A Spectroscopic Study of Structural Heterogeneity and Carbon Monoxide Binding in Neuroglobin

Neuroglobin (Ngb) is a small globular protein that binds diatomic ligands like oxygen, carbon monoxide (CO) and nitric oxide at a heme prosthetic group. We have performed FTIR spectroscopy in the infrared stretching bands of CO and flash photolysis with monitoring in the electronic heme absorption bands to investigate structural heterogeneity at the active site of Ngb and its effects on CO binding and migration at cryogenic temperatures. Four CO stretching bands were identified; they correspond to discrete conformations that differ in structural details and CO binding properties. Based on a comparison of bound-state and photoproduct IR spectra of the wild-type protein, Ngb distal pocket mutants and myoglobin, we have provided structural interpretations of the conformations associated with the different CO bands. We have also studied ligand migration to the primary docking site, B. Rebinding from this site is governed by very low enthalpy barriers (∼1 kJ/mol), indicating an extremely reactive heme iron. Moreover, we have observed ligand migration to a secondary docking site, C, from which CO rebinding involves higher enthalpy barriers.     

Study of potential binding of biologically important sugars with a dinuclear cobalt(II) complex

The investigation of the sugar–metal ion interactions remains one of the main objectives of carbohydrate coordination chemistry because the interactions between metal ions and carbohydrates are involved in many biochemical processes. The potential binding interaction between a five-coordinate dinuclear cobalt(II) complex, Na₂[Co₂(tcdc)(μ-OAc)] (1) [Na₅tcdc = Sodium-N,N,N′,N′-tetrakis(sodium carboxylate methyl)-2,6-diaminocresolate] and biologically important sugar substrates (d-glucose, d-xylose, and d-mannose) has been studied. In alkaline media, the complex 1 shows an excellent chelating ability toward these sugar substrates. A combined approach of FTIR and UV–vis spectroscopic investigations shows that the complex forms a 1:1 complex/substrate-bound product. UV–vis spectra indicate a significant blue-shift of the absorption maximum of metal complex during carbohydrate coordination highlighting the sugar binding ability of complex 1. The apparent binding constants of the substrate-bound cobalt(II) complexes have been determined from the UV–vis titration experiments.     

傅里叶变换红外光谱(FTIR)技术对滇龙胆组织培养的研究

野生药用植物资源的不断减少,使得寻找其原植物的合适替代品显得尤为重要。利用组培材料代替野生药用植物作为药源已取得重大进展,但利用傅里叶变换红外光谱(Fourier transform infrared spectroscopy,FTIR)技术筛选合适的组培材料作为野生药用植物替代资源方面的应用鲜有报道。本研究采用FTIR结合偏最小二乘判别分析(partial least squares discriminant analysis,PLS-DA)对滇龙胆组织培养形成的愈伤组织(肉质部、茎、叶)、增殖苗(肉质部、茎、叶)、生根苗(根、茎、叶)进行比较。结果显示:(1)从原始FTIR光谱图上看,滇龙胆肉质部和根部峰形相似,茎和叶峰形相似;(2)二阶导数光谱图扩大了样品间的差异。在龙胆苦苷的主要吸收峰1612 cm-1附近,吸收峰强度依次为:生根苗叶增殖苗叶和生根苗茎增殖苗茎愈伤组织叶,愈伤组织茎及肉质部、增殖苗肉质部和生根苗根部在该处无吸收峰;(3)PLS-DA得分图表明,同一组培阶段相同组织部位样品聚集在一起,而愈伤组织、增殖苗、生根苗及其各组织部位能够较好的分开。其中:肉质部、根部与茎叶之间距离较远,表明其化学成分和含量可能差异较大;肉质部和根部样品间距离较近,茎和叶样品间距离也较近。二阶导数光谱图显示,组培材料有望代替其原植物满足药用需求;若以龙胆苦苷含量为评价对象,生根苗叶则可能具有更大的开发潜能,有望代替野生滇龙胆以缓解其资源稀缺局面。本研究结果表明,采用傅里叶变换红外光谱法可以简便有效地对药用植物不同组培阶段不同组织部位的替代潜力及开发利用进行初步评估。     

An engineered heme–copper center in myoglobin: CO migration and binding

We have investigated CO migration and binding in Cu_BMb, a copper-binding myoglobin double mutant (L29H–F43H), by using Fourier transform infrared spectroscopy and flash photolysis over a wide temperature range. This mutant was originally engineered with the aim to mimic the catalytic site of heme–copper oxidases. Comparison of the wild-type protein Mb and Cu_BMb shows that the copper ion in the distal pocket gives rise to significant effects on ligand binding to the heme iron. In Mb and copper-free Cu_BMb, primary and secondary ligand docking sites are accessible upon photodissociation. In copper-bound Cu_BMb, ligands do not migrate to secondary docking sites but rather coordinate to the copper ion. Ligands entering the heme pocket from the outside normally would not be captured efficiently by the tight distal pocket housing the two additional large imidazole rings. Binding at the Cu ion, however, ensures efficient trapping in Cu_BMb. The Cu ion also restricts the motions of the His64 side chain, which is the entry/exit door for ligand movement into the active site, and this restriction results in enhanced geminate and slow bimolecular CO rebinding. These results support current mechanistic views of ligand binding in hemoglobins and the role of the Cu_B in the active of heme–copper oxidases. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Influence of proline on the thermostability of the active site and membrane arrangement of transmembrane proteins

Proline residues play a fundamental and subtle role in the dynamics, structure, and function in many membrane proteins. Temperature derivative spectroscopy and differential scanning calorimetry have been used to determine the effect of proline substitution in the structural stability of the active site and transmembrane arrangement of bacteriorhodopsin. We have analyzed the Pro-to-Ala mutation for the helix-embedded prolines Pro⁵⁰, Pro⁹¹, and Pro¹⁸⁶ in the native membrane environment. This information has been complemented with the analysis of the respective crystallographic structures by the FoldX force field. Differential scanning calorimetry allowed us to determine distorted membrane arrangement for P50A and P186A. The protein stability was severely affected for P186A and P91A. In the case of Pro⁹¹, a single point mutation is capable of strongly slowing down the conformational diffusion along the denaturation coordinate, becoming a barrier-free downhill process above 371 K. Temperature derivative spectroscopy, applied for first time to study thermal stability of proteins, has been used to monitor the stability of the active site of bacteriorhodopsin. The mutation of Pro⁹¹ and Pro¹⁸⁶ showed the most striking effects on the retinal binding pocket. These residues are the Pro in closer contact to the active site (activation energies for retinal release of 60.1 and 76.8 kcal/mol, respectively, compared to 115.8 kcal/mol for WT). FoldX analysis of the protein crystal structures indicates that the Pro-to-Ala mutations have both local and long-range effects on the structural stability of residues involved in the architecture of the protein and the active site and in the proton pumping function. Thus, this study provides a complete overview of the substitution effect of helix-embedded prolines in the thermodynamic and dynamic stability of a membrane protein, also related to its structure and function.