Theoretical and experimental studies of biotin analogues that bind almost as tightly to streptavidin as biotin

We have used a newly developed qualitative computational approach, PROFEC (Pictorial Representation of Free Energy Changes), to visualize the areas of the ligand biotin where modifications of its structure might lead to tighter binding to the protein streptavidin. The PROFEC analysis, which includes protein flexibility and ligand solvation/desolvation, led to the suggestion that the pro-9R hydrogen atom of biotin, which is in alpha-position to the CO(2)(-) group, might be changed to a larger group and lead to better binding with streptavidin and avidin. Free energy calculations supported this suggestion and predicted that the methyl analogue should bind approximately 3 kcal/mol more tightly to streptavidin, with this difference coming exclusively from the relative desolvation free energy of the ligand. The PROFEC analysis further suggested little or no improvement for changing the pro-9S hydrogen atom to a methyl group, and great reduction in changing the ureido N-H groups to N-CH(3). Stimulated by these results, we synthesized 9R-methylbiotin and 9S-methylbiotin, and their binding free energies and enthalpies were measured for interaction with streptavidin and avidin, respectively. In contrast to the calculated results, experiments found both 9-methylbiotin isomers to bind more weakly to streptavidin than biotin. The calculated preference for the binding of the 9R- over the 9S-stereoisomer was observed. In addition, 9-methylbiotin is considerably less soluble in water than biotin, as predicted by the calculation, and the 9R isomer is, to our knowledge, thus far the tightest binding analogue of biotin to streptavidin. Subsequently, X-ray structures of the complexes between streptavidin and both 9R- and 9S-methylbiotin were determined, and the structures were consistent with those used in the free energy calculations. Thus, the reason for the discrepancy between the calculated and experimental binding free energy does not lie in unusual binding modes for the 9-methylbiotins.     

Synthesis of 3-alkyl enol mimics inhibitors of type II dehydroquinase: factors influencing their inhibition potency

Several 3-alkylaryl mimics of the enol intermediate in the reaction catalyzed by type II dehydroquinase were synthesized to investigate the effect on the inhibition potency of replacing the oxygen atom in the side chain by a carbon atom. The length and the rigidity of the spacer was also studied. The inhibitory properties of the reported compounds against type II dehydroquinase from Mycobacterium tuberculosis and Helicobacter pylori are also reported. The binding modes of these analogs in the active site of both enzymes were studied by molecular docking using GOLD 5.0 and dynamic simulations studies.     

Electrochemical Shell-Isolated Nanoparticle-Enhanced Raman Spectroscopy of Imidazole Ring Functionalized Monolayer on Smooth Gold Electrode

The imidazole ring (Im) of histidine side chains plays a unique role in the function of proteins through covalent bonding with metal ions and hydrogen bonding interactions with adjusted biomolecules and water. At biological interfaces, these interactions are modified because of the presence of an electric field. Self-assembled monolayers (SAMs) with the functional Im group mimic the histidine side chain at electrified interfaces. In this study, we applied in-situ shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS) to probe the structure and hydrogen bonding of Im-functionalized SAM on smooth Au at the electrochemical interface. The self-assembly of molecules on the Au induced the proton shift from N1 atom (Tautomer-I), which is the dominant form of Im in the bulk sample, to N3 atom (Tautomer-II). The impact of electrode potential on the hydrogen bonding interaction strength of the Im ring was identified by SHINERS. Temperature-Raman measurements and density functional theory (DFT) analysis revealed the spectral marker for Im ring packing (mode near 1496–1480 cm⁻¹) that allowed us to associate the confined and strongly hydrogen bonded interfacial Im groups with electrode polarization at −0.8 V. Reflection adsorption IR (RAIR) spectra of SAMs with and without Im revealed that the bulky ring prevented the formation of a strongly hydrogen bonded amide group network.     

Ferreting out correlations from trajectory data

Thermally driven materials characterized by complex energy landscapes, such as proteins, exhibit motions on a broad range of space and time scales. Principal component analysis (PCA) is often used to extract modes of motion from protein trajectory data that correspond to coherent, functional motions. In this work, two other methods, maximum covariance analysis (MCA) and canonical correlation analysis (CCA) are formulated in a way appropriate to analyze protein trajectory data. Both methods partition the coordinates used to describe the system into two sets (two measurement domains) and inquire as to the correlations that may exist between them. MCA and CCA provide rotations of the original coordinate system that successively maximize the covariance (MCA) or correlation (CCA) between modes of each measurement domain under suitable constraint conditions. We provide a common framework based on the singular value decomposition of appropriate matrices to derive MCA and CCA. The differences between and strengths and weaknesses of MCA and CCA are discussed and illustrated. The application presented here examines the correlation between the backbone and side chain of the peptide met-enkephalin as it fluctuates between open conformations, found in solution, to closed conformations appropriate to when it is bound to its receptor. Difficulties with PCA carried out in Cartesian coordinates are found and motivate a formulation in terms of dihedral angles for the backbone atoms and selected atom distances for the side chains. These internal coordinates are a more reliable basis for all the methods explored here. MCA uncovers a correlation between combinations of several backbone dihedral angles and selected side chain atom distances of met-enkephalin. It could be used to suggest residues and dihedral angles to focus on to favor specific side chain conformers. These methods could be applied to proteins with domains that, when they rearrange upon ligand binding, may have correlated functional motions or, for multi-subunit proteins, may exhibit correlated subunit motions.     

Theoretical study of cooperativity in biotin

To give a deeper insight into the widely discussed catalytic mechanism of biotin, four representative model molecules and their aggregates hydrogen bonding (H-bonding) to water molecules were investigated by means of ab initio calculations and compared with molecular dynamics simulations. The roles of the ureido group, the sulfur atom, and the side chain of biotin are examined and discussed, respectively. Some significant H-bonding cooperativities are theoretically demonstrated in the ureido group of biotin. The pi-electron delocalization of the ureido group makes the system a good candidate for the H-bonding cooperativities, which in turn increases the covalent character of the corresponding H-bonds and facilitates the electrophilic substitution of the nitrogen atoms in the ureido group. The sulfur of biotin may participate in the delocalized pi-electron system of the ureido group via special sulfur-nitrogen bonding interactions, which reinforces the H-bonding cooperativities of the ureido group. The side chain of biotin not only reduced the accessibility of 3-NH due to steric hindrance but also enhanced the H-bonding cooperativities of the ureido group by folding over to hydrogen bond to more water molecules. The folded states are a probable way of activating 1-NH by strong cooperative effects. In addition, the H-bonding cooperativities may be a significant reason for the strong and specific binding between biotin and streptavidin.     

EGFR和4-苯胺喹唑啉类抑制剂之间相互作用模式的研究

采用分子动力学和MM/PBSA相结合的方法预测了表皮生长因子受体和4-苯胺喹 啉类抑制剂的相互作用模式。在分子动力学采样的基础上,采用MM/PBSA的方法分 别预测了四种可能结合模式下表皮生长因子受体和4-苯胺喹唑啉类抑制剂间的结合 自由能。在MM/PBSA计算中,受体和抑制剂之间的非键相互作用能采用分子力学 (MM)的方法得到;溶剂效应中极性部分对自由能的贡献通过解Possion- Boltzmanne (PB)方程的方法得到;溶液效应中非极性部分对自由能的贡献则通过 分子表面积计算(SA)的方法得到。计算表明,在四种结合模式下,表皮生长因子受 体和4-苯胺喹唑啉类抑制剂之间的结合自由能有较大的差别。在最佳的相互作用模 式中,抑制剂的苯胺部分位于活性口袋的底部,能够与受体残基的非极性侧链产生 很强的范德华和疏水相互作用。抑制剂喹唑啉环上的N(1)原子能够和Met-769上的 NH形成稳定的氢键,而抑制剂上的N(3)原子则和周围的一个水分子形成氢键。同时 ,抑制剂双环上的取代基团也能和活性口袋外部的部分残基形成一定的范德华和疏 水相互作用。最佳结合模式能够很好地解释已有抑制剂结构和活性间的关系。     

Steroid nomenclature

Proposals for revision of nomenclature are as follows:

1. In a sapogenin or alkaloid in which the terminal carbon is linked to a hetero atom, this carbon atom, in accordance with the Fischer convention, acquires the number 27.

2. If, in a Fischer projection of an open side chain, methyl or hydroxyl at a given center lies to the left it is β-oriented; if it lies to the right it is -oriented.

3. In a sapogenin or alkaloid the true stereochemistry at any center in the side chain is described as the configuration at this center in the alcohol or amine derived by opening the side chain and making a Fischer projection. If the orientation so defined as is down (or rear) it is described as -oriented. The symbol _F is used to indicate that the configuration by Fischer projection is but that the orientation is up (or front).     

Copper(II) complex formation with a linear peptide encompassing the putative cell binding site of angiogenin

Angiogenin is one of the more potent angiogenic factors known, whose activity may be affected by the presence of copper ions. Copper(II) complexes with the peptides encompassing the putative endothelial cell binding domain of angiogenin, Ac-KNGNPHREN-NH(2) and Ac-PHREN-NH(2), have been characterized by potentiometric, UV-vis, CD and EPR spectroscopic methods. The coordination features of all the copper complex species derived by both peptides are practically the same, as predictable because of the presence of a proline residue within their aminoacidic sequence. In particular, Ac-PHREN-NH(2) is really the aminoacidic sequence involved in the binding to copper(II). Thermodynamic and spectroscopic evidence are given that side chain oxygen donor atom of glutamyl residue is involved in the copper binding up to physiological pH. EPR parameters suggest that the carboxylate group is still involved also in the predominant species [Cu(L)H(-2)], the metal coordination environment being probably formed by N(Im), 2N(-), H(2)O in equatorial plane and an oxygen atom from COO(-) in apical position, or vice versa, with the carboxylate oxygen atom in the copper coordination plane and the water molecule confined to one of the apical positions. Moreover, the comparison with the thermodynamic and spectroscopic results in the case of the copper(ii) complex species formed by the single point mutated peptide, Ac-PHRQN-NH(2), provides further evidence of the presence of carboxylate oxygen atom in the copper coordination sphere.     

catena‐Poly­[[di­aqua­(nicotinato‐κ2O,O′)­cadmium(II)]‐μ‐nicotinato‐κ3N:O,O′]

The title compound, [Cd(C₆H₄NO₂)₂(H₂O)₂]_n, forms a one‐dimensional chain structure based on a Cd atom with approximate pentagonal bipyramidal coordination geometry and two nicotinate ligands in different coordination modes. One acts as a tridentate ligand, chelating one Cd atom through the carboxyl­ate group while simultaneously binding to a second symmetry‐related Cd atom through the pyridine N atom; the other acts only as a bidentate ligand through its carboxyl­ate group. Hydro­gen bonds utilizing the coordinated water mol­ecules, uncoordinated nitro­gen and carboxyl­ate O atoms as acceptors link the chains.     

Theoretical improvement of the specific inhibitor of human carbonic anhydrase VII

The selectivity of a known arylsulfonamides inhibitor for two isozymes II and VII of human carbonic anhydrases (hCAs) was studied by homology modeling, molecular docking and molecular dynamics methods. The results show that the selectivity of the inhibitor for two isozymes is due to the different side chain lengths between N67 of hCA II and Q64 of hCA VII. One more methene group in the side chain of Q64 of hCA VII makes it possible to form the hydrogen bond with the bromide atom of the known inhibitor. From the point of view, the modification to the known inhibitor was performed to obtain an inhibitor with higher selectivity. The complex conformations of the new designed inhibitor and two isozymes designate the formation of the hydrogen bond between the newly added group (hydroxypropyl group) and Q64 of hCA VII but N67 of hCA II. The results of the binding free energy from the MM/PBSA approach also prove the selectivity improvement of the new inhibitor in comparison with the known inhibitor. The work will help the design of the isozyme-specific inhibitors of hCA VII.     

NMR investigation of the electrostatic effect in binding of a neuropeptide, achatin-I, to phosphatidylcholine bilayers

Achatin-I (Gly1-d-Phe2-Ala3-Asp4), known as a neuropeptide containing a d-amino acid, binds to the surface of a zwitterionic phosphatidylcholine (PC) membrane only when the peptide N-terminal amino group is in the ionized state, NH3+ (Kimura, T.; Okamura, E.; Matubayasi, N.; Asami, K.; Nakahara, M. Biophys. J. 2004, 87, 375-385). To gain mechanistic insights into how the binding equilibrium is delicately controlled by the ionization state of the N-terminal amino group, peptide-lipid binding interactions are investigated by selectively enriched 15N (at the N-terminus) and natural-abundance 13C NMR spectroscopy. Upon binding to the PC membrane, the 15N NMR of the N-terminal NH3(+) shifts upfield. This observation supports a mechanism that the role of the N-terminal NH3(+) in stabilizing the binding state is through electrostatic attraction with a headgroup negative charge, i.e., PO4(-). Interestingly, when the side chain beta-carboxyl group in Asp4 is deionized at acidic pH, the 15N signal of the N-terminal NH3(+) exhibits no significant chemical-shift change upon membrane binding of achatin-I. The Asp4 side chain thus regulates efficiency of the electrostatic binding between the peptide N-terminal NH3(+) and the lipid headgroup PO4(-). 13C chemical shifts in the hydrophobic D-Phe2 residue are largely perturbed upon membrane binding, in the case where the side chain beta-CO2(-) in Asp4 is deionized; the deionization of Asp4 beta-CO2(-) increases the net hydrophobicity of achatin-I with a reduction of both the electrostatic hydration and the electrostatic attraction with the headgroup N(CH3)3(+) in the most superficial region of the PC membrane, resulting in deeper anchoring of the phenyl ring. Hence, the electrostatic effect of the side chain beta-CO2(-) in Asp4 floats achatin-I on the PC membrane surface, and the binding equilibrium is sensitively controlled by the ionization state of the N-terminal NH3(+).     

Light-emitting molecular machines: pH-induced intramolecular motions in a fluorescent nickel(II) scorpionate complex

A cyclam-like macrocycle has been synthesized with a pendant arm containing a dansylamide group. In the corresponding nickel(II) complex, binding of the pendant arm to the metal is pH controlled. In particular, at pH 4.3, the sulfonamide group deprotonates and coordinates the NiII center, giving rise to a complex of trigonal bipyramidal stereochemistry, as shown by X-ray diffraction studies performed on the crystalline complex salt. At pH 7.5, an OH- ion binds the metal and a six-coordinate species forms. The binding-detachment of the pendant arm to/from the NiII center is signaled by changes in the emission properties of the dansyl subunit in the side chain; the fluorescence of this side chain is high when the pendant arm is not coordinated and low when the sulfonamide group is bound to the metal. The system investigated represents the prototype of a light-emitting molecular machine, driven by a pH change.     

The mechanism of nucleophilic substitution at silicon. Action of organometallic reagents on chiral bifunctional silanes

The study of nucelophilic substitution of monofunctional organosilanes has been extended to bifunctional derivatives. The reactions are both selective and stereoselective. Our results can be interpreted in terms of leaving group polarisability and the softness/hardness of the attacking nucleophile.The presence of two electronegative substituents only slightly modifies the stereochemistry. A pseudorotation mechanism could not explain the retention observed in certain cases. We envisage two possible modes of attack, either with axial or equatorial entry, leding respectively to inversion or retention at the silicon atom.     

Polymerization of avidin and streptavidin with aromatic bisbiotin ligands

The synthesis of a range of aromatic bisbiotin ligands is described and their ability to polymerize the proteins avidin and streptavidin is investigated. The formation of protein polymers is confirmed by gel chromatography and transmission electron microscopy. It is shown that the degree of polymerization depends not only on the length of the ligand but also on the stereochemistry of the group linking the distal biotin moieties. © 1994 John Wiley & Sons, Inc.     

Tuning the Binding Affinity and Selectivity of Perfluoroaryl-Stapled Peptides by Cysteine-Editing

A growing number of approaches to “staple” α-helical peptides into a bioactive conformation using cysteine cross-linking are emerging. Here, the replacement of l -cysteine with “cysteine analogues” in combinations of different stereochemistry, side chain length and beta-carbon substitution, is explored to examine the influence that the thiol-containing residue(s) has on target protein binding affinity in a well-explored model system, p53–MDM2/MDMX, which is constituted by the interaction of the tumour suppressor protein p53 and proteins MDM2 and MDMX, which regulate p53 activity. In some cases, replacement of one or more l -cysteine residues afforded significant changes in the measured binding affinity and target selectivity of the peptide. Computationally constructed homology models indicate that some modifications, such as incorporating two d -cysteine residues, favourably alter the positions of key functional amino acid side chains, which is likely to cause changes in binding affinity, in agreement with measured surface plasmon resonance data.     

The biotin-streptavidin interaction can be reversibly broken using water at elevated temperatures

The biotin-streptavidin system is the strongest noncovalent biological interaction known, having a dissociation constant, K(d), in the order of 4x10(-14) M. The strength and specificity of the interaction has led it to be one of the most widely used affinity pairs in molecular, immunological, and cellular assays. However, it has previously been impossible to re-use any streptavidin solid support, since the conditions needed to break the interaction with biotin has led to the denaturation of the streptavidin. Here, we show that a short incubation in nonionic aqueous solutions at temperatures above 70 degrees C can efficiently break the interaction without denaturing the streptavidin tetramer. Both biotin and the streptavidin remain active after dissociation and both molecules can therefore be re-used. The efficiency of the regeneration allowed solid supports with streptavidin to be used many times, here exemplified with the multiple re-use of streptavidin beads used for sample preparation prior to automated DNA sequencing. The results suggest that streptavidin regeneration can be introduced as an improvement in existing methods and assays based on the streptavidin system as well as emerging solid phase applications in fields, such as microfluidics and nanotechnology.     

Reversible immobilization of proteins with streptavidin affinity tags on a surface plasmon resonance biosensor chip

Dissociation of biotin from streptavidin is very difficult due to their high binding affinity. The re-use of streptavidin-modified surfaces is therefore almost impossible, making devices containing them (e.g. surface plasmon resonance (SPR) sensor chips) expensive. This paper describes a new protocol for reversible and site-directed immobilization of proteins with streptavidin affinity tags on the streptavidin-coated SPR biosensor chip (SA chip). Two streptavidin affinity tags, nano-tag and streptavidin-binding peptide (SBP tag), were applied. They both can specifically interact with streptavidin but have weaker binding force compared to the biotin–streptavidin system, thus allowing association and dissociation under controlled conditions. The SA chip surface could be regenerated repeatedly without loss of activity by injection of 50 mM NaOH solution. The fusion construct of a SBP tag and a single-chain antibody to mature bovine prion protein (scFv-Z186-SBP) interacts with the SA chip, resulting in a single-chain-antibody-modified surface. The chip showed kinetic response to the prion antigen with equilibrium dissociation constant K _D≈4.01×10⁻⁷. All results indicated that the capture activity of the SA chip has no irreversible loss after repeated immobilization and regeneration cycles. The method should be of great benefit to various biosensors, biochips and immunoassay applications based on the streptavidin capture surface.     

Glycoluril dimers bearing hydrogen atoms on their convex face and their self-assembly in the solid state

A selective method for the synthesis of 1,6-substituted glycolurils has been developed. The glycolurils have been used for the synthesis of methylene-bridged glycoluril dimers bearing hydrogen atoms on their convex face. Depending on the side walls of the dimers, different modes of self-assembly in the solid state have been described using X-ray crystallography.     

Structural and energetic effects in the molecular recognition of protonated peptidomimetic bases by 18-crown-6

Absolute 18-crown-6 (18C6) affinities of nine protonated peptidomimetic bases are determined using guided ion beam tandem mass spectrometry techniques. The bases (B) included in this work are mimics for the n-terminal amino group and the side chains of the basic amino acids, i.e., the favorable sites for binding of 18C6 to peptides and proteins. Isopropylamine is chosen as a mimic for the n-terminal amino group, imidazole and 4-methylimidazole are chosen as mimics for the side chain of histidine (His), 1-methylguanidine is chosen as a mimic for the side chain of arginine (Arg), and several primary amines including methylamine, ethylamine, n-propylamine, n-butylamine, and 1,5-diamino pentane as mimics for the side chain of lysine (Lys). Theoretical electronic structure calculations are performed to determine stable geometries and energetics for neutral and protonated 18C6 and the peptidomimetic bases, as well as the proton bound complexes comprised of these species, (B)H(+)(18C6). The measured 18C6 binding affinities of the Lys side chain mimics are larger than the measured binding affinities of the mimics for Arg and His. These results suggest that the Lys side chains should be the preferred binding sites for 18C6 complexation to peptides and proteins. Present results also suggest that competition between Arg or His and Lys for 18C6 is not significant. The mimic for the n-terminal amino group exhibits a measured binding affinity for 18C6 that is similar to or greater than that of the Lys side chain mimics. However, theory suggests that binding to n-terminal amino group mimic is weaker than that to all of the Lys mimics. These results suggest that the n-terminal amino group may compete with the Lys side chains for 18C6 complexation.