Structural and biochemical characterization of native and recombinant single insulin-like growth factor-binding domain protein (SIBD-1) from the Central American hunting spider Cupiennius salei (Ctenidae)

Cupiennius salei single insulin-like growth factor binding domain protein (SIBD-1) is an 8.6 kDa Cys-, Pro-, and Gly-rich protein, discovered in the hemocytes of the Central American hunting spider Cupiennius salei. SIBD-1 exhibits high sequence similarity to the N-terminal domain of the insulin-like growth factor-binding protein superfamily and has been reported to play an important role in the spider's immune system. Here, the recombinant expression and the elucidation of the three-dimensional structure of recombinant SIBD-1 and the characterization of the sugar moiety at Thr2 of native SIBD-1 is described in detail.     

Isolation and characterization of antibody fragments selective for specific protein morphologies from nanogram antigen samples

We developed atomic force microscope (AFM)‐based protocols that enable isolation and characterization of antibody‐based reagents that selectively bind target protein variants using low nanogram amounts or less of unpurified starting material. We isolated single‐chain antibody fragments (scFvs) that specifically recognize an oligomeric beta‐amyloid (Aβ) species correlated with Alzheimer's disease (AD) using only a few nanograms of an enriched but not purified sample obtained from human AD brain tissue. We used several subtractive panning steps to remove all phage binding nondesired antigens and then used a single positive panning step using minimal antigen. We also used AFM to characterize the specificity of the isolated clones, again using minimal material, selecting the C6 scFv based on expression levels. We show that C6 selectively binds cell and brain‐derived oligomeric Aβ. The protocols described are readily adapted to isolating antibody‐based reagents against other antigenic targets with limited availability. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 463–471, 2013     

Structural analysis of the Ras-like G protein MglA and its cognate GAP MglB and implications for bacterial polarity

The bacterium Myxococcus xanthus uses a G protein cycle to dynamically regulate the leading/lagging pole polarity axis. The G protein MglA is regulated by its GTPase-activating protein (GAP) MglB, thus resembling Ras family proteins. Here, we show structurally and biochemically that MglA undergoes a dramatic, GDP-GTP-dependent conformational change involving a screw-type forward movement of the central β2-strand, never observed in any other G protein. This movement and complex formation with MglB repositions the conserved residues Arg53 and Gln82 into the active site. Residues required for catalysis are thus not provided by the GAP MglB, but by MglA itself. MglB is a Roadblock/LC7 protein and functions as a dimer to stimulate GTP hydrolysis in a 2:1 complex with MglA. In vivo analyses demonstrate that hydrolysis mutants abrogate Myxococcus' ability to regulate its polarity axis changing the reversal behaviour from stochastic to oscillatory and that both MglA GTPase activity and MglB GAP catalysis are essential for maintaining a proper polarity axis.     

Recombinant production of a VL single domain antibody in Escherichia coli and analysis of its interaction with peptostreptococcal protein L

A kappa-light chain from a Fab expression system was truncated by the insertion of a stop codon in the gene sequence to produce a variable light (VL) single domain antibody (dAb). Here, we describe the expression of dAb in the periplasm of Escherichia coli through fermentation in a defined media. Immunoglobulin binding domains from peptostreptococcal protein L (PpL) have been shown to bind specifically to kappa-light chains. We have produced recombinant PpL, at high yield, and this was used to custom-produce PpL-Sepharose affinity columns. Here, we show that the affinity purification of VL dAb by this method is simple and efficient with no apparent loss in protein at any stage. The truncated dAb protein product was confirmed by electrospray mass spectrometry and N-terminal sequencing. When analyzed by SDS-PAGE it was shown to be over 95% pure and produced at yields of 35-65 mg/L of culture medium. The dAb protein produced was shown by NMR and CD to be a folded beta-sheet domain. This domain is bound by PpL with a Kd of approximately 50 nM as determined by stopped-flow fluorimetry.     

Redesigning of anti‐c‐Met single chain Fv antibody for the cytoplasmic folding and its structural analysis

Typically, single chain Fv antibodies are unable to fold properly under a reducing cytoplasm because of the reduction of disulfide bonds. The inability to fold limits both the production of the functional scFvs and their targeting against antigens, which are generally executed in a reducing cytoplasm. In this study, the target scFv CDR was grafted with stable human consensus framework sequences, which enabled the generation of a foldable scFv in a reducing cytoplasm of Escherichia coli. Additionally, the structural features affecting the folding efficiency of the engineered scFv were identified by analyzing the predicted structure. An anti‐c‐Met scFv, which was a cytoplasmic non‐foldable protein, was redesigned as the model system. This study confirmed that the engineered anti‐c‐Met scFv was folded into its native form in the cytoplasm of E. coli BL21(DE3) without a significant loss in the specific binding activity against c‐Met antigen. The structures of the wild‐type anti‐c‐Met scFv and the engineered scFv were predicted using homology modeling. A comparative analysis based on the sequence and structure showed that the hydrophobicity of 12 solvent exposed residues decreased, and two newly formed salt bridges might have improved the folding efficiency of the engineered scFv under the reducing condition. Biotechnol. Bioeng. 2010; 106: 367–375. © 2010 Wiley Periodicals, Inc.     

Transition state of a SH3 domain detected with principle component analysis and a charge-neutralized all-atom protein model

The src SH3 domain has been known to be a two-state folder near room temperature. However, in a previous study with an all-atom model simulation near room temperature, the transition state of this protein was not successfully detected on a free-energy profile using two axes: the radius of gyration (R(g)) and native contact reproduction ratio (Q value). In this study, we focused on an atom packing effect to characterize the transition state and tried another analysis to detect it. To explore the atom packing effect more efficiently, we introduced a charge-neutralized all-atom model, where all of the atoms in the protein and water molecules were treated explicitly, but their partial atomic charges were set to zero. Ten molecular dynamics simulations were performed starting from the native structure at 300 K, where the simulation length of each run was 90 ns, and the protein unfolded in all runs. The integrated trajectories (10 x 90 = 900 ns) were analyzed by a principal component analysis (PCA) and showed a clear free-energy barrier between folded- and unfolded-state conformational clusters in a conformational space generated by PCA. There were segments that largely deformed when the conformation passed through the free-energy barrier. These segments correlated well with the structural core regions characterized by large phi-values, and the atom-packing changes correlated with the conformational deformations. Interestingly, using the same simulation data, no significant barrier was found in a free-energy profile using the R(g) and Q values for the coordinate axes. These results suggest that the atom packing effect may be one of the most important determinants of the transition state.     

Solution structure of the dimeric SAM domain of MAPKKK Ste11 and its interactions with the adaptor protein Ste50 from the budding yeast: implications for Ste11 activation and signal transmission through the Ste50-Ste11 complex

Ste11, a homologue of mammalian MAPKKKs, together with its binding partner Ste50 works in a number of MAPK signaling pathways of Saccharomyces cerevisiae. Ste11/Ste50 binding is mediated by their sterile alpha motifs or SAM domains, of which homologues are also found in many other intracellular signaling and regulatory proteins. Here, we present the solution structure of the SAM domain or residues D37-R104 of Ste11 and its interactions with the cognate SAM domain-containing region of Ste50, residues M27-Q131. NMR pulse-field-gradient (PFG) and rotational correlation time measurements (tauc) establish that the Ste11 SAM domain exists predominantly as a symmetric dimer in solution. The solution structure of the dimeric Ste11 SAM domain consists of five well-defined helices per monomer packed into a compact globular structure. The dimeric structure of the SAM domain is maintained by a novel dimer interface involving interactions between a number of hydrophobic residues situated on helix 4 and at the beginning of the C-terminal long helix (helix 5). The dimer structure may also be stabilized by potential salt bridge interactions across the interface. NMR H/2H exchange experiments showed that binding of the Ste50 SAM to the Ste11 SAM very likely involves the positively charged extreme C-terminal region as well as exposed hydrophobic patches of the dimeric Ste11 SAM domain. The dimeric structure of the Ste11 SAM and its interactions with the Ste50 SAM may have important roles in the regulation and activation of the Ste11 kinase and signal transmission and amplifications through the Ste50-Ste11 complex.     

NMR analysis of partially folded states and persistent structure in the alpha subunit of tryptophan synthase: implications for the equilibrium folding mechanism of a 29-kDa TIM barrel protein

Structural insights into the equilibrium folding mechanism of the alpha subunit of tryptophan synthase (αTS) from Escherichia coli, a (βα)₈ TIM barrel protein, were obtained with a pair of complementary nuclear magnetic resonance (NMR) spectroscopic techniques. The secondary structures of rare high-energy partially folded states were probed by native-state hydrogen-exchange NMR analysis of main-chain amide hydrogens. 2D heteronuclear single quantum coherence NMR analysis of several ¹⁵N-labeled nonpolar amino acids was used to probe the side chains involved in stabilizing a highly denatured intermediate that is devoid of secondary structure. The dynamic broadening of a subset of isoleucine and leucine side chains and the absence of protection against exchange showed that the highest energy folded state on the free-energy landscape is stabilized by a hydrophobic cluster lacking stable secondary structure. The core of this cluster, centered near the N-terminus of αTS, serves as a nucleus for the stabilization of what appears to be nonnative secondary structure in a marginally stable intermediate. The progressive decrease in protection against exchange from this nucleus toward both termini and from the N-termini to the C-termini of several β-strands is best described by an ensemble of weakly coupled conformers. Comparison with previous data strongly suggests that this ensemble corresponds to a marginally stable off-pathway intermediate that arises in the first few milliseconds of folding and persists under equilibrium conditions. A second, more stable intermediate, which has an intact β-barrel and a frayed α-helical shell, coexists with this marginally stable species. The conversion of the more stable intermediate to the native state of αTS entails the formation of a stable helical shell and completes the acquisition of the tertiary structure.