Proteomic analysis of the Cdc48/Ubx network identifies a role for Ubx2 in the regulation of lipid biosynthesis

Cdc48/p97 is an essential, highly abundant hexameric member of the AAA (ATPase associated with various cellular activities) family. It has been linked to a variety of processes throughout the cell but it is best known for its role in the ubiquitin proteasome pathway. In this system it is believed that Cdc48 behaves as a segregase, transducing the chemical energy of ATP hydrolysis into mechanical force to separate ubiquitin-conjugated proteins from their tightly-bound partners.

Current models posit that Cdc48 is linked to its substrates through a variety of adaptor proteins, including a family of seven proteins (13 in humans) that contain a Cdc48-binding UBX domain. As such, due to the complexity of the network of adaptor proteins for which it serves as the hub, Cdc48/p97 has the potential to exert a profound influence on the ubiquitin proteasome pathway. However, the number of known substrates of Cdc48/p97 remains relatively small, and smaller still is the number of substrates that have been linked to a specific UBX domain protein. As such, the goal of this dissertation research has been to discover new substrates and better understand the functions of the Cdc48 network. With this objective in mind, we established a proteomic screen to assemble a catalog of candidate substrate/targets of the Ubx adaptor system.

Here we describe the implementation and optimization of a cutting-edge quantitative mass spectrometry method to measure relative changes in the Saccharomyces cerevisiae proteome. Utilizing this technology, and in order to better understand the breadth of function of Cdc48 and its adaptors, we then performed a global screen to identify accumulating ubiquitin conjugates in cdc48-3 and ubxΔ mutants. In this screen different ubx mutants exhibited reproducible patterns of conjugate accumulation that differed greatly from each other, pointing to various unexpected functional specializations of the individual Ubx proteins.

As validation of our mass spectrometry findings, we then examined in detail the endoplasmic-reticulum bound transcription factor Spt23, which we identified as a putative Ubx2 substrate. In these studies ubx2Δ cells were deficient in processing of Spt23 to its active p90 form, and in localizing p90 to the nucleus. Additionally, consistent with reduced processing of Spt23, ubx2Δ cells demonstrated a defect in expression of their target gene OLE1, a fatty acid desaturase. Overall, this work demonstrates the power of proteomics as a tool to identify new targets of various pathways and reveals Ubx2 as a key regulator lipid membrane biosynthesis.

Engineering multi-step electron tunneling systems in proteins

Multi-step electron tunneling, or “hopping,” has become a fast-developing research field with studies ranging from theoretical modeling systems, inorganic complexes, to biological systems. In particular, the field is exploring hopping mechanisms in new proteins and protein complexes, as well as further understanding the classical biological hopping systems such as ribonuclease reductase, DNA photolyases, and photosystem II. Despite the plethora of natural systems, only a few biologically engineered systems exist. Engineered hopping systems can provide valuable information on key structural and electronic features, just like other kinds of biological model systems. Also, engineered systems can harness common biologic processes and utilize them for alternative reactions. In this thesis, two new hopping systems are engineered and characterized.

The protein Pseudomonas aeruginosa azurin is used as a building block to create the two new hopping systems. Besides being well studied and amenable to mutation, azurin already has been used to successfully engineer a hopping system. The two hopping systems presented in this thesis have a histidine-attached high potential rhenium 4,7-dimethyl-1,10-phenanthroline tricarbonyl [Re(dmp)(CO)3] + label which, when excited, acts as the initial electron acceptor. The metal donor is the type I copper of the azurin protein. The hopping intermediates are all tryptophan, an amino acid mutated into the azurin at select sites between the photoactive metal label and the protein metal site. One system exhibits an inter-molecular hopping through a protein dimer interface; the other system undergoes intra-molecular multi-hopping utilizing a tryptophan “wire.” The electron transfer reactions are triggered by excitation of the rhenium label and monitored by UV-Visible transient absorption, luminescence decays measurements, and time-resolved Infrared spectroscopy (TRIR). Both systems were structurally characterized by protein X-ray crystallography.

Cooperative properties of lipid bilayers : collective director fluctuations and the effects of hydrophobic mismatch in protein/lipid membrane systems

Cooperative director fluctuations in lipid bilayers have been postulated for many years. ^2H-NMR T_1^(-1), T_(1P)^(-1) , and T_2^(-1); measurements have been used identify these motions and to determine the origin of increased slow bilayer motion upon addition of unlike lipids or proteins to a pure lipid bilayer.

The contribution of cooperative director fluctuations to NMR relaxation in lipid bilayers has been expressed mathematically using the approach of Doane et al.^1 and Pace and Chan.^2 The T_2^(-1)’s of pure dimyristoyllecithin (DML) bilayers deuterated at the 2, 9 and 10, and all positions on both lipid hydrocarbon chains have been measured. Several characteristics of these measurements indicate the presence of cooperative director fluctuations. First of all, T_2^(-1) exhibits a linear dependence on S2/CD. Secondly, T_2^(-1) varies across the ^2H-NMR powder pattern as sin^2 (2, β), where , β is the angle between the average bilayer director and the external magnetic field. Furthermore, these fluctuations are restricted near the lecithin head group suggesting that the head group does not participate in these motions but, rather, anchors the hydrocarbon chains in the bilayer.

T_2^(-1)has been measured for selectively deuterated liquid crystalline DML hilayers to which a host of other lipids and proteins have been added. The T_2^(-1) of the DML bilayer is found to increase drastically when chlorophyll a (chl a) and Gramicidin A' (GA') are added to the bilayer. Both these molecules interfere with the lecithin head group spacing in the bilayer. Molecules such as myristic acid, distearoyllecithin (DSL), phytol, and cholesterol, whose hydrocarbon regions are quite different from DML but which have small,neutral polar head groups, leave cooperative fluctuations in the DML bilayer unchanged.

The effect of chl a on cooperative fluctuations in the DML bilayer has been examined in detail using ^2H-NMR T_1^(-1), T_(1P)^(-1) , and T_2^(-1); measurements. Cooperative fluctuations have been modelled using the continuum theory of the nematic state of liquid crystals. Chl a is found to decrease both the correlation length and the elastic constants in the DML bilayer.

A mismatch between the hydrophobic length of a lipid bilayer and that of an added protein has also been found to change the cooperative properties of the lecithin bilayer. Hydrophobic mismatch has been studied in a series GA' / lecithin bilayers. The dependence of 2H-NMR order parameters and relaxation rates on GA' concentration has been measured in selectively deuterated DML, dipalmitoyllecithin (DPL), and DSL systems. Order parameters, cooperative lengths, and elastic constants of the DML bilayer are most disrupted by GA', while the DSL bilayer is the least perturbed by GA'. Thus, it is concluded that the hydrophobic length of GA' best matches that of the DSL bilayer. Preliminary Raman spectroscopy and Differential Scanning Calorimetry experiments of GA' /lecithin systems support this conclusion. Accommodation of hydrophobic mismatch is used to rationalize the absence of H_(II) phase formation in GA' /DML systems and the observation of H_(II) phase in GA' /DPL and GA' /DSL systems.

1. J. W. Doane and D. L. Johnson, Chem. Phy3. Lett., 6, 291-295 (1970). 2. R. J. Pace and S. I. Chan, J. Chem. Phy3., 16, 4217-4227 (1982).

Phospholipids and a protein-conducting channel regulate cotranslational protein targeting

Signal recognition particle (SRP) and signal recognition particle receptor (SR) are evolutionarily conserved GTPases that deliver secretory and membrane proteins to the protein-conducting channel Sec61 complex in the lipid bilayer of the endoplasmic reticulum in eukaryotes or the SecYEG complex in the inner membrane of bacteria. Unlike the canonical Ras-type GTPases, SRP and SR are activated via nucleotide-dependent heterodimerization. Upon formation of the SR•SRP targeting complex, SRP and SR undergo a series of discrete conformational changes that culminate in their reciprocal activation and hydrolysis of GTP. How the SR•SRP GTPase cycle is regulated and coupled to the delivery of the cargo protein to the protein-conducting channel at the target membrane is not well-understood. Here we examine the role of the lipid bilayer and SecYEG in regulation of the SRP-mediated protein targeting pathway and show that they serve as important biological cues that spatially control the targeting reaction.

In the first chapter, we show that anionic phospholipids of the inner membrane activate the bacterial SR, FtsY, and favor the late conformational states of the targeting complex conducive to efficient unloading of the cargo. The results of our studies suggest that the lipid bilayer acts as a spatial cue that weakens the interaction of the cargo protein with SRP and primes the complex for unloading its cargo onto SecYEG.

In the second chapter, we focus on the effect of SecYEG on the conformational states and activity of the targeting complex. While phospholipids prime the complex for unloading its cargo, they are insufficient to trigger hydrolysis of GTP and the release of the cargo from the complex. SecYEG modulates the conformation of the targeting complex and triggers the GTP hydrolysis from the complex, thus driving the targeting reaction to completion. The results of this study suggest that SecYEG is not a passive recipient of the cargo protein; rather, it actively releases the cargo from the targeting complex. Together, anionic phospholipids and SecYEG serve distinct yet complementary roles. They spatially control the targeting reaction in a sequential manner, ensuring efficient delivery and unloading of the cargo protein.

In the third chapter, we reconstitute the transfer reaction in vitro and visualize it in real time. We show that the ribosome-nascent chain complex is transferred to SecYEG via a stepwise mechanism with gradual dissolution and formation of the contacts with SRP and SecYEG, respectively, explaining how the cargo is kept tethered to the membrane during the transfer and how its loss to the cytosol is avoided.

In the fourth chapter, we examine interaction of SecYEG with secretory and membrane proteins and attempt to address the role of a novel insertase YidC in this interaction. We show that detergent-solubilized SecYEG is capable of discriminating between the nascent chains of various lengths and engages a signal sequence in a well-defined conformation in the absence of accessory factors. Further, YidC alters the conformation of the signal peptide bound to SecYEG. The results described in this chapter show that YidC affects the SecYEG-nascent chain interaction at early stages of translocation/insertion and suggest a YidC-facilitated mechanism for lateral exit of transmembrane domains from SecYEG into the lipid bilayer.

Chemical studies of viral entry mechanisms: I. Hydrophobic protein-lipid interactions during sendai virus membrane fusion. II. Kinetics of bacteriophage λ DNA injection.

Viruses possess very specific methods of targeting and entering cells. These methods would be extremely useful if they could also be applied to drug delivery, but little is known about the molecular mechanisms of the viral entry process. In order to gain further insight into mechanisms of viral entry, chemical and spectroscopic studies in two systems were conducted, examining hydrophobic protein-lipid interactions during Sendai virus membrane fusion, and the kinetics of bacteriophage λ DNA injection.

Sendai virus glycoprotein interactions with target membranes during the early stages of fusion were examined using time-resolved hydrophobic photoaffinity labeling with the lipid-soluble carbene generator3-(trifluoromethyl)-3-(m-^(125 )I] iodophenyl)diazirine (TID). The probe was incorporated in target membranes prior to virus addition and photolysis. During Sendai virus fusion with liposomes composed of cardiolipin (CL) or phosphatidylserine (PS), the viral fusion (F) protein is preferentially labeled at early time points, supporting the hypothesis that hydrophobic interaction of the fusion peptide at the N-terminus of the F_1 subunit with the target membrane is an initiating event in fusion. Correlation of the hydrophobic interactions with independently monitored fusion kinetics further supports this conclusion. Separation of proteins after labeling shows that the F_1 subunit, containing the putative hydrophobic fusion sequence, is exclusively labeled, and that the F_2 subunit does not participate in fusion. Labeling shows temperature and pH dependence consistent with a need for protein conformational mobility and fusion at neutral pH. Higher amounts of labeling during fusion with CL vesicles than during virus-PS vesicle fusion reflects membrane packing regulation of peptide insertion into target membranes. Labeling of the viral hemagglutinin/neuraminidase (HN) at low pH indicates that HN-mediated fusion is triggered by hydrophobic interactions, after titration of acidic amino acids. HN labeling under nonfusogenic conditions reveals that viral binding may involve hydrophobic as well as electrostatic interactions. Controls for diffusional labeling exclude a major contribution from this source. Labeling during reconstituted Sendai virus envelope-liposome fusion shows that functional reconstitution involves protein retention of the ability to undergo hydrophobic interactions.

Examination of Sendai virus fusion with erythrocyte membranes indicates that hydrophobic interactions also trigger fusion between biological membranes, and that HN binding may involve hydrophobic interactions as well. Labeling of the erythrocyte membranes revealed close membrane association of spectrin, which may play a role in regulating membrane fusion. The data show that hydrophobic fusion protein interaction with both artificial and biological membranes is a triggering event in fusion. Correlation of these results with earlier studies of membrane hydration and fusion kinetics provides a more detailed view of the mechanism of fusion.

The kinetics of DNA injection by bacteriophage λ. into liposomes bearing reconstituted receptors were measured using fluorescence spectroscopy. LamB, the bacteriophage receptor, was extracted from bacteria and reconstituted into liposomes by detergent removal dialysis. The DNA binding fluorophore ethidium bromide was encapsulated in the liposomes during dialysis. Enhanced fluorescence of ethidium bromide upon binding to injected DNA was monitored, and showed that injection is a rapid, one-step process. The bimolecular rate law, determined by the method of initial rates, revealed that injection occurs several times faster than indicated by earlier studies employing indirect assays.

It is hoped that these studies will increase the understanding of the mechanisms of virus entry into cells, and to facilitate the development of virus-mimetic drug delivery strategies.

Electron tunneling in proteins and water

The subject of this thesis is electronic coupling in donor-bridge-acceptor systems. In Chapter 2, ET properties of cyanide-bridged dinuclear ruthenium complexes were investigated. The strong interaction between the mixed-valent ruthenium centers leads to intense metal-to-metal charge transfer bands (MMCT). Hush analysis of the MMCT absorption bands yields the electronic-coupling strength between the metal centers (H_(AB)) and the total reorganization energy (λ). Comparison of ET kinetics to calculated rates shows that classical ET models fail to account for the observed kinetics and nuclear tunneling must be considered.

In Chapter 3, ET rates were measured in four ruthenium-modified highpotential iron-sulfur proteins (HiPIP), which were modified at position His50, His81, His42 and His18, respectively. ET kinetics for the His50 and His81 mutants are a factor of 300 different, while the donor-acceptor separation is nearly identical. PATHWAY calculations corroborate these measurements and highlight the importance of structural detail of the intervening protein matrix.

In Chapter 4, the distance dependence of ET through water bridges was measured. Photoinduced ET measurements in aqueous glasses at 77 K show that water is a poor medium for ET. Luminescence decay and quantum yield data were analyzed in the context of a quenching model that accounts for the exponential distance dependence of ET, the distance distribution of donors and acceptors embedded in the glass and the excluded volumes generated by the finite sizes of the donors and acceptors.

In Chapter 5, the pH-dependent excited state dynamics of ruthenium-modified amino acids were measured. The [Ru(bpy)_(3)] ^(2+) chromophore was linked to amino acids via an amide linkage. Protonation of the amide oxygen effectively quenches the excited state. In addition. time-resolved and steady-state luminescence data reveal that nonradiative rates are very sensitive to the protonation state and the structure of the amino acid moiety.

Electron transfer in chemically and genetically modified myoglobins

The temperature dependences of the reduction potentials (E^o') of wildtype human myoglobin (Mb) and three site-directed mutants have been measured by using thin-layer spectroelectrochemistry. Residue Val68, which is in van der Waals contact with the heme in Mb, has been replaced by Glu, Asp, and Asn. At pH 7.0, reduction of the heme iron (III) in the former two proteins is accompanied by uptake of a proton by the protein. The changes in E^o', and the standard entropy (ΔS^o') and enthalpy (ΔH^o') of reduction in the mutant proteins were determined relative to values for wild-type; the change in E^o' at 25°C was about -200 millivolts for the Glu and Asp mutants, and about -80 millivolts for the Asn mutant. Reduction of Fe(III) to Fe(II) in the Glu and Asp mutants is accompanied by uptake of a proton. These studies demonstrate that Mb can tolerate substitution of a buried hydrophobic group by potentially charged and polar residues, and that such amino acid replacements can lead to substantial changes in the redox thermodynamics of the protein.

Through analysis of the temperature dependence and shapes of NMR dispersion signals, it is determined that a water molecule is bound to the sixth coordination site of the ferric heme in the Val68Asp and in the Val68Asn recombinant proteins while the carboxyl group of the sidechain of Glu68 occupies this position in Val68Glu. The relative rhombic distortions in the ESR spectra of these mutant proteins combined with H₂¹⁷O and spin interconversion experiments performed on them confirm the conclusions of the NMRD study.

The rates of intramolecular electron transfer (ET) of (NH₃)₅Ru-His48 (Val68Asp, His81GIn, Cys110AIa)Mb and (NH₃)₅Ru-His48 (Val68GIu,His81GIn,Cys110Ala)Mb were measured to be .85(3)s^-1 and .30(2)s^-1, respectively. This data supports the hypothesis that entropy of 111 reduction and reorganization energy of ET are inversely related. The rates of forward and reverse ET for (NH₃)₅ Ru-His48 (Val68GIu, His81 GIn, Cys110AIa)ZnMb -7.2(5)•10⁴s^-1and 1.4(2)•10⁵s^-1, respectively- demonstrate that the placement of a highly polar residue nearby does not significantly change the reorganization energy of the photoactive Zn porphyrin.

The distal histidine imidazoles of (NH₃)₄isnRu-His48 SWMb and (NH₃)₅Ru-His48 SWMb were cyanated with BrCN. The intramolecular ET rates of these BrCN-modified Mb derivatives are 5.5(6)s^-1 and 3.2(5)s^-1, respectively. These respective rates are 20 and 10 times faster than those of their noncyanated counterparts after the differences in ET rate from driving force are scaled according to the Marcus equation. This increase in ET rate of the cyanated Mb derivatives is attributed to lower reorganization energy since the cyanated Mb heme is pentacoordinate in both oxidation states; whereas, the native Mb heme loses a water molecule upon reduction so that it changes from six to five coordinate. The reorganization energy from Fe-OH₂ dissociation is estimated to be .2eV. This conclusion is used to reconcile data from previous experiments in our lab. ET in photoactive porphyrin-substituted myoglobins proceed faster than predicted by Marcus Theory when it is assumed that the only difference in ET parameters between photoactive porphyrins and native heme systems is driving force. However, the data can be consistently fit to Marcus Theory if one corrects for the smaller reorganization in the photoactive porphyrin systems since they do not undergo a coordination change upon ET.

Finally, the intramolecular ET rate of (NH₃)₄isnRu-His48 SWMb was measured to be 3.0(4)s^-1. This rate is within experimental error of that for (NH₃)₄pyrRu-His48 SWMb even though the former has 80mV more driving force. One likely possibility for this observation is that the tetraamminepyridineruthenium group undergoes less reorganization upon ET than the tetraammineisonicotinamideruthenium group. Moreover, analysis of the (NH₃)₄isnRu-His48 SWMb experimental system gives a likely explanation of why ET was not observed previously in (NH₃)₄isnRu-Cytochrome C.

Mechanisms of Transposable Element Repression by Piwi Proteins in the piRNA Pathway of Drosophila Germ Cells

The ability to reproduce is a defining characteristic of all living organisms. During reproduction, the integrity of genetic material transferred from one generation to the next is of utmost importance. Organisms have diverse strategies to ensure the fidelity of genomic information inherited between generations of individuals. In sexually reproducing animals, the piRNA pathway is an RNA-interference (RNAi) mechanism that protects the genomes of germ cells from the replication of ‘selfish’ genetic sequences called transposable elements (TE). When left unabated, the replication of TE sequences can cause gene disruption, double-stranded DNA breaks, and germ cell death that results in sterility of the organism. In Drosophila, the piRNA pathway is divided into a cytoplasmic and nuclear branch that involves the functions of three Piwi-clade Argonaute proteins—Piwi, Aubergine (Aub) and Argonaute-3 (Ago3)—which bind piwi-interacting RNA (piRNA) to form the effector complexes that represses deleterious TE sequences.

The work presented in this thesis examines the function and regulation of Piwi proteins in Drosophila germ cells. Chapter 1 presents an introduction to piRNA biogenesis and to the essential roles occupied by each Piwi protein in the repression of TE. We discuss the architecture and function of germ granules as the cellular compartments where much of the piRNA pathway operates. In Chapter 2, we present how Piwi in the nucleus co-transcriptionally targets genomic loci expressing TE sequences to direct the deposition of repressive chromatin marks. Chapter 3 examines the cytoplasmic function of the piRNA pathway, where we find that the protein Krimper coordinates Aub and Ago3 in the piRNA ping-pong pathway to adaptively target and destroy TE transcripts. Chapter 4 explores how interactions of Piwis with associated proteins are modulated by arginine methylation modifications. Lastly, in Chapter 5 I present evidence that the cytoplasmic branch of the piRNA pathway can potentially ‘cross-talk’ with the nuclear branch to transfer sequence information to better target and co-transcriptionally silence the genomic loci coding active TE sequences. Overall, the work presented in this thesis constitutes a part of the first steps in understanding the molecular mechanisms that protect germ cells from invasion by TE sequences.

Photochemical electron transfer at fixed distance : a synthetic model of the photosynthetic primary process

A series of meso-phenyloctamethylporphyrins covalently bonded at the 4'phenyl position to quinones via rigid bicyclo[2.2.2]octane spacers were synthesized for the study of the dependence of electron transfer reaction rate on solvent, distance, temperature, and energy gap. A general and convergent synthesis was developed based on the condensation of ac-biladienes with masked quinonespacer-benzaldehydes. From picosecond fluorescence spectroscopy emission lifetimes were measured in seven solvents of varying polarity. Rate constants were determined to vary from 5.0x10⁹sec^-1 in N,N-dimethylformamide to 1.15x10¹⁰ Sec^-1 in benzene, and were observed to rise at most by about a factor of three with decreasing solvent polarity. Experiments at low temperature in 2-MTHF glass (77K) revealed fast, nearly temperature-independent electron transfer characterized by non-exponential fluorescence decays, in contrast to monophasic behavior in fluid solution at 298K. This example evidently represents the first photosynthetic model system not based on proteins to display nearly temperature-independent electron transfer at high temperatures (nuclear tunneling). Low temperatures appear to freeze out the rotational motion of the chromophores, and the observed nonexponential fluorescence decays may be explained as a result of electron transfer from an ensemble of rotational conformations. The nonexponentiality demonstrates the sensitivity of the electron transfer rate to the precise magnitude of the electronic matrix element, which supports the expectation that electron transfer is nonadiabatic in this system. The addition of a second bicyclooctane moiety (15 Å vs. 18 Å edge-to-edge between porphyrin and quinone) reduces the transfer rate by at least a factor of 500-1500. Porphyrinquinones with variously substituted quinones allowed an examination of the dependence of the electron transfer rate constant κ_ET on reaction driving force. The classical trend of increasing rate versus increasing exothermicity occurs from 0.7 eV≤ |ΔG^0'(R)| ≤ 1.0 eV until a maximum is reached (κ_ET = 3 x 10⁸ sec^-1 rising to 1.15 x 10¹⁰ sec^-1 in acetonitrile). The rate remains insensitive to ΔG⁰ for ~ 300 mV from 1.0 eV≤ |ΔG^0’(R)| ≤ 1.3 eV, and then slightly decreases in the most exothermic case studied (cyanoquinone, κ_ET = 5 x 10⁹ sec^-1).

A bifunctional structure for the direct electron transfer of Cytochrome c

It was found that silicon dioxide (SiO2) nanoparticles modified onto glassy carbon (GC) electrode exhibited a dramatic promotion on the direct electron transfer of Cytochrome c (Cyt c). The corresponding mechanism was discussed based on the electrochemical characteristics and a spatial geometrical model of the bifunctional structure. The model could offer insight to the study of biosensors and bioreactors without chemical mediator and serve as a basis for their fabrication. (c) 2008 Elsevier Ltd. All rights reserved.

A study on the interaction between ibuprofen and bilayer lipid membrane

Ibuprofen is a well-known nonsteroidal anti-inflammatory drug, which can interact with lipid membranes. In this paper, the interaction of ibuprofen with bilayer lipid membrane was studied by UV-vis spectroscopy, cyclic voltammetry and AC impedance spectroscopy. UV-vis spectroscopy data indicated directly that ibuprofen could interact with lipid vesicles. In electrochemical experiments, ibuprofen displayed a biphasic behavior on bilayer lipid membrane supported on a glassy carbon electrode. It could stabilize the lipid membrane in low concentration, while it induced defects formation, even removed off bilayer lipid membrane from the surface of the electrode with increasing concentration. The mechanism about the interaction between ibuprofen and supported bilayer lipid membrane was discussed.

Hydrogen peroxide biosensor based on microperoxidase-11 entrapped in lipid membrane

A highly catalytic activity microperoxidase-11 (MP-11) biosensor for H2O2 was developed to immobilizing the heme peptide in didodecyldimethylammonium bromide (DDAB) lipid membrane. The enzyme electrode thus obtained responded to H2O2 without electron mediator or promoter, at a potential of +0.10 V versus Ag \ AgCl. A linear calibration curve is obtained over the range from 2.0 x 10(-5) to 2.4 x 10(-3) M. The biosensor responds to hydrogen peroxide in 15 s and has a detection limit of 8 x 10(-7) M (S/N = 3) Providing a natural environment with lipid membrane for protein immobilization and maintenance of protein functions is a suitable option for the design of biosensors.

Study of the ion channel behavior of didodecyldimethylammonium bromide formed bilayer lipid membrane stimulated by PF6-

Bilayer lipid membranes ( BLM) formed from didode-cyldimethylammonium bromide were made on the freshly exposed surface of a glassy carbon (GC) and were demonstrated by the ac impedance spectroscopy. The ion channels of membrane properties induced by PF6- were studied by the cyclic voltammetric methods. Experimental results indicated that the ion channel of BLM was open in the presence of the PF6- due to the interaction of PF6- with the BLM, while it was switched off in the absence of PF6-. Because the ion channel behavior was affected by the concentration of PF6-, a sensor for PF6- can be developed.

Electrochemical and spectroscopic study on the interaction of cytochrome c with anionic lipid vesicles

The structure and the electron-transfer of cytochrome c binding on the anionic lipid vesicles were analyzed by electrochemical and various spectroscopic methods. It was found that upon binding to anionic lipid membrane, the formal potential of. cytochrome c shifted 30 mV negatively indicating an eager redox interaction than that in its native state. This is due to the local alteration of the coordination and the heme crevice. The structural Perturbation in which a molten globule-like state is formed during binding to anionic lipid vesicles is more important. This study may help to understand the mechanism of the electron-transfer reactions of cytochrome c at the mitochondrial membrane.

Conformation change of horseradish peroxidase in lipid membrane

The electrochemical behavior of horseradish peroxidase (HRP) in the dimyristoyl phosphatidylcholine (DMPC) bilayer on the glassy carbon (GC) electrode was studied by cyclic voltammetry. The direct electron transfer of HRP was observed in the DMPC bilayer. Only a small cathodic peak was observed for HRP on the bare GC electrode. The electron transfer of HRP in the DMPC membrane is facilitated by DMPC membrane. UV-Vis and circular dichroism (CD) spectroscopy were used to study the interaction between HRP and DMPC membrane. On binding to the DMPC membrane the secondary structure of HRP remains unchanged while there is a substantial change in the conformation of the heme active site. Tapping mode atomic force microscopy (AFM) was first applied for the investigation on the structure of HRP adsorbed on supported phospholipid bilayer on the mica and on the bare mica. HRP molecules adsorb and aggregate on the mica without DMPC bilayer. The aggregation indicates an attractive interaction among the adsorbed molecules. The molecules are randomly distributed in the DMPC bilayer. The adsorption of HRP in the DMPC bilayer changes drastically the domains and defects in the DMPC bilayer due to a strong interaction between HRP and DMPC films.