A central goal of protein design is to devise novel proteins for applications in biotechnology and medicine. Many applications, including those focused on sensing and catalysis, will require proteins that recognize and bind to small molecules. Here we show that stably folded a-helical proteins isolated from a binary patterned library of designed sequences can be mutated to produce binding sites capable of binding a range of small aromatic compounds. Specifically, we mutated two phenylalanine side chains to alanine in the known structure of de novo protein S-824 to create buried cavities in the core of this 4-helix bundle. The parental protein and the Phe?Ala variants were exposed to mixtures of compounds, and selective binding was assessed by saturation transfer difference (STD) NMR. The affinities of benzene and a number of its derivatives were determined by pulse field gradient spin echo (PFGSE) NMR, and several of the compounds were shown to bind the mutated protein with micromolar dissociation constants. These studies suggest that stably folded de novo proteins from binary patterned libraries are well-suited as scaffolds for the design of binding sites.
Copyright © 2011 The Protein Society.mandag 28. februar 2011
Binding of small molecules to cavity forming mutants of a de novo designed protein.
Crystal structure of a soluble form of human monoglyceride lipase in complex with an inhibitor at 1.35 A resolution.
A high-resolution structure of a ligand-bound, soluble form of human monoglyceride lipase is presented. The structure highlights a novel conformation of the regulatory lid-domain present in the lipase family as well as the binding mode of a pharmaceutically relevant reversible inhibitor. Analysis of the structure lacking the inhibitor indicates that the closed conformation can accommodate the native substrate 2 arachidonoyl glycerol. A model is proposed in which monoglyceride lipase undergoes conformational and electrostatic changes during the catalytic cycle ultimately resulting in its dissociation from the membrane upon completion of the cycle. In addition, the study outlines a successful approach to transform membrane associated proteins, which tend to aggregate upon purification, into a monomeric and soluble form.
Copyright © 2011 The Protein Society.PMID: 21308848 [PubMed - as supplied by publisher]søndag 27. februar 2011
2-Methyl-2,4-pentanediol induces spontaneous assembly of staphylococcal alpha-hemolysin into heptameric pore structure.
Staphylococcal a-hemolysin is expressed as a water-soluble monomeric protein and assembles on membranes to form a heptameric pore structure. The heptameric pore structure of a-hemolysin can be prepared from monomer in vitro only in the presence of deoxycholate detergent micelles, artificially constructed phospholipid bilayers, or erythrocytes. Here, we succeeded in preparing crystals of the heptameric form of a-hemolysin without any detergent but with 2-methyl-2,4-pentanediol (MPD), and determined its structure. The structure of the heptameric pore was similar to that reported previously. In the structure, two molecules of MPD were bound around Trp179, around which phospholipid head groups were bound in the heptameric pore structure reported previously. Size exclusion chromatography showed that a-hemolysin did not assemble spontaneously even when stored for 1 year. SDS-PAGE analysis revealed that, among the compounds in the crystallizing buffer, MPD could induce heptamer formation. The concentration of MPD that most efficiently induced oligomerization was between 10 and 30%. Based on these observations, we propose MPD as a reagent that can facilitate heptameric pore formation of a-hemolysin without membrane binding.
Copyright © 2011 The Protein Society.Insights into the conformational flexibility of Bruton's tyrosine kinase from multiple ligand complex structures.
Bruton's tyrosine kinase (BTK) plays a key role in B cell receptor signaling and is considered a promising drug target for lymphoma and inflammatory diseases. We have determined the X-ray crystal structures of BTK kinase domain in complex with six inhibitors from distinct chemical classes. Five different BTK protein conformations are stabilized by the bound inhibitors, providing insights into the structural flexibility of the Gly-rich loop, helix C, the DFG sequence, and activation loop. The conformational changes occur independent of activation loop phosphorylation and do not correlate with the structurally unchanged WEI motif in the Src homology 2-kinase domain linker. Two novel activation loop conformations and an atypical DFG conformation are observed representing unique inactive states of BTK. Two regions within the activation loop are shown to structurally transform between 3(10) - and a-helices, one of which collapses into the adenosine-5'-triphosphate binding pocket. The first crystal structure of a Tec kinase family member in the pharmacologically important DFG-out conformation and bound to a type II kinase inhibitor is described. The different protein conformations observed provide insights into the structural flexibility of BTK, the molecular basis of its regulation, and the structure-based design of specific inhibitors.
Copyright © 2010 The Protein Society.Structures of domains I and IV from YbbR are representative of a widely distributed protein family.
YbbR domains are widespread throughout Eubacteria and are expressed as monomeric units, linked in tandem repeats or cotranslated with other domains. Although the precise role of these domains remains undefined, the location of the multiple YbbR domain-encoding ybbR gene in the Bacillus subtilis glmM operon and its previous identification as a substrate for a surfactin-type phosphopantetheinyl transferase suggests a role in cell growth, division, and virulence. To further characterize the YbbR domains, structures of two of the four domains (I and IV) from the YbbR-like protein of Desulfitobacterium hafniense Y51 were solved by solution nuclear magnetic resonance and X-ray crystallography. The structures show the domains to have nearly identical topologies despite a low amino acid identity (23%). The topology is dominated by ß-strands, roughly following a "figure 8" pattern with some strands coiling around the domain perimeter and others crossing the center. A similar topology is found in the C-terminal domain of two stress-responsive bacterial ribosomal proteins, TL5 and L25. Based on these models, a structurally guided amino acid alignment identifies features of the YbbR domains that are not evident from naïve amino acid sequence alignments. A structurally conserved cis-proline (cis-Pro) residue was identified in both domains, though the local structure in the immediate vicinities surrounding this residue differed between the two models. The conservation and location of this cis-Pro, plus anchoring Val residues, suggest this motif may be significant to protein function.
Copyright © 2011 The Protein Society.lørdag 26. februar 2011
Exploring sparsely populated states of macromolecules by diamagnetic and paramagnetic NMR relaxation.
Sparsely populated states of macromolecules, characterized by short lifetimes and high free-energies relative to the predominant ground state, often play a key role in many biological, chemical, and biophysical processes. In this review, we briefly summarize various new developments in NMR spectroscopy that permit these heretofore invisible, sparsely populated states to be detected, characterized, and in some instances visualized. Relaxation dispersion spectroscopy yields detailed kinetic information on processes involving species characterized by distinct chemical shifts with lifetimes in the ~50 µs-10 ms range and populations as low as 0.5%. In the fast exchange regime (time scale less than ~250-500 µs), the footprint of sparsely populated states can be observed on paramagnetic relaxation enhancement profiles measured on the resonances of the major species, thereby yielding structural information that is directly related to paramagnetic center-nuclei distances from which it is possible, under suitable circumstances, to compute a structure or ensemble of structures for the minor species. Finally, differential transverse relaxation measurements can be used to detect lifetime broadening effects that directly reflect the unidirectional rates for the conversion of NMR-visible into high-molecular weight NMR-invisible species. Examples of these various approaches are presented.
Copyright © 2010 The Protein Society.Calorimetric study of a series of designed repeat proteins: Modular structure and modular folding.
Repeat proteins comprise tandem arrays of a small structural motif. Their structure is defined and stabilized by interactions between residues that are close in the primary sequence. Several studies have investigated whether their structural modularity translates into modular thermodynamic properties. Tetratricopeptide repeat proteins (TPRs) are a class in which the repeated unit is a 34 amino acid helix-turn-helix motif. In this work, we use differential scanning calorimetry (DSC) to study the equilibrium stability of a series of TPR proteins with different numbers of an identical consensus repeat, from 2 to 20, CTPRa2 to CTPRa20. The DSC data provides direct evidence that the folding/unfolding transition of CTPR proteins does not fit a two-state folding model. Our results confirm and expand earlier studies on TPR proteins, which showed that apparent two-state unfolding curves are better fit by linear statistical mechanics models: 1D Ising models in which each repeat is treated as an independent folding unit.
Copyright © 2010 The Protein Society.fredag 25. februar 2011
Binding of small molecules to cavity forming mutants of a de novo designed protein.
A central goal of protein design is to devise novel proteins for applications in biotechnology and medicine. Many applications, including those focused on sensing and catalysis, will require proteins that recognize and bind to small molecules. Here we show that stably folded a-helical proteins isolated from a binary patterned library of designed sequences can be mutated to produce binding sites capable of binding a range of small aromatic compounds. Specifically, we mutated two phenylalanine side chains to alanine in the known structure of de novo protein S-824 to create buried cavities in the core of this 4-helix bundle. The parental protein and the Phe?Ala variants were exposed to mixtures of compounds, and selective binding was assessed by saturation transfer difference (STD) NMR. The affinities of benzene and a number of its derivatives were determined by pulse field gradient spin echo (PFGSE) NMR, and several of the compounds were shown to bind the mutated protein with micromolar dissociation constants. These studies suggest that stably folded de novo proteins from binary patterned libraries are well-suited as scaffolds for the design of binding sites.
Copyright © 2011 The Protein Society.Loss of recognition by cross-reactive T cells and its relation to a C-terminus-induced conformational reorientation of an HLA-B*2705-bound peptide.
The human major histocompatibility complex class I antigen HLA-B*2705 binds several sequence-related peptides (pVIPR, RRKWRRWHL; pLPM2, RRRWRRLTV; pGR, RRRWHRWRL). Cross-reactivity of cytotoxic T cells (CTL) against these HLA-B*2705:peptide complexes seemed to depend on a particular peptide conformation that is facilitated by the engagement of a crucial residue within the binding groove (Asp116), associated with a noncanonical bulging-in of the middle portion of the bound peptide. We were interested whether a conformational reorientation of the ligand might contribute to the lack of cross-reactivity of these CTL with a peptide derived from voltage-dependent calcium channel a1 subunit (pCAC, SRRWRRWNR), in which the C-terminal peptide residue pArg9 could engage Asp116. Analyses of the HLA-B*2705:pCAC complex by X-ray crystallography at 1.94 Å resolution demonstrated that the peptide had indeed undergone a drastic reorientation, leading it to adopt a canonical binding mode accompanied by the loss of molecular mimicry between pCAC and sequence-related peptides such as pVIPR, pLMP2, and pGR. This was clearly a consequence of interactions of pArg9 with Asp116 and other F-pocket residues. Furthermore, we observed an unprecedented reorientation of several additional residues of the HLA-B*2705 heavy chain near the N-terminal region of the peptide, including also the presence of double conformations of two glutamate residues, Glu63 and Glu163, on opposing sides of the peptide binding groove. Together with the Arg-Ser exchange at peptide position 1, there are thus multiple structural reasons that may explain the observed failure of pVIPR-directed, HLA-B*2705-restricted CTL to cross-react with HLA-B*2705:pCAC complexes.
Copyright © 2010 The Protein Society.torsdag 24. februar 2011
Crystal structures of CmeR-bile acid complexes from Campylobacter jejuni.
The TetR family of transcription regulators are diverse proteins capable of sensing and responding to various structurally dissimilar antimicrobial agents. Upon detecting these agents, the regulators allow transcription of an appropriate array of resistance markers to counteract the deleterious compounds. Campylobacter jejuni CmeR is a pleiotropic regulator of multiple proteins, including the membrane-bound multidrug efflux transporter CmeABC. CmeR represses the expression of CmeABC and is induced by bile acids, which are substrates of the CmeABC tripartite pump. The multiligand-binding pocket of CmeR has been shown to be very extensive and consists of several positively charged and multiple aromatic amino acids. Here we describe the crystal structures of CmeR in complexes with the bile acids, taurocholate and cholate. Taurocholate and cholate are structurally related, differing by only the anionic charged group. However, these two ligands bind distinctly in the binding tunnel. Taurocholate spans the novel bile acid binding site adjacent to and without overlapping with the previously determined glycerol-binding site. The anionic aminoethanesulfonate group of taurocholate is neutralized by a charge-dipole interaction. Unlike taurocholate, cholate binds in an anti-parallel orientation but occupies the same bile acid-binding site. Its anionic pentanoate moiety makes a water-mediated hydrogen bond with a cationic residue to neutralize the formal negative charge. These structures underscore the promiscuity of the multifaceted binding pocket of CmeR. The capacity of CmeR to recognize bile acids was confirmed using isothermal titration calorimetry and fluorescence polarization. The results revealed that the regulator binds these acids with dissociation constants in the micromolar region.
Copyright © 2011 The Protein Society.Potenspiller
Vel, veldig mange voksne menn sliter med å få ereksjon når de skal penetrere den deiligste dama de vet om, nemlig kona si. Og da kan det være et problem at man faktisk sliter med å få bennern når man skal ut i ilden.
Mange ender opp i bingen når de skal ha sex, og det kan være som i gode gamle dager når man får bennern så og si når som helst. Vel, potenspiller er så definitivt fordelen med å ha et godt erotisk liv, rett og slett fordi man på død og liv blir så mye bedre i senga.
Skaff deg potenspiller i dag og bli bedre i senga.
The role of the local environment of engineered Tyr to Trp substitutions for probing the denaturation mechanism of FIS.
Factor for inversion stimulation (FIS), a 98-residue homodimeric protein, does not contain tryptophan (Trp) residues but has four tyrosine (Tyr) residues located at positions 38, 51, 69, and 95. The equilibrium denaturation of a P61A mutant of FIS appears to occur via a three-state (N(2) ? I(2) ? 2U) process involving a dimeric intermediate (I(2) ). Although it was suggested that this intermediate had a denatured C-terminus, direct evidence was lacking. Therefore, three FIS double mutants, P61A/Y38W, P61A/Y69W, and P61A/Y95W were made, and their denaturation was monitored by circular dichroism and Trp fluorescence. Surprisingly, the P61A/Y38W mutant best monitored the N(2) ? I(2) transition, even though Trp38 is buried within the dimer removed from the C-terminus. In addition, although Trp69 is located on the protein surface, the P61A/Y69W FIS mutant exhibited clearly biphasic denaturation curves. In contrast, P61A/Y95W FIS was the least effective in decoupling the two transitions, exhibiting a monophasic fluorescence transition with modest concentration-dependence. When considering the local environment of the Trp residues and the effect of each mutation on protein stability, these results not only confirm that P61A FIS denatures via a dimeric intermediate involving a disrupted C-terminus but also suggest the occurrence of conformational changes near Tyr38. Thus, the P61A mutation appears to compromise the denaturation cooperativity of FIS by failing to propagate stability to those regions involved mostly in intramolecular interactions. Furthermore, our results highlight the challenge of anticipating the optimal location to engineer a Trp residue for investigating the denaturation mechanism of even small proteins.
Copyright © 2010 The Protein Society.onsdag 23. februar 2011
Metals affect the structure and activity of human plasminogen activator inhibitor-1. I. Modulation of stability and protease inhibition.
Human plasminogen activator inhibitor type 1 (PAI-1) is a serine protease inhibitor with a metastable active conformation. Under physiological conditions, half of the inhibitor transitions to a latent state within 1-2 h. The interaction between PAI-1 and the plasma protein vitronectin prolongs this active lifespan by ~50%. Previously, our group demonstrated that PAI-1 binds to resins using immobilized metal affinity chromatography (Day, U.S. Pat. 7,015,021 B2, March 21, 2006). In this study, the effect of these metals on function and stability was investigated by measuring the rate of the transition from the active to latent conformation. All metals tested showed effects on stability, with the majority falling into one of two types depending on their effects. The first type of metal, which includes magnesium, calcium and manganese, invoked a slight stabilization of the active conformation of PAI-1. A second category of metals, including cobalt, nickel and copper, showed the opposite effects and a unique vitronectin-dependent modulation of PAI-1 stability. This second group of metals significantly destabilized PAI-1, although the addition of vitronectin in conjunction with these metals resulted in a marked stabilization and slower conversion to the latent conformation. In the presence of copper and vitronectin, the half-life of active PAI-1 was extended to 3 h, compared to a half-life of only ~30 min with copper alone. Nickel had the largest effect, reducing the half-life to ~5 min. Together, these data demonstrate a heretofore-unknown role for metals in modulating PAI-1 stability.
Copyright © 2010 The Protein Society.Crystal structure of the sensory domain of Escherichia coli CadC, a member of the ToxR-like protein family.
The membrane-integral transcriptional activator CadC comprises sensory and transcriptional regulatory functions within one polypeptide chain. Its C-terminal periplasmic domain, CadC(pd) , is responsible for sensing of environmental pH as well as for binding of the feedback inhibitor cadaverine. Here we describe the crystal structure of CadC(pd) (residues 188-512) solved at a resolution of 1.8 Å via multiple wavelength anomalous dispersion (MAD) using a ReCl(6) (2-) derivative. CadC(pd) reveals a novel fold comprising two subdomains: an N-terminal subdomain dominated by a $\tilde \beta$sheet in contact with three a-helices and a C-terminal subdomain formed by a ten-membered a-helical bundle, which is oriented almost perpendicular to the helices in the first subdomain. Further to the native protein, crystal structures were also solved for its variants D471N and D471E, which show functionally different behavior in pH sensing. Interestingly, in the heavy metal derivative of CadC(pd) used for MAD phasing a ReCl(6) (2-) ion was found in a cavity located between the two subdomains. Amino acid side chains that coordinate this complex ion are conserved in CadC homologues from various bacterial species, suggesting a function of the cavity in the binding of cadaverine, which was supported by docking studies. Notably, CadC(pd) and its variants form dimers in solution, which can be explained by an extended, albeit rather polar interface between two symmetry-related monomers in the crystal structure. The occurrence of several acidic residues in this region suggests protonation-dependent changes in the mode of dimerization, which could eventually trigger transcriptional activation by CadC in the bacterial cytoplasm.
Copyright © 2011 The Protein Society.Characterizing diffusion dynamics of a membrane protein associated with nanolipoproteins using fluorescence correlation spectroscopy.
Nanolipoprotein particles (NLPs) represent a unique nanometer-sized scaffold for supporting membrane proteins (MP). Characterization of their dynamic shape and association with MP in solution remains a challenge. Here, we present a rapid method of analysis by fluorescence correlation spectroscopy (FCS) to characterize bacteriorhodopsin (bR), a membrane protein capable of forming a NLP complex. By selectively labeling individual components of NLPs during cell-free synthesis, FCS enabled us to measure specific NLP diffusion times and infer size information for different NLP species. The resulting bR-loaded NLPs were shown to be dynamically discoidal in solution with a mean diameter of 7.8 nm. The insertion rate of bR in the complex was ~55% based on a fit model incorporating two separate diffusion properties to best approximate the FCS data. More importantly, based on these data, we infer that membrane protein associated NLPs are thermodynamically constrained as discs in solution, while empty NLPs appear to be less constrained and dynamically spherical.
Copyright © 2010 The Protein Society.