Structural Transformation Detection Contributes to Screening of Behaviorally Active Compounds: Dynamic Binding Process Analysis of DhelOBP21 from <Emphasis Type="Italic">Dastarcus helophoroides</Emphasis>

In light of reverse chemical ecology, the fluorescence competitive binding assays of functional odorant binding proteins (OBPs) is a recent advanced approach for screening behaviorally active compounds of insects. Previous research on Dastareus helophoroides identified a minus-C OBP, DhelOBP21, which preferably binds to several ligands. In this study, only (+)-β-pinene proved attractive to unmated adult beetles. To obtain a more in-depth explanation of the lack of behavioral activity of other ligands we selected compounds with high (camphor) and low (β-caryophyllene) binding affinities. The structural transformation of OBPs was investigated using well-established approaches for studying binding processes, such as fluorescent quenching assays, circular dichroism, and molecular dynamics. The dynamic binding process revealed that the flexibility of DhelOBP21 seems conducive to binding specific ligands, as opposed to broad substrate binding. The compound (+)-β-pinene and DhelOBP21 formed a stable complex through a secondary structural transformation of DhelOBP21, in which its amino-terminus transformed from random coil to an α-helix to cover the binding pocket. On the other hand, camphor could not efficiently induce a stable structural transformation, and its high binding affinities were due to strong hydrogen-bonding, compromising the structure of the protein. The other compound, β-caryophyllene, only collided with DhelOBP21 and could not be positioned in the binding pocket. Studying structural transformation of these proteins through examining the dynamic binding process rather than using approaches that just measure binding affinities such as fluorescence competitive binding assays can provide a more efficient and reliable approach for screening behaviorally active compounds.     

Conformation changes of polyphenol oxidase and lipoxygenase induced by PEF treatment

Inactivation of polyphenol oxidase (PPO) and lipoxygenase (LOX) by pulsed electric fields (PEFs) has been investigated using a coaxial treatment chamber. Circular dichroism (CD) and fluorescence analysis have been used to study conformation changes in the protein. The experimental results show that PPO and LOX can be effectively deactivated by the PEF treatment and that the effect on PPO and LOX increases with the increase of the applied electric field and the number of pulses. The activity of PPO and LOX can be reduced by 69 and 88% when fields of 24 kV/cm were applied for 320 and 962 μs, respectively. The CD analysis showed that the PEF treatment caused a loss of α-helix and increase of β-sheet content, indicating that conformation changes occur in the secondary structure of the PPO and LOX enzymes. The fluorescence intensity of LOX increases after the PEF treatment while, at the same time, increases in the applied electric field increases the intensity of the fluorescence emitted. These results prove the occurrence of local tertiary structure changes in the LOX protein.     

Impact of Azidohomoalanine Incorporation on Protein Structure and Ligand Binding

The impact of the incorporation of a non‐natural amino acid (NNAA) on protein structure, dynamics, and ligand binding has not been studied rigorously so far. NNAAs are regularly used to modify proteins post‐translationally in vivo and in vitro through click chemistry. Herein, structural characterisation of the impact of the incorporation of azidohomoalanine (AZH) into the model protein domain PDZ3 is examined by means of NMR spectroscopy and X‐ray crystallography. The structure and dynamics of the apo state of AZH‐modified PDZ3 remain mostly unperturbed. Furthermore, the binding of two PDZ3 binding peptides are unchanged upon incorporation of AZH. The interface of the AZH‐modified PDZ3 and an azulene‐linked peptide for vibrational energy transfer studies has been mapped by means of chemical shift perturbations and NOEs between the unlabelled azulene‐linked peptide and the isotopically labelled protein. Co‐crystallisation and soaking failed for the peptide‐bound holo complex. NMR spectroscopy, however, allowed determination of the protein–ligand interface. Although the incorporation of AZH was minimally invasive for PDZ3, structural analysis of NNAA‐modified proteins through the methodology presented herein should be performed to ensure structural integrity of the studied target.     

Design of Lanthanide Fingers: Compact Lanthanide‐Binding Metalloproteins

Lanthanides have interesting chemical properties; these include luminescent, magnetic, and catalytic functions. Toward the development of proteins incorporating novel functions, we have designed a new lanthanide‐binding motif, lanthanide fingers. These were designed based on the Zif268 zinc finger, which exhibits a ββα structural motif. Lanthanide fingers utilize an Asp₂Glu₂ metal‐coordination environment to bind lanthanides through a tetracarboxylate peptide ligand. The iterative design of a general lanthanide‐binding peptide incorporated the following key elements: 1) residues with high α‐helix and β‐sheet propensities in the respective secondary structures; 2) an optimized big box α‐helix N‐cap; 3) a Schellman α‐helix C‐cap motif; and 4) an optional D ‐Pro‐Ser type II’ β‐turn in the β‐hairpin. The peptides were characterized for lanthanide binding by circular dichroism (CD), NMR, and fluorescence spectroscopy. In all instances, stabilization of the peptide secondary structures resulted in an increase in metal affinity. The optimized protein design was a 25‐residue peptide that was a general lanthanide‐binding motif; this binds all lanthanides examined in a competitive aqueous environment, with a dissociation constant of 9.3 μM for binding Er³⁺. CD spectra of the peptide‐lanthanide complexes are similar to those of zinc fingers and other ββα proteins. Metal binding involves residues from the N‐terminal β‐hairpin and the C terminal α‐helical segments of the peptide. NMR data indicated that metal binding induced a global change in the peptide structure. The D ‐Pro‐Ser type II’ β‐turn motif could be replaced by Thr–Ile to generate genetically encodable lanthanide fingers. Replacement of the central Phe with Trp generated genetically encodable lanthanide fingers that exhibited terbium luminescence greater than that of an EF‐hand peptide.     

Nucleotide-induced conformational transitions in the CBS domain protein MJ0729 of Methanocaldococcus jannaschii

Nucleotide-binding cystathionine β-synthase (CBS) domains function as regulatory motifs in several proteins distributed through all kingdoms of life. This function has been proposed based on their affinity for adenosyl-derivatives, although the exact binding mechanisms remain largely unknown. The question of how CBS domains exactly work is relevant because in humans, several genetic diseases have been associated with mutations in those motifs. In this work, we describe the adenosyl-ligand (AMP, ATP, NADP and SAM) properties of the wild-type CBS domain protein MJ0729 from Methanocaldococcus jannaschii by using a combination of spectroscopic techniques (fluorescence, FTIR and FRET). The fluorescence results show that binding to AMP and ATP occurs with an apparent dissociation constant of ~10 μM, and interestingly enough, binding induces protein conformational changes, as shown by FTIR. On the other hand, fluorescence spectra (FRET and steady-state) did not change upon addition of NADP and SAM to MJ0729, suggesting that tryptophan and/or tyrosine residues were not involved in the recognition of those ligands; however, there were changes in the secondary structure of the protein upon addition of NADP and SAM, as shown by FTIR (thus, indicating binding to the nucleotide). Taken together, these results suggest that: (i) the adenosyl ligands bind to MJ0729 in different ways, and (ii) there are changes in the protein secondary structure upon binding of the nucleotides.     

Peptides and proteins as a continuing exciting source of inspiration for peptidomimetics

Despite their enormous diversity in biological function and structure, peptides and proteins are endowed with properties that have induced and stimulated the development of peptidomimetics. Clearly, peptides can be considered as the "stem" of a phylogenetic molecular development tree from which branches of oligomeric peptidomimetics such as peptoids, peptidosulfonamides, urea peptidomimetics, as well as β-peptides have sprouted. It is still a challenge to efficiently synthesize these oligomeric species, and study their structural and biological properties. Combining peptides and peptidomimetics led to the emergence of peptide-peptidomimetic hybrids in which one or more (proteinogenic) amino acid residues have been replaced with these mimetic residues. In scan-like approaches, the influence of these replacements on biological activity can then be studied, to evaluate to what extent a peptide can be transformed into a peptidomimetic structure while maintaining, or even improving, its biological properties. A central issue, especially with the smaller peptides, is the lack of secondary structure. Important approaches to control secondary structure include the introduction of α,α-disubstituted amino acids, or (di)peptidomimetic structures such as the Freidinger lactam. Apart from intra-amino acid constraints, inter-amino acid constraints for formation of a diversity of cyclic peptides have shaped a thick branch. Apart from the classical disulfide bridges, the repertoire has been extended to include sulfide and triazole bridges as well as the single-, double- and even triple-bond replacements, accessible by the extremely versatile ring-closing alkene/alkyne metathesis approaches. The latter approach is now the method of choice for the secondary structure that presents the greatest challenge for structural stabilization: the α-helix.     

Direct electron transfer and conformational change of glucose oxidase on carbon nanotube-based electrodes

To gain insights into the direct electron transfer (DET) mechanism of multi-walled carbon nanotubes (MWCNTs), we investigated the conformational changes that occur in proteins when they interact with MWCNTs. We used glucose oxidase (GOD) as an example. Using cyclic voltammetry measurements, the GOD that was immobilized on the MWCNT-modified carbon paper electrode exhibited apparent direct electrochemistry compared to that on the bare electrode without MWCNTs. The structural transformation of GOD upon adsorption on the MWCNTs was characterized spectrally. GOD was not denatured, and only small shifts of the wavenumber of the β-sheet structure were observed. There was a consistent tendency for the amount of α-helix to decrease and the β-sheet to increase. The α-helix content dropped from 21.2% to 19.6% as measured using Fourier transform infrared spectroscopy and from 27.1% to 25.9% as measured using circular dichroism. The reduction in the amount of α-helix led to a less shielded GOD active site and weakened the resistance of the electron transfer. These MWCNT-induced conformational changes could account for the DET between GOD and the MWCNT-modified electrode surface.     

Hetero‐bivalent GLP‐1/Glibenclamide for Targeting Pancreatic β‐Cells

G protein‐coupled receptor (GPCR) cell signalling cascades are initiated upon binding of a specific agonist ligand to its cell surface receptor. Linking multiple heterologous ligands that simultaneously bind and potentially link different receptors on the cell surface is a unique approach to modulate cell responses. Moreover, if the target receptors are selected based on analysis of cell‐specific expression of a receptor combination, then the linked binding elements might provide enhanced specificity of targeting the cell type of interest, that is, only to cells that express the complementary receptors. Two receptors whose expression is relatively specific (in combination) to insulin‐secreting pancreatic β‐cells are the sulfonylurea‐1 (SUR1) and the glucagon‐like peptide‐1 (GLP‐1) receptors. A heterobivalent ligand was assembled from the active fragment of GLP‐1 (7–36 GLP‐1) and glibenclamide, a small organic ligand for SUR1. The synthetic construct was labelled with Cy5 or europium chelated in DTPA to evaluate binding to β‐cells, by using fluorescence microscopy or time‐resolved saturation and competition binding assays, respectively. Once the ligand binds to β‐cells, it is rapidly capped and presumably removed from the cell surface by endocytosis. The bivalent ligand had an affinity approximately fivefold higher than monomeric europium‐labelled GLP‐1, likely a result of cooperative binding to the complementary receptors on the βTC3 cells. The high‐affinity binding was lost in the presence of either unlabelled monomer, thus demonstrating that interaction with both receptors is required for the enhanced binding at low concentrations. Importantly, bivalent enhancement was accomplished in a cell system with physiological levels of expression of the complementary receptors, thus indicating that this approach might be applicable for β‐cell targeting in vivo.     

Protein flexibility and ligand rigidity: a thermodynamic and kinetic study of ITAM-based ligand binding to Syk tandem SH2

The Syk tandem Src homology 2 domain (Syk tSH2) constitutes a flexible protein module involved in the regulation of Syk kinase activity. The Syk tSH2 domain is assumed to function by adapting the distance between its two SH2 domains upon bivalent binding to diphosphotyrosine ligands. A thermodynamic and kinetic analysis of ligand binding was performed by using surface plasmon resonance (SPR). Furthermore, the effect of binding on the Syk tSH2 structural dynamics was probed by hydrogen/deuterium exchange and electrospray mass spectrometry (ESI-MS). Two ligands were studied: 1, a flexible peptide derived from the tSH2 recognition ITAM sequence at the gamma chain of the FcepsilonRI-receptor, and 2, a ligand in which the amino acids between the two SH2 binding motifs in ligand 1 have been replaced by a rigid linker of comparable length. Both ligands display comparable affinity for Syk tSH2 at 25 degrees C, yet a major difference in thermodynamics is observed. Upon binding of the rigid ligand, 2, the expected entropy advantage is not realized. On the contrary, 2 binds with a considerably higher entropy price of approximately 9 kcal mol-1, which is attributed to a further decrease in protein flexibility upon binding to this rigid ligand. The significant reduction in deuterium incorporation in the Syk tSH2 protein upon binding of either 1 or 2, as monitored by ESI-MS, indicates a major reduction in protein dynamics upon binding. The results are consistent with a two-step binding model: after an initial binding step, a rapid structural change of the protein occurs, followed by a second binding step. Such a bivalent binding model allows high affinity and fast dissociation kinetics, which are very important in transient signal-transduction processes.     

NMR chemical shift perturbation study of the N-terminal domain of Hsp90 upon binding of ADP,AMP-PNP,geldanamycin, and radicicol

Hsp90 is one of the most abundant chaperone proteins in the cytosol. In an ATP-dependent manner it plays an essential role in the folding and activation of a range of client proteins involved in signal transduction and cell cycle regulation. We used NMR shift perturbation experiments to obtain information on the structural implications of the binding of AMP-PNP (adenylyl-imidodiphosphate-a non-hydrolysable ATP analogue), ADP and the inhibitors radicicol and geldanamycin. Analysis of (1)H,(15)N correlation spectra showed a specific pattern of chemical shift perturbations at N210 (ATP binding domain of Hsp90, residues 1-210) upon ligand binding. This can be interpreted qualitatively either as a consequence of direct ligand interactions or of ligand-induced conformational changes within the protein. All ligands show specific interactions in the binding site, which is known from the crystal structure of the N-terminal domain of Hsp90. For AMP-PNP and ADP, additional shift perturbations of residues outside the binding pocket were observed and can be regarded as a result of conformational rearrangement upon binding. According to the crystal structures, these regions are the first alpha-helix and the "ATP-lid" ranging from amino acids 85 to 110. The N-terminal domain is therefore not a passive nucleotide-binding site, as suggested by X-ray crystallography, but responds to the binding of ATP in a dynamic way with specific structural changes required for the progression of the ATPase cycle.     

Analysis of Metal Effects on C-Peptide Structure and Internalization

The connecting peptide (C-peptide) has received increased attention for its potential therapeutic effects in ameliorating illnesses such as kidney disease and diabetes. Although the mechanism of C-peptide signaling remains elusive, evidence supports its internalization and intracellular function. Emerging research is uncovering the diverse biological roles metals play in controlling and affecting the function of bioactive peptides. The work presented herein investigates interactions between C-peptide and first-row d-block transition metals, as well as their effects on C-peptide internalization into cells. Through spectroscopic techniques, it is demonstrated that Cr^III, Cu^II, and Zn^II bind to C-peptide with differing stoichiometries and biologically relevant affinities. In addition, metal binding elicits both subtle changes in secondary structure and inhibits adoption of an α-helical character in environments where the dielectric constants are reduced. This study shows how metal ions can modulate peptide hormone activity through subtle structural changes to disrupt cellular uptake.     

Structure‐Guided Rational Design of α/β‐Peptide Foldamers with High Affinity for BCL‐2 Family Prosurvival Proteins

We have used computational methods to improve the affinity of a foldamer ligand for its target protein. The effort began with a previously reported α/β‐peptide based on the BH3 domain of the proapoptotic protein Puma; this foldamer binds tightly to Bcl‐x_L but weakly to Mcl‐1. The crystal structure of the Puma‐derived α/β‐peptide complexed to Bcl‐x_L was used as the basis for computational design of variants intended to display improved binding to Mcl‐1. Molecular modelling suggested modification of three α residues of the original α/β backbone. Individually, each substitution caused only a modest (4‐ to 15‐fold) gain in affinity; however, together the three substitutions led to a 250‐fold increase in binding to Mcl‐1. These modifications had very little effect on affinity for Bcl‐x_L. Crystal structures of a number of the new α/β‐peptides bound to either Mcl‐1 or Bcl‐x_L validated the selection of each substitution. Overall, our findings demonstrate that structure‐guided rational design can be used to improve affinity and alter partner selectivity of peptidic ligands with unnatural backbones that bind to specific protein partners.     

黄鳝血清转铁蛋白的提取及表征

对从黄鳝血液中提取的蛋白层析纯化后进行生物质谱测试,确定为血清转铁蛋白. 利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、原子力显微镜、圆二色谱法对蛋白的分子量、表面形貌、二级结构进行了表征. 结果表明,该转铁蛋白分子量为74 kDa,含量为0.750 mg/mL,含a-螺旋29.8%,b-折叠6.6%.     

Structural and conformational changes of regenerated Antheraea pernyi silk fibroin films treated with methanol solution

The regenerated Antheraea pernyi silk fibroin films were prepared from calcium nitrate solution and crystallized with aqueous methanol solution. The structural and conformational changes were investigated by the X-ray diffraction method and infrared spectroscopy upon methanol treatment. The concentration and treatment time of aqueous methanol solution have a great influence on the conformation of regenerated films. The conformational change from a random coil to β-sheet structure was occurred within 5 min by the treatment of 40–60% methanol solution. As the methanol concentration increases up to 100%, the transition time of conformational change is delayed, and the structural change is not occurred in the case of 100% methanol used. The content of a α-helix, β-sheet, and random coil conformation was calculated and examined to find out the effect of methanol treatment on the conformation changes. The β-sheet structure can be transformed from a random coil, while the content of α-helix structure is not changed, regardless of methanol treatment. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 73: 2887–2894, 1999     

Molecular modeling of interleukin-8 receptor beta and analysis of the receptor-ligand interaction

A structural model of interleukin-8 receptor type beta (IL-8R-beta) was constructed based on the structure of bacteriorhodopsin. High temperature molecular dynamics simulations were performed to search the possible conformations of loop regions in IL-8R-beta which recognize the ligand. The crystal structure of interleukin 8 (IL-8) was used as a geometric constraint of the extracellular loop regions of IL-8R-beta in the conformational search. 500 complex structures were extracted from the dynamics trajectory and five plausible models were selected based on the binding energy and known experimental data. To study further the interaction between IL-8R-beta and its ligands, the complex of IL-8R- beta and platelet factor 4 (PF4) C-terminal peptide was also modeled by molecular dynamics simulations. From these models, the N-terminus, extracellular domain 3 and extracellular domain 4 of IL-8R-beta were found to be important for ligand binding. Key residues of these regions involved in ligand binding were characterized. These models provide insight into the structural basis of biological activity of IL-8 and PF4 and may guide the design of potential therapeutic agents targeting IL-8 receptors. Furthermore, the approach developed from this study may have implications for the understanding of other chemokine receptor- ligand interactions that have been recently suggested to be involved in HIV infection.      

几种不同来源食用明胶的蛋白二级结构分析及安全性能比较

用傅里叶变换红外光谱仪和圆二色谱仪对四种不同来源食用明胶的蛋白二级结构组成、重金属含量进行了分析。结果表明,四种食用明胶的二级结构均以β-sheet为主,且含有β-turn结构,其中鱼鳞明胶和猪皮明胶还含有大量的310-helix结构,鱼皮明胶和猪皮明胶含有α-helix结构,鱼鳞明胶和牛骨明胶的无序结构含量较高。四个明胶样品的重金属指标均符合国内相关标准,其中牛骨明胶的重金属含量最低,安全性最高。     

3- Instead of 4-helix formation in a de novo designed protein in solution revealed by small-angle X-ray scattering

De novo design and chemical synthesis of proteins and their mimics are central approaches for understanding protein folding and accessing proteins with novel functions. We have previously described carbohydrates as templates for the assembly of artificial proteins, so-called carboproteins. Here, we describe the preparation and structural studies of three alpha-helical bundle carboproteins, which were assembled from three different carbohydrate templates and one amphiphilic hexadecapeptide sequence. This heptad repeat peptide sequence has been reported to lead to 4-alpha-helix formation. The low resolution solution structures of the three carboproteins were analyzed by means of small-angle X-ray scattering (SAXS) and synchrotron radiation circular dichroism (SRCD). The ab initio SAXS data analysis revealed that all three carboproteins adopted an unexpected 3+1-helix folding topology in solution, while the free peptide formed a 3-helix bundle. This finding is consistent with the calculated alpha-helicities based on the SRCD data, which are 72 and 68 % for two of the carboproteins. The choice of template did not affect the overall folding topology (that is for the 3+1 helix bundle) the template did have a noticeable impact on the solution structure. This was particularly evident when comparing 4-helix carboprotein monomers with the 2x2-helix carboprotein dimer as the latter adopted a more compact conformation. Furthermore, the clear conformational differences observed between the two 4-helix (3+1) carboproteins based on D-altropyranoside and D-galactopyranoside support the notion that folding is affected by the template, and subtle variations in template distance-geometry design may be exploited to control the solution fold. In addition, the SRCD data show that template assembly significantly increases thermostability.     

An Engineered Calmodulin‐Based Allosteric Switch for Peptide Biosensing

This work describes the development of a new platform for allosteric protein engineering that takes advantage of the ability of calmodulin to change conformation upon binding to peptide and protein ligands. The switch we have developed consists of a fusion protein in which calmodulin is genetically inserted into the sequence of TEM1 β‐lactamase. In this approach, calmodulin acts as the input domain, whose ligand‐dependent conformational changes control the activity of the β‐lactamase output domain. The new allosteric enzyme exhibits up to 120 times higher catalytic activity in the activated (peptide bound) state compared to the inactive (no peptide bound) state in vitro. Activation of the enzyme is ligand‐dependent—peptides with higher affinities for wild‐type calmodulin exhibit increased switch activity. Calmodulin's ability to “turn on” the activity of β‐lactamase makes this a potentially valuable scaffold for the directed evolution of highly specific biosensors for detecting toxins and other clinically relevant biomarkers.     

Trastuzumab-Peptide Interactions: Mechanism and Application in Structure-Based Ligand Design

Understanding of protein-ligand interactions and its influences on protein stability is necessary in the research on all biological processes and correlative applications, for instance, the appropriate affinity ligand design for the purification of bio-drugs. In this study, computational methods were applied to identify binding site interaction details between trastuzumab and its natural receptor. Trastuzumab is an approved antibody used in the treatment of human breast cancer for patients whose tumors overexpress the HER2 (human epidermal growth factor receptor 2) protein. However, rational design of affinity ligands to keep the stability of protein during the binding process is still a challenge. Herein, molecular simulations and quantum mechanics were used on protein-ligand interaction analysis and protein ligand design. We analyzed the structure of the HER2-trastuzumab complex by molecular dynamics (MD) simulations. The interaction energies of the mutated peptides indicate that trastuzumab binds to ligand through electrostatic and hydrophobic interactions. Quantitative investigation of interactions shows that electrostatic interactions play the most important role in the binding of the peptide ligand. Prime/MM-GBSA calculations were carried out to predict the binding affinity of the designed peptide ligands. A high binding affinity and specificity peptide ligand is designed rationally with equivalent interaction energy to the wild-type octadecapeptide. The results offer new insights into affinity ligand design.