Synthesis of functionalized rab GTPases by a combination of solution- or solid-phase lipopeptide synthesis with expressed protein ligation

Prenylated proteins with non-native functionalities are generally very difficult to obtain by recombinant or enzymatic means. The semisynthesis of preparative amounts of prenylated Rab guanosine triphosphatases (GTPases) from recombinant proteins and synthetic prenylated peptides depends largely on the availability of functionalised prenylated peptides corresponding to the proteins' native structure or modifications thereof. Here, we describe and compare solution-phase and solid-phase strategies for the generation of peptides corresponding to the prenylated C terminus of Rab7 GTPase. The solid-phase with utilisation of a hydrazide linker emerges as the more favourable approach. It allows a fast and practical synthesis of pure peptides and gives a high degree of flexibility in their modification. To facilitate the analysis of semisynthetic proteins, the synthesised peptides were equipped with a fluorescent group. Using the described approach, we introduced fluorophores at several different positions of the Rab7 C terminus. The position of the incorporated fluorescent groups in the peptides did not influence the protein-ligation reaction, as the generated peptides could be ligated onto thioester-tagged Rab7. However, it was found that the positioning of the fluorescent group had an influence on the functionality of the Rab7 proteins; analysis of the interaction of the semisynthetic Rab7 proteins with REP (Rab escort protein) and GDI (guanosine diphosphate dissociation inhibitor) molecules revealed that modification of the peptide side chains or of the C-terminal isoprenoid did not significantly interfere with complex formation. However, functionalisation of the C terminus was found to have an adverse effect on complex formation and stability, possibly reflecting low structural flexibility of the Rab GDI/REP molecules in the vicinity of the lipid-binding site.     

On the High Viscosity of Aqueous Solution of Lysozyme Induced by Some Organic Solvents

A remarkably high viscosity has been induced in protein aqueous solutions by the addition of certain structurally related organic solvents. The effect has been observed for lysozyme aqueous solutions containing tetramethylurea (TMU), dimethylsulfoxide (DMSO), dimethylformamide, and hexamethylphosphortriamide. The effect has also been induced in ferrocytochrome c aqueous solutions by TMU. Critical concentrations for both the protein and organic solvent were verified for the onset of the viscosity increase. A common feature of the solvents which were able to induce the effect is a dipolar moiety (C=O, S → O and P → O) and a nonpolar region represented by the methyl groups. The resulting fluids show an extremely restricted flow and a typical non-Newtonian, pseudoplastic behavior. Use was made of¹H nuclear magnetic resonance (NMR) and Raman spectroscopy to characterize protein structural modifications and of¹³C NMR to investigate changes in relaxation times and chemical shifts in the solvent/water solutions. A systematic rheological characterization of the systems was undertaken for some of the solvents, and unusual patterns of viscous effects were identified for the solvent/water systems both with and without protein. The process was found to be at least partially reversible, as concluded from the recovered original solution rheological characteristics and the original protein¹H NMR spectrum, after eliminating the organic solvent by ultrafiltration. The whole process was characterized as consisting of two mutually independent stages. The first involves an extensive conformational transition of the polypeptide backbone, from a predominantly α-helical to increased random coiled and β-sheet structures, with the occurrence of nonorthodox protein secondary structures at regions above the solvent critical point. The second stage consists of short-lived interchain contacts leading to an entanglement of the macromolecular system as a whole. A microphase reversion in the organic solvent/water mixture, supported by¹³C NMR and rheological results, is proposed as the driving force causing the observed behavior.     

Effects of medium and chemical modification on thermal characteristics of Β-lactoglobulin

The thermal properties of -lactoglobulin (-LG) were studied by differential scanning calorimetry (DSC) under different medium conditions.pH, neutral salts, protein perturbants, and polyols all affected the DSC characteristics of -LG. Acylation with fatty acids also changed the thermal properties, particularly peak width at half-height. The results suggest that the structural stability of -LG is controlled by non-covalent forces, particularly electrostatic and hydrophobic interactions. Disulfide bonds did not contribute to the thermal response of -LG. Fatty N-acyl-amino acids caused marked increases in thermal stability and decreases in denaturation enthalpy, and additional peaks were observed in the presence of some palmitoyl derivatives.Contribution No. 2310, Centre for Food and Animal Research.We thank D. Raymond for her excellent technical assistance.     

Further solvent-free reactions of ferrocenylaldehydes: Synthesis of 1,1′-ferrocenyldiimines and ferrocenylacrylonitriles

Grinding of 1,1′-ferrocenedicarboxaldehyde with a 2.2 molar equivalent of an aromatic amine in a solvent-free environment provided excellent yields of 1,1′-ferrocenyldiimines. After mixing the aldehyde and amines, a gum or melt formed which eventually solidified to the product. An analytically pure sample of the product was obtained by cold recrystallization. Grinding of ferrocenecarboxaldehyde and 4-substituted phenylacetonitriles under solvent-free conditions provided good yields of the corresponding ferrocenylacrylonitriles. The yield in this reaction was very low when the substituent group para to the acetonitrile group was electron-donating.     

Relative stability of major types of beta-turns as a function of amino acid composition: a study based on Ab initio energetic and natural abundance data

Folding properties of small globular proteins are determined by their amino acid sequence (primary structure). This holds both for local (secondary structure) and for global conformational features of linear polypeptides and proteins composed from natural amino acid derivatives. It thus provides the rational basis of structure prediction algorithms. The shortest secondary structure element, the beta-turn, most typically adopts either a type I or a type II form, depending on the amino acid composition. Herein we investigate the sequence-dependent folding stability of both major types of beta-turns using simple dipeptide models (-Xxx-Yyy-). Gas-phase ab initio properties of 16 carefully selected and suitably protected dipeptide models (for example Val-Ser, Ala-Gly, Ser-Ser) were studied. For each backbone fold most probable side-chain conformers were considered. Fully optimized 321G RHF molecular structures were employed in medium level [B3LYP/6-311++G(d,p)//RHF/3-21G] energy calculations to estimate relative populations of the different backbone conformers. Our results show that the preference for beta-turn forms as calculated by quantum mechanics and observed in Xray determined proteins correlates significantly.     

Expression and Characterization of Catalytic Domain of T Cell Protein Tyrosine Phosphatase（△TC-PTP） —— Immunohistochemical Study of △TC-PTP Expression in Non-small Cell Lung Carcinomas

This study objective was to express and characterize the catalytic domain of the human T cell protein tyrosine phosphatase（△TC-PTP） and to study immunohistochemically the expression of △TC-PTP in human non-small cell lung cancers. △TC-PTP gene was PCR amplified with the cDNA of human TC-PTP as template, and cloned into the pT7 expression vector. The recombinant pT7-△TC-PTP was expressed in E. coli Rosetta （ DE3 ） host cells and puri- fied. The enzymatic characteristics of △TC-PTP including enzyme activity and kinetics assay were measured. The antiserum was prepared by immunizing rabbit with the purified recombinant △TC-PTP. Rabbit polyclonal antibody against △TC-PTP was purified by PVDF immobilized antigen affinity chromatography. Immunohistochemical staining of lung cancer tissues was performed with antibody against △TC-PTP protein. △TC-PTP gene was correctly cloned, expressed, and purified. The recombinant △TC-PTP had a highly catalytic activity of PTPase. Squamous cell lung carcinoma showed a significantly higher expression rate of △TC-PTP （76. 92%, 10/13 ） than adenocarcinoma （57.14%, 4/7） and normal lung tissue（20%, 1/5 ）. This study represents the first demonstration that △TC-PTP is highly expressed in human squamous cell lung carcinomas. In addition, this study provides an important basis for further studying the biological function of TC-PTP and its relationship with lung carcinomas and other diseases.     

氟代二苯乙烯氨基酸的合成及性能

以4-碘-L-苯丙氨酸为初始原料,经羧基和氨基的保护得到化合物2;化合物2与4-氟苯乙烯进行Heck反应得化合物3;化合物3脱去保护基得氟代二苯乙烯氨基酸(4,47.4%);其结构经1H NMR,13C NMR,IR和MS(ESI-TOF)确证。光学性质研究结果表明:4的激发、发射波长分别为314 nm和355 nm,斯托克斯位移为41 nm,荧光量子产率为0.22。化合物4在甲醇溶液中存在光致顺反异构现象,反式→顺式结构转变速率常数为0.026 min^-1。     

Rapid Protein Hydrolysis by Microwave Irradiation Using Heat-Resistant Teflon-Pyrex Tubes

A rapid peptide-bond hydrolysis by means of microwave irradiation is introduced for the facile preparation of protein hydrolysates used for amino acid analysis. The optimal hydrolysis condition has been determined using several enzymes with known amino acid compositions. The effects of hydrolysis time on the recovery of various labile and hydrophobic amino acids are also exemplified in the microwave heating of standard amino acids. The method has been applied to the complete amino acid analysis with a single nonvolatile solvent of methanesulfonic acid with good recovery of tryptophan and half-cystine. It provides a radical expedition of protein and peptide hydrolysis via commercial microwave ovens and specially-designed Teflon-Pyrex tubes, circumventing the tedious procedures using vacuum-sealed pyrex lubes heating at 110°C for more than 24 h. This novel type of microwave chemistry associated with rapid peptide-bond cleavage is of great potential in the automation of the complete process of amino acid analysis starting from the preparation of protein hydrolysates.     

New methods for data analysis of isothermal titration calorimetry

Heat divided by ligand concentration vs. heat, similar to the Scatchard plot, was introduced to obtain the equilibrium constant (K) and the enthalpy of binding (DH) using isothermal titration calorimetry data. Values of K and DH obtained by this linear pseudo-Scatchard plot for a system with a set of independent binding sites (such as binding fluoride ions on urease and monosaccharide methyl a-D-mannopyranoside on concavalin A) were remarkably like that obtained from a normal fitting Wiseman method and other our technical methods. On applying this graphical method to study the binding of copper ion on myelin basic protein (MBP), a concave downward curve obtained was consistent with the positive cooperativity in the binding. A graphical fitting by simple method for determination of thermodynamic parameters was also introduced. This method is general, without any assumption and restriction made in previous method. This general method was applied to the product inhibition study of adenosine deaminase. This revised version was published online in July 2006 with corrections to the Cover Date.