Physicochemical factors for discriminating between soluble and membrane proteins: hydrophobicity of helical segments and protein length

The average hydrophobicity of a polypeptide segment is consideredto be the most important factor in the formation of transmembranehelices, and the partitioning of the most hydrophobic (MH) segmentinto the alternative nonpolar environment, a membrane or hydrophobiccore of a globular protein may determine the type of proteinproduced. In order to elucidate the importance of the MH segmentin determining which of the two types of protein results froma given amino acid sequence, we statistically studied the characteristicsof MH helices, longer than 19 residues in length, in 97 membraneproteins whose three-dimensional structure or topology is known,as well as 397 soluble proteins selected from the Protein DataBank. The average hydrophobicity of MH helices in membrane proteinshad a characteristic relationship with the length of the protein.All MH helices in membrane proteins that were longer than 500residues had a hydrophobicity greater than 1.75 (Kyte and Doolittlescale), while the MH helices in membrane proteins smaller than100 residues could be as hydrophilic as 0.1. The possibilityof developing a method to discriminate membrane proteins fromsoluble ones, based on the effect of size on the type of proteinproduced, is discussed.     

Prodrugs of anthracyclines for use in antibody-directed enzyme prodrug therapy

A series of new prodrugs of daunorubicin and doxorubicin which are candidates for antibody-directed enzyme prodrug therapy (ADEPT) is reported. These compounds (25a,b,c and 32a,b,c) have been designed to generate cytotoxic drugs after activation with beta-glucuronidase. As expected, recovery of the active drug was observed after enzymatic cleavage by Escherichia coli beta-glucuronidase as well as by a fusion protein which has been obtained from human beta-glucuronidase and humanized CEA-specific binding region. The six prodrugs are highly stable and are more than 100-fold less cytotoxic than doxorubicin against murine L1210 cell lines. The ortho-substituted phenyl carbamates 25a,b,c are better substrates for beta-glucuronidase than the corresponding para-substituted analogues. After taking into account additional factors such as stability in plasma and kinetics of enzymatic cleavage, we selected the o-nitro prodrug 25c for clinical trials.     

Composite torsional quartz transducer for shear ultrasonic measurements of aqueous liquids

A composite torsional quartz transducer has been developed for the shear ultrasonic measurements of aqueous solutions in the frequency range from 10 to 100 kHz. In order to prevent the electric interaction and low contact angle between the transducer and water, a torsional quartz crystal transducer is attached to a fused quartz rod whose surface is partly gilded by evaporation. The accuracy of +/-60 dyn/cm2 for the rigidity and +/-0.03 cP for the viscosity is attained by use of this transducer.     

Synthesis and cytotoxic activity of pyranocoumarins of the seselin and xanthyletin series

This article illustrates how thermodynamic functions can be calculated from moisture sorption isotherms for water-solid systems. The materials evaluated include the drugs albuterol sulfate and indomethacin, the carbohydrates sucrose, raffinose, and trehalose, and the polymers poly(vinyl pyrrolidone) and microcrystalline cellulose. The results demonstrated that significant positive and negative deviations from ideality occurred for water and solid components, respectively, producing higher free energy materials than for the ideal mixtures. The calculations quantitatively discriminated major differences in thermodynamic activity and free energy for all of the solid phases studied, arising from the uniqueness in chemical interactions. This approach to analyzing moisture sorption data is invaluable in advancing our understanding of the physical chemistry of water-solid systems.     

Silicalite-l Synthesis from Silicate Aqueous Solutions Including Amines as a Base

Aqueous silicate solutions could be prepared from a silicon alkoxide using amines as a base. The synthesis of silicalite-1 conducted by hydrothermally treating the solutions after the addition of NPrⁿ₄Br (TPABr), was examined focusing on triethylamine (NEt₃) as the amine. The aqueous solutions with chemical compositions of Si(OEt)₄ (TEOS) : NEt₃ : TPABr : H₂O = 1 : ≥0.5 : ≥0.8 : 200 led to silicalite-1 crystallization upon hydrothermal treatment at 130C for 4 days. The resulting crystals had a small thin coffin shape. With increasing the added amounts of NEt₃ and TPABr, the crystal size decreased. Among the investigated conditions, the smallest silicalite-1 crystal measuring about 1.0 × 0.7 × 0.4 um was obtained at starting compositions of NEt₃/TEOS ≥2.0 and TPABr/TEOS ≥3.2. The increase in the TPA content of the starting solutions accelerated the crystal nucleation of silicalite-1, so that the obtained crystals became smaller. During the hydrothermal synthesis, an amorphous silica gel was formed before the crystallization commenced. The increase in the NEt₃ content may suppress the formation of the silica gel, and/or facilitate its dissolution, thereby the supersaturation degree of silicate species in the solution was increased, giving small silicalite-1 crystals. When an aqueous suspension of the smallest silicalite-1 crystals was spread onto a glass substrate by a dip-coating method, an ordered mono-grain layer of the silicalite-1 crystals oriented with the straight channels normal to the substrate surface was obtained.     

Optimal configuration of pyrene excimer induced by the excimer-forming two-probe nucleic acid hybridization method

Excimer-forming two-probe nucleic acid hybridization (ETPH) method with pyrene as a fluorophore enables homogeneous hybridization assays. We examined the effect of linker length between a pyrene residue and a terminal sugar moiety on Tm of hybrids in the presence of 20% dimethylformamide (DMF). The results including those of CD measurements indicated no interaction of pyrene residues with the duplex formed between a target 32-mer and a pyrenemethyliodoacetamide-introduced 16-mer probe (PMIA-P5)/a pyrenebutanoic acid-introduced 16-mer probe (PBuA-P3), which is the best pair of probes for intense excimer emission. This was also supported by a computer-assisted molecular modeling using Insight II and Discover software.     

Anomalies of nanosecond ultrasonic relaxation in the lipid bilayer transition

The frequency dependence of ultrasonic velocity as well as absorption in a suspension of sonicated dipalmitoylphosphatidylcholine vesicles was measured by a differential ultrasonic resonator. The frequency was scanned between 1.3 and 13 MHz and the temperature was varied from 25 to 47 degrees C. A pronounced relaxation was observed in the time range of 10 ns. The data were analyzed assuming a single relaxation which appeared to be a good approximation. The relaxation time as well as relaxation strength increased anomalously in the vicinity of the gel-to-liquid crystal transition of 41.5 degrees C. This result represents the first definite evidence of the critical slowing down in the lipid bilayer and is discussed in terms of the Landau theory of phase transition. The possible biological significance of the mechanical relaxation is also presented.     

Light-induced denaturation of bacteriorhodopsin solubilized by octyl-{beta}-glucoside

The structural stability of bacteriorhodopsin (bR) solubilizedby octyl-ß-glucoside was studied by measuring the denaturationkinetics under visible light irradiation and in the dark. Thedenaturation of bR solubilized by 50 mM octyl-ß-glucosidewas very slow at room temperature when it was left in the dark.However, its spontaneous denaturation was accelerated when thesolubilized bR was irradiated by visible light. The denaturationkinetics under visible light irradiation and in the dark couldbe well described by a single decay constant. The activationenergy for the denaturation of bR was estimated from the temperaturedependence of decay time constants. The activation energy undervisible light irradiation was 12.5 kcal/mol, which was muchsmaller than the corresponding value in the dark, 26.2 kcal/mol.These results strongly suggest that some of the photointermediatestates are less stable than the ground state of bR. The criticaltemperature and the activation energy for denaturation of bRin the solubilized state were much lower than those in the 2Dcrystalline state. Comparing the denaturation behavior in the2D crystalline state and that in the octyl-ß-glucoside-solubilizedstate, our findings suggest that protein–protein interactioncontributes to the stability of this protein.     

A triangle lattice model that predicts transmembrane helix configuration using a polar jigsaw puzzle

We developed a method of predicting the tertiary structuresof seven transmembrane helical proteins in triangle latticemodels, assuming that the configuration of helices is stabilizedby polar interactions. Triangle lattice models having 12 or11 nearest neighbor pairs were used as general templates ofa seven-helix system, then the orientation angles of all heliceswere varied at intervals of 15°. The polar interaction energyfor all possible positions of each helix was estimated usingthe calculated polar indices of transmembrane helices. An automatedsystem was constructed and applied to bacteriorhodopsin, a typicalmembrane protein with seven transmembrane helices. The predictedoptimal and actual structures were similar. The top 100 predictedhelical configurations indicated that the helix-triangle, CFG,occurred at the highest frequency. In fact, this helix-triangleof bacteriorhodopsin forms an active proton-pumping site, suggestingthat the present method can identify functionally importanthelices in membrane proteins. The possibility of studying thestructure change of bacteriorhodopsin during the functionalprocess by this method is discussed, and may serve to explainthe experimental structures of photointermediate states.