SNAC-tag for sequence-specific chemical protein cleavage.

Site-specific protein cleavage is essential for many protein-production protocols and typically requires proteases. We report the development of a chemical protein-cleavage method that is achieved through the use of a sequence-specific nickel-assisted cleavage (SNAC)-tag. We demonstrate that the SNAC-tag can be inserted before both water-soluble and membrane proteins to achieve fusion protein cleavage under biocompatible conditions with efficiency comparable to that of enzymes, and that the method works even when enzymatic cleavages fail

Two models of the influenza A M2 channel domain: verification by comparison

Background: The influenza M2 protein is a simple membrane protein, containing a single transmembrane helix. It is representative of a very large family of single-transmembrane helix proteins. The functional protein is a tetramer, with the four transmembrane helices forming a proton-permeable channel across the bilayer. Two independently derived models of the M2 channel domain are compared, in order to assess the success of applying molecular modelling approaches to simple membrane proteins.Results: The Cα RSMD between the two models is 1.7 å. Both models are composed of a left-handed bundle of helices, with the helices tilted roughly 15° relative to the (presumed) bilayer normal. The two models have similar pore radius profiles, with a pore cavity lined by the Ser31 and Gly34 residues and a pore constriction formed by the ring of His37 residues.Conclusions:Independent studies of M2 have converged on the same structural model for the channel domain. This model is in agreement with solid state NMR data. In particular, both model and NMR data indicate that the M2 helices are tilted relative to the bilayer normal and form a left-handed bundle. Such convergence suggests that, at least for simple membrane proteins, restraints-directed modelling might yield plausible models worthy of further computational and experimental investigation

Structural organization and interactions of transmembrane domains in tetraspanin proteins

BACKGROUND: Proteins of the tetraspanin family contain four transmembrane domains (TM1-4) linked by two extracellular loops and a short intracellular loop, and have short intracellular N- and C-termini. While structure and function analysis of the larger extracellular loop has been performed, the organization and role of transmembrane domains have not been systematically assessed. RESULTS: Among 28 human tetraspanin proteins, the TM1-3 sequences display a distinct heptad repeat motif (abcdefg)(n). In TM1, position a is occupied by structurally conserved bulky residues and position d contains highly conserved Asn and Gly residues. In TM2, position a is occupied by conserved small residues (Gly/Ala/Thr), and position d has a conserved Gly and two bulky aliphatic residues. In TM3, three a positions of the heptad repeat are filled by two leucines and a glutamate/glutamine residue, and two d positions are occupied by either Phe/Tyr or Val/Ile/Leu residues. No heptad motif is apparent in TM4 sequences. Mutations of conserved glycines in human CD9 (Gly25 and Gly32 in TM1; Gly67 and Gly74 in TM2) caused aggregation of mutant proteins inside the cell. Modeling of the TM1-TM2 interface in CD9, using a novel algorithm, predicts tight packing of conserved bulky residues against conserved Gly residues along the two helices. The homodimeric interface of CD9 was mapped, by disulfide cross-linking of single-cysteine mutants, to the vicinity of residues Leu14 and Phe17 in TM1 (positions g and c) and Gly77, Gly80 and Ala81 in TM2 (positions d, g and a, respectively). Mutations of a and d residues in both TM1 and TM2 (Gly25, Gly32, Gly67 and Gly74), involved in intramolecular TM1-TM2 interaction, also strongly diminished intermolecular interaction, as assessed by cross-linking of Cys80. CONCLUSION: Our results suggest that tetraspanin intra- and intermolecular interactions are mediated by conserved residues in adjacent, but distinct regions of TM1 and TM2. A key structural element that defines TM1-TM2 interaction in tetraspanins is the specific packing of bulky residues against small residues

Computational Design and Elaboration of a De Novo Heterotetrameric α-Helical Protein that Selectively Binds an Emissive Abiological (Porphinato)zinc Chromophore

The first example of a computationally de novo designed protein that binds an emissive abiological chromophore is presented, in which a sophisticated level of cofactor discrimination is pre-engineered. This heterotetrameric, C(2)-symmetric bundle, A(His):B(Thr), uniquely binds (5,15-di[(4-carboxymethyleneoxy)phenyl]porphinato)zinc [(DPP)Zn] via histidine coordination and complementary noncovalent interactions. The A(2)B(2) heterotetrameric protein reflects ligand-directed elements of both positive and negative design, including hydrogen bonds to second-shell ligands. Experimental support for the appropriate formulation of [(DPP)Zn:A(His):B(Thr)](2) is provided by UV/visible and circular dichroism spectroscopies, size exclusion chromatography, and analytical ultracentrifugation. Time-resolved transient absorption and fluorescence spectroscopic data reveal classic excited-state singlet and triplet PZn photophysics for the A(His):B(Thr):(DPP)Zn protein (k(fluorescence) = 4 x 10(8) s(-1); tau(triplet) = 5 ms). The A(2)B(2) apoprotein has immeasurably low binding affinities for related [porphinato]metal chromophores that include a (DPP)Fe(III) cofactor and the zinc metal ion hemin derivative [(PPIX)Zn], underscoring the exquisite active-site binding discrimination realized in this computationally designed protein. Importantly, elements of design in the A(His):B(Thr) protein ensure that interactions within the tetra-alpha-helical bundle are such that only the heterotetramer is stable in solution; corresponding homomeric bundles present unfavorable ligand-binding environments and thus preclude protein structural rearrangements that could lead to binding of (porphinato)iron cofactors

Crystallization of a designed peptide from a molten globule ensemble

Backgound:The design of amino acid sequences that adopt a desired three-dimensional fold has been of keen interest over the past decade. However, the design of proteins that adopt unique conformations is still a considerable problem. Until very recently, all of the designed proteins that have been extensively characterized possess the hallmarks of the molten globular state. Molten globular intermediates have been observed in both equilibrium and kinetic protein folding/stability studies, and understanding the forces that determine compact non-native states is critical for a comprehensive understanding of proteins. This paper describes the solution and early solid state characterization of peptides that form molten globular ensembles.Results & Conclusions:Crystals diffracting to 3.5å resolution have been grown of a 16-residue peptide (α1A) designed to form a tetramer of α-helices. In addition, a closely related peptide, α1, has previously been shown to yield crystals that diffract to 1.2å resolution. The solution properties of these two peptides were examined to determine whether their well defined crystalline conformations were retained in solution. On the basis of an examination of their NMR spectra, sedimentation equilibria, thermal unfolding, and ANS binding, it is concluded that the peptides form α-helical aggregates with properties similar to those of the molten globule state. Thus, for these peptides, the process of crystallization bears many similarities to models of protein folding. Upon dissolution, the peptides rapidly assume compact molten globular states similar to the molten globule like intermediates that are formed at short times after refolding is initiated. Following a rate-determining nucleation step, the peptides crystallize into a single or a small number of conformations in a process that mimics the formation of native structure in proteins

Self-assembling dipeptide antibacterial nanostructures with membrane disrupting activity.

Peptide-based supramolecular assemblies are a promising class of nanomaterials with important biomedical applications, specifically in drug delivery and tissue regeneration. However, the intrinsic antibacterial capabilities of these assemblies have been largely overlooked. The recent identification of common characteristics shared by antibacterial and self-assembling peptides provides a paradigm shift towards development of antibacterial agents. Here we present the antibacterial activity of self-assembled diphenylalanine, which emerges as the minimal model for antibacterial supramolecular polymers. The diphenylalanine nano-assemblies completely inhibit bacterial growth, trigger upregulation of stress-response regulons, induce substantial disruption to bacterial morphology, and cause membrane permeation and depolarization. We demonstrate the specificity of these membrane interactions and the development of antibacterial materials by integration of the peptide assemblies into tissue scaffolds. This study provides important insights into the significance of the interplay between self-assembly and antimicrobial activity and establishes innovative design principles toward the development of antimicrobial agents and materials

Spectroscopic and metal binding properties of a de novo metalloprotein binding a tetrazinc cluster

De novo design provides an attractive approach, which allows one to test and refine the principles guiding metalloproteins in defining the geometry and reactivity of their metal ion cofactors. Although impressive progress has been made in designing proteins that bind transition metal ions including iron–sulfur clusters, the design of tetranuclear clusters with oxygen‐rich environments remains in its infancy. In previous work, we described the design of homotetrameric four‐helix bundles that bind tetra‐Zn2+ clusters. The crystal structures of the helical proteins were in good agreement with the overall design, and the metal‐binding and conformational properties of the helical bundles in solution were consistent with the crystal structures. However, the corresponding apo‐proteins were not fully folded in solution. In this work, we design three peptides, based on the crystal structure of the original bundles. One of the peptides forms tetramers in aqueous solution in the absence of metal ions as assessed by CD and NMR. It also binds Zn2+ in the intended stoichiometry. These studies strongly suggest that the desired structure has been achieved in the apo state, providing evidence that the peptide is able to actively impart the designed geometry to the metal cluster

Engineered single- and multi-cell chemotaxis pathways in E. coli

We have engineered the chemotaxis system of Escherichia coli to respond to molecules that are not attractants for wild-type cells. The system depends on an artificially introduced enzymatic activity that converts the target molecule into a ligand for an E. coli chemoreceptor, thereby enabling the cells to respond to the new attractant. Two systems were designed, and both showed robust chemotactic responses in semisolid and liquid media. The first incorporates an asparaginase enzyme and the native E. coli aspartate receptor to produce a response to asparagine; the second uses penicillin acylase and an engineered chemoreceptor for phenylacetic acid to produce a response to phenylacetyl glycine. In addition, by taking advantage of a ‘hitchhiker' effect in which cells producing the ligand can induce chemotaxis of neighboring cells lacking enzymatic activity, we were able to design a more complex system that functions as a simple microbial consortium. The result effectively introduces a logical ‘AND' into the system so that the population only swims towards the combined gradients of two attractants

Affinity of Talin-1 for the β3-Integrin Cytosolic Domain is Modulated by its Phospholipid Bilayer Environment

Binding of the talin-1 FERM (4.1/ezrin/radixin/moesin) domain to the β3 cytosolic tail causes activation of the integrin αIIbβ3. The FERM domain also binds to acidic phospholipids. Although much is known about the interaction of talin-1 with integrins and lipids, the relative contribution of each interaction to integrin regulation and possible synergy between them remain to be clarified. Here, we examined the thermodynamic interplay between FERM domain binding to phospholipid bilayers and to its binding sites in the β3 tail. We found that although both the F0F1 and F2F3 subdomains of the talin-1 FERM domain bind acidic bilayers, the full-length FERM domain binds with an affinity similar to F2F3, indicating that F0F1 contributes little to the overall interaction. When free in solution, the β3 tail has weak affinity for the FERM domain. However, appending the tail to acidic phospholipids increased its affinity for the FERM domain by three orders of magnitude. Nonetheless, the affinity of the FERM for the appended tail was similar to its affinity for binding to bilayers alone. Thus, talin-1 binding to the β3 tail is a ternary interaction dominated by a favorable surface interaction with phospholipid bilayers and set by lipid composition. Nonetheless, interactions between the FERM domain, the β3 tail, and lipid bilayers are not optimized for a high-affinity synergistic interaction, even at the membrane surface. Instead, the interactions appear to be tuned in such a way that the equilibrium between inactive and active integrin conformations can be readily regulated