based on force-field potentials, one of its advantages is that it provides global binding free energy surface profiles for further analysis. The efficiency of the program is also comparable with that of other FFT based protein-protein docking programs. SDOCK is available for noncommercial applications at http://mdl.ipc.pku.edu.cn/cgi-bin/down.cgi.     

Crystal Structure of Carboxypeptidase a Complexed with an Inactivator in a New Crystalline Form

1INTR0DUCTIONInrecentyears,zinc-containingmeta1loenzymeshavereceivedspecialattentionbecauseoftheroletheyplayintheetiologyofmanyseriousdiseases.Theyserveastargetsinthedesignoftherapeuticallyusefulinhibitorsoftheseenzymes.Inthisre-spect,carboxypeptidaseA(CPA),awell-studiedzinc-containingmetalloex-opeptidase,bearsanunusualimportancebecauseitservesasamodelformanymetal-loenzymes.Thetertiarystructure0fCPAinc1udingthatoftheactivesitehasbeenwellcharacterizedalth0ughthecataIyticmechanismatthemo…     

Multiple Stable Conformations Account for Reversible Concentration‐Dependent Oligomerization and Autoinhibition of a Metamorphic Metallopeptidase

Molecular plasticity controls enzymatic activity: the native fold of a protein in a given environment is normally unique and at a global free‐energy minimum. Some proteins, however, spontaneously undergo substantial fold switching to reversibly transit between defined conformers, the “metamorphic” proteins. Here, we present a minimal metamorphic, selective, and specific caseinolytic metallopeptidase, selecase, which reversibly transits between several different states of defined three‐dimensional structure, which are associated with loss of enzymatic activity due to autoinhibition. The latter is triggered by sequestering the competent conformation in incompetent but structured dimers, tetramers, and octamers. This system, which is compatible with a discrete multifunnel energy landscape, affords a switch that provides a reversible mechanism of control of catalytic activity unique in nature.