Biomedical subjects

Y K Shin

Publications and source records attributed to Y K Shin.

At least 37 records · Page 2Linked to original sources

PubMed2000

Amitriptyline modulation of Na(+) channels in rat dorsal root ganglion neurons.

The effects of amitriptyline, a tricyclic antidepressant, on tetrodotoxin-sensitive and tetrodotoxin-resistant Na(+) currents in rat dorsal root ganglion neurons were studied using the whole-cell patch clamp method. Amitriptyline blocked both types of Na(+)currents in a dose-and holding potential-dependent manner. At the holding potential of -80 mV, the apparent dissociation constants (K(d)) for amitriptyline to block tetrodotoxin-sensitive and tetrodotoxin-resistant Na(+) channels were 4.7 and 105 microM, respectively. These values increased to 181 and 193 microM, respectively, when the membrane was held at a potential negative enough to remove the steady-state inactivation. Amitriptyline dose-dependently shifted the steady-state inactivation curves in the hyperpolarizing direction and increased the values of the slope factors for both types of Na(+) channels. The voltage dependence of the activation of both types of Na(+) channels was shifted in the depolarizing direction. It was concluded that amitriptyline blocked the two types of Na(+) channels in rat sensory neurons by modulating the activation and the inactivation kinetics.

Amitriptyline↗

PubMed2000

ATP evokes different currents in TTX-sensitive and TTX-resistant cells of dorsal root ganglia.

The relationship between the level of expression of tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium currents and the occurrence of two kinetically different ATP-induced currents in rat dorsal root ganglion neurons was studied. ATP evoked two distinct types of currents, one with fast activation and desensitization (I-fast) and the other with slow and persistent development (I-slow). In all TTX-S cells which expressed solely TTX-S sodium currents ATP evoked I-fast. The other cells expressed a variable proportion of TTX-S and TTX-R sodium currents. Only 15% of these TTX-R+S cells responded to ATP with I-fast. I-slow was evoked in both cell types but the magnitude of response was much greater in TTX-R+S cells. This result suggests that a different array of ion channels is equipped in different types of sensory neurons.

Adenosine Triphosphate↗

PubMed2000

Inhibitory action of thimerosal, a sulfhydryl oxidant, on sodium channels in rat sensory neurons.

The effects of thimerosal, a sulfhydryl oxidizing agent, on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channels in rat dorsal root ganglion neurons were studied using the whole-cell patch clamp technique. Thimerosal blocked the two types of sodium channels in a dose-dependent manner. The inhibitory effect of thimerosal was much more pronounced in TTX-R sodium channels than TTX-S sodium channels. The effect of thimerosal was irreversible upon wash-out with thimerosal-free external solution. However, dithiothreitol, a reducing agent, partially reversed it. Thimerosal shifted the steady-state inactivation curves for both types of sodium channels in the hyperpolarizing direction. The voltage dependence of activation of both types of sodium channels was shifted in the depolarizing direction by thimerosal. The inactivation rate in both types of sodium channels increased after thimerosal treatment. All these effects of thimerosal would add up to cause a depression of sodium channel function leading to a diminished neuronal excitability.

Animals↗

PubMed2000

Factors determining vesicular lipid mixing induced by shortened constructs of influenza hemagglutinin.

The HA2 subunit of influenza hemagglutinin is responsible for fusion of the viral and host-cell membranes during infection. An N-terminal 127 amino acid construct of HA2, FHA2-127, is shown to induce lipid mixing of large unilamellar vesicles under endosomal low pH conditions. Thus, FHA2 could serve as a good model system for biophysical studies of membrane fusion. With FHA2, we began to develop a mechanistic model which could explain how this short construct facilitates membrane fusion. In this endeavor, we studied the possible role of the kinked loop region (amino acids 105-113). A construct missing this loop, FHA2-90, although able to induce lipid mixing, has lost the sharp pH-dependent transition seen with FHA2-127 and native HA. In addition, FHA2-127 promotes extensive vesicle aggregation more effectively than FHA2-90 upon acidification. These data suggest that the kinked loop may play a pH-dependent regulatory role. To test this, we compared bis-ANS binding to the two constructs and observed that binding to FHA2-127 increases at a faster rate than FHA2-90 as the pH is decreased, indicating that the kinked loop not only is an ANS-binding site, but that it binds better at low pH. The pH dependence of this transition directly correlates with that observed in lipid mixing. Further, cysteine mutations of acidic residues in the kinked region are both fusion inactive and bind much less ANS, whereas a similar mutation of a threonine residue had little effect on fusion activity or ANS binding. This evidence lends further support to our idea that the kinked loop serves a regulatory role. To test the physiological relevance of the FHA2-127 fusion mechanism, we studied the effects of a G1E mutation, known to abolish fusion in native HA. We found that G1E-127 is fusion inactive as expected. This evidence indirectly suggests that the mechanism of FHA2-127 is perhaps physiologically relevant and from its study, we can learn much about the mechanism of native HA.

Amino Acid Sequence↗

PubMed2000

N-Ethylmaleimide modulation of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels in rat dorsal root ganglion neurons.

The effects of N-ethylmaleimide (NEM), an alkylating reagent to protein sulfhydryl groups, on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channels in rat dorsal root ganglion (DRG) neurons were studied using the whole cell configuration of patch-clamp technique. When currents were evoked by step depolarizations to 0 mV from a holding potential of -80 mV NEM decreased the amplitude of TTX-S sodium current, but exerted little or no effect on that of TTX-R sodium current. The inhibitory effect of NEM on TTX-S sodium channel was mainly due to the shift of the steady-state inactivation curve in the hyperpolarizing direction. NEM did not affect the voltage-dependence of the activation of TTX-S sodium channel. The steady-state inactivation curve for TTX-R sodium channel was shifted by NEM in the hyperpolarizing direction as that for TTX-S sodium channel. NEM caused a change in the voltage-dependence of the activation of TTX-R sodium channel unlike TTX-S sodium channel. After NEM treatment, the amplitudes of TTX-R sodium currents at test voltages below -10 mV were increased, but those at more positive voltages were not affected. This was explained by the shift in the conductance-voltage curve for TTX-R sodium channels in the hyperpolarizing direction after NEM treatment.

Animals↗

PubMed2000

Viral latent membrane protein 1 (LMP-1)-induced CD99 down-regulation in B cells leads to the generation of cells with Hodgkin's and Reed-Sternberg phenotype.

Recently we reported that the down-regulation of CD99 (Mic2) is a primary requirement for the generation of Hodgkin's and Reed-Sternberg (H-RS) cells seen in Hodgkin's disease. In this study, we provide evidence that the down-regulation of CD99 is induced by high expression of Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1), which is highly expressed in H-RS cells of EBV-associated Hodgkin's disease. To investigate the effect of LMP-1 on the expression of CD99 in vitro, we established a stable cell line by transfecting an SV40-early promoter driven-LMP-1 expression construct into a neoplastic lymphoblastoid B cell line, IM9, in which the level of endogenous LMP-1 expression is almost negligible. In this cell line, the overexpression of LMP-1 led to the down-regulation of CD99 and the acquisition of morphological and functional characteristics of H-RS cells indistinguishable from those in lymph nodes of Hodgkin's disease patients and in CD99-deficient B cells. In addition, induced LMP-1 expression in an EBV-negative B cell clone, BJAB, directly caused the down-regulation of surface CD99 expression. Northern and Western analysis data, showing that overexpression of LMP-1 negatively influenced the expression of CD99, were supported by experiments in which a CD99 promoter-driven luciferase promoter reporter construct transfected into 293T cells was down-regulated when LMP-1 was coexpressed. Therefore, our data strongly suggest that the EBV LMP-1 protein plays a pivotal role in the down-regulation of CD99 via transcriptional regulation, which leads to the generation of the H-RS cells. (Blood. 2000;95:294-300)

12E7 Antigen↗

PubMed2000

Protective effect of boldine on oxidative mitochondrial damage in streptozotocin-induced diabetic rats.

Increased oxidative stress has been suggested to be involved in the pathogenesis and progression of diabetic tissue damage. Several antioxidants have been described as beneficial for oxidative stress-associated diseases. Boldine ([s]-2,9-dihydroxy-1, 10-dimethoxyaporphine) is a major alkaloid found in the leaves and bark of boldo (Peumus boldus Molina), and has been shown to possess antioxidant activity and anti-inflammatory effects. From this point of view, the possible anti-diabetic effect of boldine and its mechanism were evaluated. The experiments were performed on male rats divided into four groups: control, boldine (100 mg kg(-1), daily in drinking water), diabetic [single dose of 80 mg kg(-1)of streptozotocin (STZ), i.p.] and diabetic simultaneously fed with boldine for 8 weeks. Diabetic status was evaluated periodically with changes of plasma glucose levels and body weight in rats. The effect of boldine on the STZ-induced diabetic rats was examined with the formation of malondialdehydes and carbonyls and the activities of endogenous antioxidant enzymes (superoxide dismutase and glutathione peroxidase) in mitochondria of the pancreas, kidney and liver. The scavenging action of boldine on oxygen free radicals and the effect on mitochondrial free-radical production were also investigated. The treatment of boldine attenuated the development of hyperglycemia and weight loss induced by STZ injection in rats. The levels of malondialdehyde (MDA) and carbonyls in liver, kidney and pancreas mitochondria were significantly increased in STZ-treated rats and decreased after boldine administration. The activities of mitochondrial manganese superoxide dismutase (MnSOD) in the liver, pancreas and kidney were significantly elevated in STZ-treated rats. Boldine administration decreased STZ-induced elevation of MnSOD activity in kidney and pancreas mitochondria, but not in liver mitochondria. In the STZ-treated group, glutathione peroxidase activities decreased in liver mitochondria, and were elevated in pancreas and kidney mitochondria. The boldine treatment restored the altered enzyme activities in the liver and pancreas, but not the kidney. Boldine attenuated both STZ- and iron plus ascorbate-induced MDA and carbonyl formation and thiol oxidation in the pancreas homogenates. Boldine decomposed superoxide anions, hydrogen peroxides and hydroxyl radicals in a dose-dependent manner. The alkaloid significantly attenuated the production of superoxide anions, hydrogen peroxide and nitric oxide caused by liver mitochondria. The results indicate that boldine may exert an inhibitory effect on STZ-induced oxidative tissue damage and altered antioxidant enzyme activity by the decomposition of reactive oxygen species and inhibition of nitric oxide production and by the reduction of the peroxidation-induced product formation. Boldine may attenuate the development of STZ-induced diabetes in rats and interfere with the role of oxidative stress, one of the pathogeneses of diabetes mellitus.

Animals↗

PubMed2000

Insights into a structure-based mechanism of viral membrane fusion.

A number of different viral spike proteins, responsible for membrane fusion, show striking similarities in their core structures. The prospect of developing a general structure-based mechanism seems plausible in light of these newly determined structures. Influenza hemagglutinin (HA) is the best-studied fusion machine, whose action has previously been described by a hypothetical "spring-loaded" model. This model has recently been extended to explain the mechanism of other systems, such as HIV gp120-gp41. However, evidence supporting this idea is insufficient, requiring re-examination of the mechanism of HA-induced membrane fusion. Recent experiments with a shortened construct of HA, which is able to induce lipid mixing, have provided evidence for an alternative scenario for HA-induced membrane fusion and perhaps that of other viral systems.

Hemagglutinins↗

PubMed2000

Gordonia nitida sp. nov., a bacterium that degrades 3-ethylpyridine and 3-methylpyridine.

A bacterial strain, LE31T, which is capable of degrading 3-ethylpyridine and 3-methylpyridine, was isolated from an industrial wastewater and was taxonomically studied by using a polyphasic approach. Strain LE31T was identified as a member of the genus Gordonia on the basis of chemotaxonomic characteristics and phylogenetic inference-based 16S rDNA sequence. The cell wall contained meso-diaminopimelic acid, arabinose and galactose (wall chemotype IV). The predominant menaquinone was MK-9(H2). The mycolic acids contained 47-55 carbon atoms. The major fatty acids were C16:0, C18:1 omega9c, 10-methyl-C18:0 (TBSA). The G+C content of DNA was 67 mol%. The 16S rDNA sequence of strain LE31T was most similar to that of the type strain of Gordonia rubropertincta. The differences in some phenotypic characteristics and the genetic distinctiveness distinguish strain LE31T from the Gordonia species described previously. Therefore it is proposed that strain LE31T should be placed in the genus Gordonia as a new species. The name Gordonia nitida is proposed for strain LE31T. The type strain of the new species is strain LE31T (= KCTC 0605BPT = KCCM 80004T).

Actinomycetales↗

PubMed2000

DNA-DNA relatedness among Thermoactinomyces species: Thermoactinomyces candidus as a synonym of Thermoactinomyces vulgaris and Thermoactinomyces thalpophilus as a synonym of Thermoactinomyces sacchari.

DNA-DNA relatedness of all validly described Thermoactinomyces species was determined to infer the genetic relationships between them. The levels of DNA-DNA relatedness among the type strains of Thermoactinomyces species ranged from 2-5 to 92.8%. Based on DNA relatedness data, the type strains of Thermoactinomyces intermedius, Thermoactinomyces putidus, Thermoactinomyces dichotomicus and Thermoactinomyces peptonophilus were considered to be distinct species of the genus Thermoactinomyces. However, the relationship between the type strains of Thermoactinomyces vulgaris and Thermoactinomyces candidus and the relationship between the type strains of Thermoactinomyces sacchari and Thermoactinomyces thalpophilus were reevaluated from levels of DNA-DNA relatedness. The independent DNA relatedness values between Thermoactinomyces vulgaris KCTC 9076T and Thermoactinomyces candidus KCTC 9557T were 90.8 and 92.8%. Thermoactinomyces thalpophilus KCTC 9789T and Thermoactinomyces sacchari KCTC 9790T exhibited independent values of 85.6 and 87.3%. Accordingly, on the basis of DNA-DNA relatedness data together with 16S rDNA sequence data determined recently, it is proposed that Thermoactinomyces candidus should be considered as a synonym of Thermoactinomyces vulgaris and Thermoactinomyces thalpophilus be considered as a synonym of Thermoactinomyces sacchari.

DNA, Bacterial↗

PubMed2000

Phylogenetic analysis of the small hydrophobic (SH) gene of mumps virus in Korea: identification of a new genotype.

Viral RNAs extracted from fifteen mumps virus isolated from throat swab, saliva, blood, urine or CSF during mumps epidemics between 1997-1998 in Korea were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) and compared by nucleotide sequencing of the small hydrophobic (SH) gene. The deduced amino acid sequences of the SH gene were aligned with the published sequences of mumps virus isolated in different geographic areas. A comparison of the SH gene of mumps viruses in Korea indicated 96.2-100% and 91.2-100% similarity at the nucleotide and amino acid levels, respectively. Phylogenetic analysis, using the neighbor-joining method, showed that Korean mumps virus strains formed a genetically distinct monophyletic group from previously reported genotypes based on the 315-bp length nucleotide and 57 deduced amino acid sequences of the SH gene, and possibly be designated as a new genotype (I).

Adolescent↗

PubMed1999

A piston model for transmembrane signaling of the aspartate receptor.

To characterize the mechanism by which receptors propagate conformational changes across membranes, nitroxide spin labels were attached at strategic positions in the bacterial aspartate receptor. By collecting the electron paramagnetic resonance spectra of these labeled receptors in the presence and absence of the ligand aspartate, ligand binding was shown to generate an approximately 1 angstrom intrasubunit piston-type movement of one transmembrane helix downward relative to the other transmembrane helix. The receptor-associated phosphorylation cascade proteins CheA and CheW did not alter the ligand-induced movement. Because the piston movement is very small, the ability of receptors to produce large outcomes in response to stimuli is caused by the ability of the receptor-coupled enzymes to detect small changes in the conformation of the receptor.

Aspartic Acid↗

PubMed1999

Simvastatin preserves the ischemic-reperfused myocardium in normocholesterolemic rat hearts.

BACKGROUND: Ischemia followed by reperfusion in the presence of polymorphonuclear leukocytes (PMNs) results in cardiac contractile dysfunction as well as cardiomyocyte injury. These deleterious effects are due in large part to endothelial dysfunction leading to the upregulation of cell adhesion molecules and subsequent neutrophil-endothelium interaction. At clinically relevant doses, simvastatin, an HMG-CoA reductase inhibitor, has been shown to lower serum cholesterol levels and normalize endothelial cell function. We wanted to test the effects of simvastatin on neutrophil-mediated cardiac dysfunction in a controlled model of myocardial ischemia-reperfusion. METHODS AND RESULTS: This study examines the effects of simvastatin in a neutrophil-dependent isolated perfused rat heart model of ischemia (I) (20 minutes) and reperfusion (R) (45 minutes) injury. Administration of simvastatin 25 micrograms/rat improved coronary flow and preserved left ventricular developed pressure (LVDP) and dP/dtmax, indexes of cardiac contractile function. Final LVDP was 95+/-5 mm Hg in I/R hearts perfused with PMNs and simvastatin, compared with 49+/-4 mm Hg in PMN-perfused I/R hearts receiving only vehicle (P<0.001). In addition, simvastatin significantly reduced PMN accumulation in the ischemic myocardium (P<0.01). In PMN-perfused rat hearts after I/R, simvastatin also significantly attenuated P-selectin expression, CD18 upregulation in rat PMNs, and PMN adherence to rat vascular endothelium. Significant, although less potent, effects were obtained with pravastatin. CONCLUSIONS: These results provide evidence that HMG-CoA reductase inhibitors are potent and effective cardioprotective agents that inhibit leukocyte-endothelial cell interactions and preserve cardiac contractile function and coronary perfusion after myocardial ischemia and reperfusion. Moreover, these effects are unrelated to the cholesterol-lowering action of this agent and appear to be mediated by enhanced endothelial release of NO.

Animals↗

PubMed1999

Identification of unacceptable background caused by non-specific protein adsorption to the plastic surface of 96-well immunoassay plates using a standardized enzyme-linked immunosorbent assay procedure.

A standardized enzyme-linked immunosorbent assay (ELISA) was used to examine the capacity of immunoassay plates to prevent non-specific protein binding under blocking conditions. Data from 16 types of 96-well microtitre plate from seven commercial sources, are described. Plates were evaluated with respect to their capacity to adsorb a conjugated antibody in diluent buffer containing non-ionic detergent Tween 20 (0.05%) and skimmed milk proteins (5%). Plates with an absorbance value of > or = 0.05, in not more than one well, were defined as within acceptable limits. Major problems were seen in high binding gamma-irradiated polystyrene plates, from all sources, where only < or = 30% of plates were acceptable. These showed high, randomly distributed, non-specific binding, with some wells showing absorbance values > 2.0. Similar results were obtained when high binding plates were repeatedly gamma-irradiated, and after gamma-irradiation of low binding polystyrene plates. For high binding, non- gamma-irradiated polystyrene plates, approximately 70% of plates were acceptable. Better results (86-100% acceptability) were observed for all low binding polystyrene plates. Only one source in three provided acceptable, low binding, polyvinylchloride plates. This paper confirms a widely held view that non-specific binding to certain plates could be a serious factor in both the development and application of ELISAs. Therefore, the test protocol described is proposed as an additional quality control method for certifying ELISA plates by commercial companies.

Adsorption↗

PubMed1999

Physical evidence for a phosphorylation-dependent conformational change in the enhancer-binding protein NtrC.

The bacterial enhancer-binding protein nitrogen regulatory protein C (NtrC) activates transcription by sigma54-containing RNA polymerase in a reaction that depends on ATP hydrolysis. Phosphorylation of an aspartate residue in the N-terminal receiver domain of NtrC induces oligomerization of the protein and activates the ATPase activity, which is a function of its central output domain. To study the role of the receiver domain of NtrC, which is known to act positively, we isolated mutant forms of the protein carrying single cysteine residues and derivatized them with a sulfhydryl-specific nitroxide reagent for electron paramagnetic resonance studies. Single cysteines were placed at four positions at which we had obtained constitutive amino acid substitutions, those that yield activity without phosphorylation. In only one case, derivatized C86 in alpha-helix 4 of the receiver domain, did the motion of the side chain become dramatically slower upon phosphorylation. Importantly, derivatized NtrCD86C (NtrCD86C*) activated transcription normally. Additional experiments indicated that the spectral change observed upon phosphorylation of NtrCD86C* was due to interdomain interactions rather than a conformational change within the N-terminal domain itself. These interactions did not appear to occur within a monomer. Although it is not clear whether the spectral change seen upon phosphorylation of NtrCD86C* is due to an interaction that occurs within a dimer of NtrC or requires the formation of higher-order oligomers, the change indicated that alpha-helix 4 of the receiver domain probably plays an important role in communication with the remainder of the protein.

Bacterial Proteins↗

PubMed1999

Biochemical and biophysical characterization of the trimerization domain from the heat shock transcription factor.

Previously, we had characterized a 91 amino acid fragment of the heat shock transcription factor from the yeast Kluyveromyces lactis and had shown it to be highly alpha-helical and sufficient for formation of homotrimers [Peteranderl, R., and Nelson, H. C. M. (1992) Biochemistry 31, 12272-12276]. Based on those data, as well as the presence of hydrophobic heptad repeats, we postulated that the trimerization domain contains a three-stranded coiled-coil and that it might resemble the trimerization domain found in influenza hemagglutinin. Here, we further characterize the trimerization domain and show that the minimal domain needs 71 residues to remain trimeric and highly alpha-helical. 19F NMR spectroscopy suggests that the structure contains three parallel strands that are in register along the long axis of the coiled-coil. Electron paramagnetic resonance spectroscopy studies show that the C-termini of the subunits are in close proximity; this is in contrast to the topology of the hemaglutinin trimerization domain where the C-termini form buttressing helices. Analytical ultracentrifugation also confirms that the structure is elongated and unlikely to have buttressing helices. Additional experiments suggest that the trimerization domain has at least two subdomains. The first subdomain has the potential to form trimers independently, though not as stably as the complete domain. The second subdomain is quite helical, forms large oligomers, and appears to provide stability to the complete domain. Our current model for the heat shock transcription factor trimerization domain is a highly elongated coiled-coil structure, with a potential break in the coiled-coil region located between the two subdomains.

Amino Acid Sequence↗

PubMed1999

The ectodomain of HA2 of influenza virus promotes rapid pH dependent membrane fusion.

To better understand the roles of different regions of influenza hemagglutinin in membrane fusion, we have studied the fusion properties of large unilamellar vesicles in the presence of constructs comprising the 127 amino acid ectodomain of the HA2 fragment (FHA2) as well as mutated forms of FHA2 containing single amino acid substitutions, the 95 amino acid truncated form of FHA2 lacking the N-terminal fusion peptide (SHA2), the 20 amino acid N-terminal fusion peptide and the ten amino acid peptide corresponding to the kinked loop region of FHA2. The 100 nm liposomes were made from dioleoylphosphatidylethanolamine, dioleoylphosphatidylcholine and cholesterol in equimolar ratio. At pH 5 a high rate of lipid mixing was observed with FHA2 present, even at very low molar concentrations, whereas much lower rates were observed using the shorter constructs: SHA2, the fusion peptide, and the loop peptide. Concentrations of FHA2 which promoted extensive lipid mixing also induced leakage of aqueous contents. Marked effects of FHA2 were also observed with liposomes of egg phosphatidylcholine. All of the changes observed with the liposomes were highly pH-dependent, with only negligible changes occurring at pH 7. The results demonstrate the potent action of FHA2 in promoting lipid mixing and demonstrate the contribution of other regions of the ectodomain of FHA2, in addition to the fusion peptide, to the mechanism of acceleration of membrane fusion. The results also indicate that the pH dependence of fusion is not due solely to changes in the interactions between the HA1 and HA2 subunits. Thus, the "spring loaded energy" is not required to bring about the apposition of the two membranes, considering that FHA2 is already in its thermostable conformation. The acidic amino acid residues in the kinked loop region appear to play a particularly important role in the pH-dependent fusion process as demonstrated by the marked loss of lipid mixing activity of mutant forms of FHA2.

Amino Acid Sequence↗

PubMed1999

The membrane affinities of the aliphatic amino acid side chains in an alpha-helical context are independent of membrane immersion depth.

Understanding, predicting, and designing the binding of peptides and proteins to bilayers require quantifying the intrinsic propensities of individual amino acid residues to bind membranes as a function of structural context and bilayer depth. A host-guest study was performed using the peptide host named helix5 in order to determine the membrane affinities of the aliphatic side chains both in an alpha-helical context and as a function of bilayer depth. Use of the alpha-helical host with a constrained geometry allowed the placement of guest sites at three different depths in bilayers and minimized secondary structural changes due to guest substitutions. Circular dichroism and electron paramagnetic resonance (EPR) were used to characterize the aqueous and bilayer-bound structures of the peptide variants. EPR was also used to measure the bilayer-water partition constants of the peptide variants, and the Delta DeltaGtr values (relative to Gly) of the aliphatic amino acid side chains were subsequently calculated. Surprisingly, the DeltaDeltaGtr values did not significantly vary as a function of the guest site depth in bilayers. In addition, the Delta DeltaGtr values determined in an alpha-helical context are reduced to approximately two-thirds of Delta DeltaGtr values determined in other studies for the bilayer-water and octanol-water partitioning of amino acid side chains in extended and unstructured hosts. Both the relative reduction in Delta DeltaGtr values in the context of an alpha-helical host and the invariance of Delta DeltaGtr values with respect to bilayer depth are consistent with the membrane affinities of the aliphatic residues being largely determined by the classical hydrophobic effect.

Amino Acid Sequence↗