Solid Phase Synthesis and Application of Labeled Peptide Derivatives: Probes of Receptor-Opioid Peptide Interactions

Solid phase synthetic methodology has been developed in our laboratory to incorporate an affinity label (a reactive functionality such as isothiocyanate or bromoacetamide) into peptides (Leelasvatanakij and Aldrich J Peptide Res 56, 80, 2000), and we have used this synthetic strategy to prepare affinity label derivatives of a variety of opioid peptides. To date side reactions have been detected only in two cases, both involving intramolecular cyclization. We have identified several peptide-based affinity labels for δ opioid receptors that exhibit wash-resistant inhibition of binding to these receptors and are valuable pharmacological tools to study opioid receptors. Even in cases where the peptide derivatives do not bind covalently to their target receptor, studying their binding has revealed subtle differences in receptor interactions with particular opioid peptide residues, especially Phe residues in the N-terminal “message” sequences. Solid phase synthetic methodology for the incorporation of other labels (e.g. biotin) into the C-terminus of peptides has also been developed in our laboratory (Kumar and Aldrich Org Lett 5, 613, 2003). These two synthetic approaches have been combined to prepare peptides containing multiple labels that can be used as tools to study peptide ligand-receptor interactions. These solid phase synthetic methodologies are versatile strategies that are applicable to the preparation of labeled peptides for a variety of targets in addition to opioid receptors.     

HIV-1 Gag P17m的融合表达、纯化及NMT有效底物

 利用PCR技术扩增编码HIV 1GagP17m的DNA片段 ,进而构建了T7启动子控制下的C端His Tag融合表达质粒pMF P17mHT .SDS PAGE分析结果表明 ,融合蛋白 (约 2 1kD)在大肠杆菌BL2 1(DE3)中得到高表达 ,表达产物约占全菌蛋白 2 0 % .该融合蛋白主要以可溶性形式存在 ,通过金属离子 (Ni2 +)螯合亲和层析予以纯化 ,纯度在 90 %以上 .体外标记结果表明 ,融合表达的P17mHT能被人NMT有效地N端豆蔻酰化 .这些结果为深入研究筛选HIV 1GagP17或P55的豆蔻酰化专一性抑制剂 ,奠定了良好的基础     

Stereoselectivity of Mucorales lipases toward triradylglycerols--a simple solution to a complex problem

The lipases from Rhizopus and Rhizomucor are members of the family of Mucorales lipases. Although they display high sequence homology, their stereoselectivity toward triradylglycerols (sn-2 substituted triacylglycerols) varies. Four different triradylglycerols were investigated, which were classified into two groups: flexible substrates with rotatable O'-C1' ether or ester bonds adjacent to C2 of glycerol and rigid substrates with a rigid N'-C1' amide bond or a phenyl ring in sn-2. Although Rhizopus lipase shows opposite stereopreference for flexible and rigid substrates (hydrolysis in sn-1 and sn-3, respectively), Rhizomucor lipase hydrolyzes both groups of triradylglycerols preferably in sn-1. To explain these experimental observations, computer-aided molecular modeling was applied to study the molecular basis of stereoselectivity. A generalized model for both lipases of the Mucorales family highlights the residues mediating stereoselectivity: (1) L258, the C-terminal neighbor of the catalytic histidine, and (2) G266, which is located in a loop contacting the glycerol backbone of a bound substrate. Interactions with triradylglycerol substrates are dominated by van der Waals contacts. Stereoselectivity can be predicted by analyzing the value of a single substrate torsion angle that discriminates between sn-1 and sn-3 stereopreference for all substrates and lipases investigated here. This simple model can be easily applied in enzyme and substrate engineering to predict Mucorales lipase variants and synthetic substrates with desired stereoselectivity.     

One-step metal-affinity purification of histidine-tagged proteins by temperature-triggered precipitation

The feature of elastin-like proteins (ELPs) to reversibly precipitate above their transition temperature was exploited as a general method for the purification of histidine (His)-tagged proteins. The principle of the single-step metal-affinity method is based on coordinated ligand-bridging between the modified ELPs and the target proteins. ELPs with repeating sequences of [(VPGVG)(2)(VPGKG)(VPGVG)(2)](21) were synthesized and the free amino groups on the lysine residues were modified by reacting with imidazole-2-carboxyaldehyde to incorporate the metal-binding ligands into the ELP bio- polymers. Biopolymers charged with Ni(2+) were able to interact with a His tag on the target proteins based on metal coordination chemistry. Purifications of two His-tagged enzymes, beta-D-galactosidase and chloramphenicol acetyltransferase, were used to demonstrate the utility of this general method and over 85% recovery was observed in both cases. The bound enzymes were easily released by addition of either EDTA or imidazole. The recovered ELPs were reused four times with no observable decrease in the purification performance.     

Functional over-expression of the Stm1 protein,a G-protein-coupled receptor,in Schizosaccharomyces pombe

We report here the first functional over-expression of the Stm1 protein, a G-protein-coupled receptor with seven-trans-membrane spanning regions, in a homologous expression system without internal modification of the open reading frame of Stm1. The entire coding sequence, except for the termination codon followed by a C-terminal His6 tag, has been cloned into the pREP1 vector. The functionally active Stm1-His6 was over-expressed in Schizosaccharomyces pombe under the control of the nmt1 (no message in thiamine) promoter. The expression after induction was 120 times as much as that of control before induction and it gave 500 ng protein/2 × 10⁷cells.     

Conformational strictness required for maximum activity and stability of bovine pancreatic ribonuclease A as revealed by crystallographic study of three Phe120 mutants at 1.4 Å resolution

The replacement of Phe120 with other hydrophobic residues causes a decrease in the activity and thermal stability in ribonuclease A (RNase A). To explain this, the crystal structures of wild-type RNase A and three mutants--F120A, F120G, and F120W--were analyzed up to a 1.4 A resolution. Although the overall backbone structures of all mutant samples were nearly the same as that of wild-type RNase A, except for the C-terminal region of F120G with a high B-factor, two local conformational changes were observed at His119 in the mutants. First, His119 of the wild-type and F120W RNase A adopted an A position, whereas those of F120A and F120G adopted a B position, but the static crystallographic position did not reflect either the efficiency of transphosphorylation or the hydrolysis reaction. Second, His119 imidazole rings of all mutant enzymes were deviated from that of wild-type RNase A, and those of F120W and F120G appeared to be "inside out" compared with that of wild-type RNase A. Only approximately 1 A change in the distance between N(epsilon2) of His12 and N(delta1) of His119 causes a drastic decrease in k(cat), indicating that the active site requires the strict positioning of the catalytic residues. A good correlation between the change in total accessible surface area of the pockets on the surface of the mutant enzymes and enthalpy change in their thermal denaturation also indicates that the effects caused by the replacements are not localized but extend to remote regions of the protein molecule.     

Elaborate manifold of short hydrogen bond arrays mediating binding of active site-directed serine protease inhibitors

An extensive structural manifold of short hydrogen bond-mediated, active site-directed, serine protease inhibition motifs is revealed in a set of over 300 crystal structures involving a large suite of small molecule inhibitors (2-(2-phenol)-indoles and 2-(2-phenol)-benzimidazoles) determined over a wide range of pH (3.5-11.4). The active site hydrogen-bonding mode was found to vary markedly with pH, with the steric and electronic properties of the inhibitor, and with the type of protease (trypsin, thrombin or urokinase type plasminogen activator (uPA)). The pH dependence of the active site hydrogen-bonding motif is often intricate, constituting a distinct fingerprint of each complex. Isosteric replacements or minor substitutions within the inhibitor that modulate the pK(a) of the phenol hydroxyl involved in short hydrogen bonding, or that affect steric interactions distal to the active site, can significantly shift the pH-dependent structural profile characteristic of the parent scaffold, or produce active site-binding motifs unique to the bound analog.Ionization equilibria at the active site associated with inhibitor binding are probed in a series of the protease-inhibitor complexes through analysis of the pH dependence of the structure and environment of the active site-binding groups involved in short hydrogen bond arrays. Structures determined at high pH (>11), suggest that the pK(a) of His57 is dramatically elevated, to a value as high as approximately 11 in certain complexes. K(i) values involving uPA and trypsin determined as a function of pH for a set of inhibitors show pronounced parabolic pH dependence, the pH for optimal inhibition governed by the pK(a) of the inhibitor phenol involved in short hydrogen bonds. Comparison of structures of trypsin, thrombin and uPA, each bound by the same inhibitor, highlights important structural variations in the S1 and active sites accessible for engineering notable selectivity into remarkably small molecules with low nanomolar K(i) values.     

Cytochrome c folds through a smooth funnel

A dominant feature of folding of cytochrome c is the presence of nonnative His-heme kinetic traps, which either pre-exist in the unfolded protein or are formed soon after initiation of folding. The kinetically trapped species can constitute the majority of folding species, and their breakdown limits the rate of folding to the native state. A temperature jump (T-jump) relaxation technique has been used to compare the unfolding/folding kinetics of yeast iso-2 cytochrome c and a genetically engineered double mutant that lacks His-heme kinetic traps, H33N,H39K iso-2. The results show that the thermodynamic properties of the transition states are very similar. A single relaxation time tau(obs) is observed for both proteins by absorbance changes at 287 nm, a measure of solvent exclusion from aromatic residues. At temperatures near Tm, the midpoint of the thermal unfolding transitions, tau(obs) is four to eight times faster for H33N,H39K iso-2 (tau(obs) approximately 4-10 ms) than for iso-2 (tau(obs) approximately 20-30 ms). T-jumps show that there are no kinetically unresolved (tau < 1-3 micros T-jump dead time) "burst" phases for either protein. Using a two-state model, the folding (k(f)) and unfolding (k(u)) rate constants and the thermodynamic activation parameters standard deltaGf, standard deltaGu, standard deltaHf, standard deltaHu, standard deltaSf, standard deltaSu are evaluated by fitting the data to a function describing the temperature dependence of the apparent rate constant k(obs) (= tau(obs)(-1)) = k(f) + k(u). The results show that there is a small activation enthalpy for folding, suggesting that the barrier to folding is largely entropic. In the "new view," a purely entropic kinetic barrier to folding is consistent with a smooth funnel folding landscape.     

Konjac ceramide (kCer) regulates keratinocyte migration by Sema3A-like repulsion mechanism

Previously, we proposed the following mechanism for konjac ceramide (kCer)-mediated neurite outgrowth inhibition: kCer binds to Nrp as a Sema3A agonist, resulting in Nrp1/PlexA complex formation and activation of the Sema3A signaling pathway to induce phosphorylation of CRMP2 and microtubule depolymerization. The Sema3A/Nrp1 signaling pathway is known to be also expressed in normal human keratinocytes. To determine whether kCer can function in human keratinocytes as it does in neurites, that is, if it can bind to Nrp1 in place of Sema3A, we studied the effect of kCer on HaCaT cell migration activity. Using a trans-well chamber assay, we compared the effects of Sema3A and kCer on serum-derived cell migration activity. kCer showed Sema3A-like suppression of cell migration activity and induction of cellular Cofilin phosphorylation. In addition, kCer and Sema3A inhibited histamine (His)-enhanced migration of immature HaCaT cells. We have demonstrated that kCer does not interact with histaime receptors H1R or H4R directly, but we speculate that kCer may transduce a signal downstream of the His signaling pathway.     

Empirical rules for rationalising visible circular dichroism of Cu2+ and Ni2+ histidine complexes: applications to the prion protein

A natively unfolded region of the prion protein, PrP(90-126) binds Cu(2+) ions and is vital for prion propagation. Pentapeptides, acyl-GGGTH(92-96) and acyl-TNMKH(107-111), represent the minimum motif for this Cu(2+) binding region. EPR and (1)H NMR suggests that the coordination geometry for the two binding sites is very similar. However, the visible CD spectra of the two sites are very different, producing almost mirror image spectra. We have used a series of analogues of the pentapeptides containing His(96) and His(111) to rationalise these differences in the visible CD spectra. Using simple histidine-containing tri-peptides we have formulated a set of empirical rules that can predict the appearance of Cu(2+) visible CD spectra involving histidine and amide main-chain coordination.