Biomedical subjects

R S Hodges

Publications and source records attributed to R S Hodges.

At least 55 records · Page 3Linked to original sources

PubMed1999

Alternative roles for putative ice-binding residues in type I antifreeze protein.

Two sets of variants of type I antifreeze protein have been synthesized to investigate the role of Leu and Asn in the activity of this 37-residue alpha-helix. Leu and Asn flank the central two of four regularly spaced ice-binding Thr in the i-1 and i + 3 positions, respectively. All three residues project from the same side of the helix to form the protein's putative ice-adsorption site and are considered in some models to act together as an "ice-binding motif". Replacement of Asn by residues with shorter side chains resulted in either a small loss (Ala) or gain (Thr) of antifreeze activity. However, substitution of Asn by its slightly larger homologue (Gln) abolished thermal hysteresis activity. The Gln-containing peptide was very soluble, largely monomeric, and fully helical. Of the three variants in which Leu was replaced by Ala, two of the three were more active than their Leu-containing counterparts, but all three variants began to precipitate as the peptide concentration increased. None of the seven variants tested showed dramatic differences in ice crystal morphology from that established by the wild type. These results are consistent with a primary role for Leu in preventing peptide aggregation at the antifreeze protein concentrations (10 mg/mL) normally present in fish serum. Similarly the role for Asn may have more to do with enhancing the solubility of these rather hydrophobic peptides than of making a stereospecific hydrogen-bonding match to the ice lattice as traditionally thought. Nevertheless, the dramatic loss of activity in the Asn-to-Gln replacement demonstrates the steric restriction on residues in or near the ice-binding site of the peptide.

Amino Acid Sequence↗

PubMed1999

Multiple distinct coiled-coils are involved in dynamin self-assembly.

Dynamin, a 100-kDa GTPase, has been implicated to be involved in synaptic vesicle recycling, receptor-mediated endocytosis, and other membrane sorting processes. Dynamin self-assembles into helical collars around the necks of coated pits and other membrane invaginations and mediates membrane scission. In vitro, dynamin has been reported to exist as dimers, tetramers, ring-shaped oligomers, and helical polymers. In this study we sought to define self-assembly regions in dynamin. Deletion of two closely spaced sequences near the dynamin-1 C terminus abolished self-association as assayed by co-immunoprecipitation and the yeast interaction trap, and reduced the sedimentation coefficient from 7.5 to 4.5 S. Circular dichroism spectroscopy and equilibrium ultracentrifugation of synthetic peptides revealed coiled-coil formation within the C-terminal assembly domain and at a third, centrally located site. Two of the peptides formed tetramers, supporting a role for each in the monomer-tetramer transition and providing novel insight into the organization of the tetramer. Partial deletions of the C-terminal assembly domain reversed the dominant inhibition of endocytosis by dynamin-1 GTPase mutants. Self-association was also observed between different dynamin isoforms. Taken altogether, our results reveal two distinct coiled-coil-containing assembly domains that can recognize other dynamin isoforms and mediate endocytic inhibition. In addition, our data strongly suggests a parallel model for dynamin subunit self-association.

Animals↗

PubMed1999

Differential scanning calorimetric study of the effect of the antimicrobial peptide gramicidin S on the thermotropic phase behavior of phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol lipid bilayer membranes.

We have studied the effects of the antimicrobial peptide gramicidin S (GS) on the thermotropic phase behavior of large multilamellar vesicles of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylethanolamine (DMPE) and dimyristoyl phosphatidylglycerol (DMPG) by high-sensitivity differential scanning calorimetry. We find that the effect of GS on the lamellar gel to liquid-crystalline phase transition of these phospholipids varies markedly with the structure and charge of their polar headgroups. Specifically, the presence of even large quantities of GS has essentially no effect on the main phase transition of zwitterionic DMPE vesicles, even after repeating cycling through the phase transition, unless these vesicles are exposed to high temperatures, after which a small reduction in the temperature, enthalpy and cooperativity of the gel to liquid-crystalline phase transitions is observed. Similarly, even large amounts of GS produce similar modest decreases in the temperature, enthalpy and cooperativity of the main phase transition of DMPC vesicles, although the pretransition is abolished at low peptide concentrations. However, exposure to high temperatures is not required for these effects of GS on DMPC bilayers to be manifested. In contrast, GS has a much greater effect on the thermotropic phase behavior of anionic DMPG vesicles, substantially reducing the temperature, enthalpy and cooperativity of the main phase transition at higher peptide concentrations, and abolishing the pretransition at lower peptide concentrations as compared to DMPC. Moreover, the relatively larger effects of GS on the thermotropic phase behavior of DMPG vesicles are also manifest without cycling through the phase transition or exposure to high temperatures. Furthermore, the addition of GS to DMPG vesicles protects the phospholipid molecules from the chemical hydrolysis induced by their repeated exposure to high temperatures. These results indicate that GS interacts more strongly with anionic than with zwitterionic phospholipid bilayers, probably because of the more favorable net attractive electrostatic interactions between the positively charged peptide and the negatively charged polar headgroup in such systems. Moreover, at comparable reduced temperatures, GS appears to interact more strongly with zwitterionic DMPC than with zwitterionic DMPE bilayers, probably because of the more fluid character of the former system. In addition, the general effects of GS on the thermotropic phase behavior of zwitterionic and anionic phospholipids suggest that it is located at the polar/apolar interface of liquid-crystalline bilayers, where it interacts primarily with the polar headgroup and glycerol-backbone regions of the phospholipid molecules and only secondarily with the lipid hydrocarbon chains. Finally, the considerable lipid specificity of GS interactions with phospholipid bilayers may prove useful in the design of peptide analogs with stronger interactions with microbial as opposed to eucaryotic membrane lipids.

Anti-Infective Agents↗

PubMed1999

De novo design of a model peptide sequence to examine the effects of single amino acid substitutions in the hydrophobic core on both stability and oligomerization state of coiled-coils.

A model peptide sequence was de novo designed to investigate the hydrophobicity, stability and oligomerization state resulting from single amino acid substitutions in the hydrophobic core a position of the central heptad of a five heptad coiled-coil. This involved selecting a hydrophobic core consisting of Val and Leu at a and d positions, respectively, known to form both two- and three-stranded coiled-coils. In addition, the sequence provided the correct overall coiled-coil stability and maximized the hydrophobicity surrounding the substitution site by having Leu in the hydrophobic core above and below the site of substitution. To control oligomerization state we exploited differential placement of an interhelical disulfide-bridge, which was placed in the coiled-coil hydrophobic core at the C-terminal d position, or alternatively outside of the core at the N-terminal via a Cys-Gly-Gly linker. We found that the Cys-Gly-Gly linker allowed assessment of both relative stability and oligomerization state after extensive biophysical characterization of the models by circular dichroism, sedimentation equilibrium, sedimentation velocity and finally high-performance size-exclusion chromatography (HPSEC) under both benign and denaturing conditions. The Cys-Gly-Gly linker was found to be unique in allowing the inherent two- or three-stranded oligomerization state to be observed in benign medium, while also allowing the stability to be determined by concentration independent chemical denaturation of a two-stranded coiled-coil. This entails a two-state transition from a folded disulfide-bridged two-stranded coiled-coil (monomeric state) to the unfolded monomer, even for analogs where the coiled-coil is a trimer of disulfide-bridged peptides in benign medium. We also developed novel HPSEC methodology for monitoring the chemical denaturation of a folded monomeric protein in fast exchange with the corresponding unfolded protein, which elutes as a single peak throughout the denaturation process.

Amino Acid Sequence↗

PubMed1999

Hydrophilic interaction/cation-exchange chromatography for the purification of synthetic peptides from closely related impurities: serine side-chain acetylated peptides.

Mixed-mode hydrophilic interaction/cation-exchange chromatography (HILIC/CEC) is a novel HPLC technique which has excellent potential for peptide separations. Separations by HILIC/CEC are carried out by subjecting peptides to linear increasing salt gradients in the presence of high levels of acetonitrile, which promotes hydrophilic interactions overlayed on ionic interactions with the cation-exchange matrix. Complex peptide mixtures produced by solid-phase synthesis are a frequently encountered and challenging purification problem. In the present study a two-step protocol, consisting of HILIC/CEC followed by RPC, was required for the successful purification of a 21-residue synthetic amphipathic alpha-helical peptide from serine side-chain acetylated impurities, with HILIC/CEC proving to be highly sensitive to subtle differences in hydrophilicities between the acetylated peptides and the desired product. Investigation of the three potential sites of serine acetylation through solid-phase synthesis of acetylated analogues of the desired peptide (peptides of the same sequence and secondary structure, but acetylated at different positions on the hydrophilic face of the alpha-helix) demonstrated that acetylation was occurring at different sites on the peptide. HILIC/CEC was able to take advantage of very subtle changes in environment around the acetylation sites and thus effect a separation of these analogues not achievable by RPC or CEC alone.

Acetylation↗

PubMed1999

13C-13C rotational resonance in a transmembrane peptide: a comparison of the fluid and gel phases.

A comparative study of two doubly 13C labeled amphiphilic transmembrane peptides was undertaken to determine the potential of rotational resonance for measuring internuclear distances through the direct dipolar coupling in the presence of motion. The two peptides, having the sequence acetyl-K2-G-L16-K2-A-amide, differed only in the position of 13C labels. The first peptide, [1-13C]leu(11):[alpha-13C]leu(12), had labels on adjacent residues, at the carbonyl of leu(11) and the alpha carbon of leu(12). The second, [1-13C]leu(8):[alpha-(13)/C]leu(11), was labeled on consecutive turns of the alpha-helical peptide. The internuclear distance between labeled positions of the first peptide, which for an ideal alpha helix has a value of 2.48 A, is relatively independent of internal flexibility or peptide conformational change. The dipolar coupling between these two nuclei is sensitive to motional averaging by molecular reorientation, however, making this peptide ideal for investigating these motions. The internuclear distance between labels on the second peptide has an expected static ideal alpha-helix value of 4.6 A, but this is sensitive to internal flexibility. In addition, the dipolar coupling between these two nuclei is much weaker because of their larger separation, making this peptide a much more difficult test of the rotational resonance technique. The dipolar couplings between the labeled nuclei of these two peptides were measured by rotational resonance in the dry peptide powders and in multilamellar dispersions with dimyristoylphosphatidylcholine in the gel phase, at -10 degrees C, and in the fluid phase, at 40 degrees C. The results for the peptide having adjacent labels can be readily interpreted in terms of a simple model for the peptide motion. The results for the second peptide show that, in the fluid phase, the motionally averaged dipolar coupling is too small to be measured by rotational resonance. Rotational resonance, rotational echo double resonance, and related techniques can be used to obtain reliable and valuable dipolar couplings in static solid and membrane systems. The interpretation of these couplings in terms of internuclear distances is straightforward in the absence of molecular motion. These techniques hold considerable promise for membrane protein structural studies under conditions, such as at low temperatures, where molecular motion does not modulate the dipolar couplings. However, a typical membrane at physiological temperatures exhibits complex molecular motions. In the absence of an accurate and detailed description of both internal and whole body molecular motions, it is unlikely that techniques of this type, which are based on extracting distances from direct internuclear dipolar couplings, can be used to study molecular structure under these conditions. Furthermore, the reduction in the strengths of the dipolar couplings by these motions dramatically reduces the useful range of distances which can be measured.

Amino Acid Sequence↗

PubMed1999

The role of position a in determining the stability and oligomerization state of alpha-helical coiled coils: 20 amino acid stability coefficients in the hydrophobic core of proteins.

We describe here a systematic investigation into the role of position a in the hydrophobic core of a model coiled-coil protein in determining coiled-coil stability and oligomerization state. We employed a model coiled coil that allowed the formation of an extended three-stranded trimeric oligomerization state for some of the analogs; however, due to the presence of a Cys-Gly-Gly linker, unfolding occurred from the same two-stranded monomeric oligomerization state for all of the analogs. Denaturation from a two-stranded state allowed us to measure the relative contribution of 20 different amino acid side chains to coiled-coil stability from chemical denaturation profiles. In addition, the relative hydrophobicity of the substituted amino acid side chains was assessed by reversed-phase high-performance liquid chromatography and found to correlate very highly (R = 0.95) with coiled-coil stability. We also determined the effect of position a in specifying the oligomerization state using ultracentrifugation as well as high-performance size-exclusion chromatography. We found that nine of the analogs populated one oligomerization state exclusively at peptide concentrations of 50 microM under benign buffer conditions. The Leu-, Tyr-, Gln-, and His-substituted analogs were found to be exclusively three-stranded trimers, while the Asn-, Lys-, Orn-, Arg-, and Trp-substituted analogs formed exclusively two-stranded monomers. Modeling results for the Leu-substituted analog showed that a three-stranded oligomerization state is preferred due to increased side-chain burial, while a two-stranded oligomerization state was observed for the Trp analog due to unfavorable cavity formation in the three-stranded state.

Amino Acid Sequence↗

PubMed1998

Orientation, positional, additivity, and oligomerization-state effects of interhelical ion pairs in alpha-helical coiled-coils.

The role of interhelical g-e' ion pairs in the dimerization specificity and stability of alpha-helical coiled-coils is highly controversial. Synthetic 35-residue coiled-coils based on the heptad repeat QgVaGbAcLdQeK f were used to investigate the effect of orientation of interhelical ion pairs between lysine and glutamic acid residues on coiled-coil stability. Stability was estimated from urea denaturation at 20 degreesC, monitoring unfolding with circular-dichroism spectroscopy. Double mutant cycles were employed to estimate the net interaction energy, Delta DeltaGuint, for the two orientations of the ion pair; Ee-Kg and Ke-Eg. Delta DeltaGuint was found to be about 1.4-fold higher for the Ee-Kg orientation in a coiled-coil containing an N-terminal disulfide bridge. The Delta DeltaGuint value was similar whether obtained from the middle heptad or averaged over all five heptads of the coiled-coil, suggesting that ion pairs contribute additively to coiled-coil stability. The effect of uncompensated charges was also illustrated by single substitutions of Gln with either Lys or Glu, resulting in Lys-Gln or Glu-Gln g-e' pairs. These substitutions were found to be twice as destabilizing at position g as at position e, and Lys was about twice as destabilizing as Glu at both positions e and g. In the absence of an interhelical disulfide bridge, Glu and Lys substitutions in the middle heptad were equally destabilizing at positions e and g (Lys continued to be more destabilizing than Glu) and the Delta DeltaGuint value for Lys-Glu ion pairs was not orientation dependent. These and previous results suggest the non-covalently-linked synthetic coiled-coils behave as molten globules, whereas a disulfide-bridge may "lock in" the structural differences between positions of the heptad repeat. Interhelical Lys-Glu ion pairs in either orientation promoted the formation of trimeric coiled-coils (in the absence of a disulfide bridge) while Gln-Gln g-e' interactions led to dimer formation. The results support a role for g-e' ionic attractions in controlling coiled-coil specificity, stability and oligomerization state, possibly through effects on the side-chain packing at the subunit interface.

Amino Acid Sequence↗

PubMed1998

Use of a heterodimeric coiled-coil system for biosensor application and affinity purification.

The two-stranded alpha-helical coiled-coil is now recognized as one of nature's favorite ways of creating a dimerization motif. Based on the knowledge of protein folding studies and de novo design model systems, a novel heterodimeric coiled-coil protein was synthesized. The heterodimeric E/K coiled-coil was constructed with two distinct peptides (E and K) that will spontaneously associate into a full helical coiled-coil structure in solution. Equilibrium CD, NMR and real time biosensor kinetics experiments showed that the E/K coiled-coil is both structurally (deltaG(unfold)=11.3 kcal/mol) and kinetically (Kd approximately 1 nM) stable in solution at neutral pH. The engineered coiled-coil had been applied as a dimerization and capture domain for biosensor based applications and used in an expression/detection/affinity chromatography system. Specific test examples demonstrated the usefulness of the E/K heterodimeric system in these applications. The universality of coiled-coil as a dimerization motif in nature and our ability to design and synthesize these proteins suggest a wide variety of applications.

Amino Acid Sequence↗

PubMed1998

Insights into the mechanism of heterodimerization from the 1H-NMR solution structure of the c-Myc-Max heterodimeric leucine zipper.

The oncoprotein c-Myc (a member of the helix-loop-helix-leucine zipper (b-HLH-LZ) family of transcription factors) must heterodimerize with the b-HLH-LZ Max protein to bind DNA and activate transcription. It has been shown that the LZ domains of the c-Myc and Max proteins specifically form a heterodimeric LZ at 20 degreesC and neutral pH. This suggests that the LZ domains of the c-Myc and Max proteins are playing an important role in the heterodimerization of the corresponding gene products in vivo. Initially, to gain an insight into the energetics of heterodimerization, we studied the stability of N-terminal disulfide-linked versions of the c-Myc and Max homodimeric LZs and c-Myc-Max heterodimeric LZ by fitting the temperature-induced denaturation curves monitored by circular dichroism spectroscopy. The c-Myc LZ does not homodimerize (as previously reported) and the c-Myc-Max heterodimeric LZ is more stable than the Max homodimeric LZ at 20 degreesC and pH 7.0. In order to determine the critical interhelical interactions responsible for the molecular recognition between the c-Myc and Max LZs, the solution structure of the disulfide-linked c-Myc-Max heterodimeric LZ was solved by two-dimensional 1H-NMR techniques at 25 degreesC and pH 4.7. Both LZs are alpha-helical and the tertiary structure depicts the typical left-handed super-helical twist of a two-stranded parallel alpha-helical coiled-coil. A buried salt bridge involving a histidine on the Max LZ and two glutamate residues on the c-Myc LZ is observed at the interface of the heterodimeric LZ. A buried H-bond between an asparagine side-chain and a backbone carbonyl is also observed. Moreover, evidence for e-g interhelical salt bridges is reported. These specific interactions give insights into the preferential heterodimerization process of the two LZs. The low stabilities of the Max homodimeric LZ and the c-Myc-Max heterodimeric LZ as well as the specific interactions observed are discussed with regard to regulation of transcription in this family of transcription factors.

Amino Acid Sequence↗

PubMed1998

Hydrophilic interaction/cation-exchange chromatography for separation of amphipathic alpha-helical peptides.

Mixed-mode hydrophilic interaction/cation-exchange chromatography (HILIC/CEX) is a novel high-performance technique which has excellent potential for peptide separations. Separations by HILIC/CEX are carried out by subjecting peptides to linear increasing salt gradients in the presence of high levels of acetonitrile, which promotes hydrophilic interactions overlaid on ionic interactions with the cation-exchange matrix. In the present study, HILIC/CEX has been applied to the separation of synthetic amphipathic alpha-helical peptides, varying in amphipathicity and the nature of side-chain substitutions in the centre of the hydrophobic or hydrophilic face. Observation of the retention behaviour of these amphipathic alpha-helical peptide analogues during HILIC/CEX and reversed-phase chromatography (RPLC) enabled the establishment of general rules concerning the applicability of these complementary HPLC techniques to peptides displaying a secondary structural motif of common occurrence.

Acetonitriles↗

PubMed1998

The entericidin locus of Escherichia coli and its implications for programmed bacterial cell death.

Antidote/toxin gene pairs known as "addiction modules" can maintain plasmids in bacterial populations by means of post-segregational killing. However, several chromosome-encoded addiction modules may provide an entirely distinct function in the programmed cell death of moribund subpopulations under starvation conditions. We now report a novel chromosomal bacteriolytic module of Escherichia coli called the entericidin locus, which is activated in stationary phase under high osmolarity conditions by sigmaS and simultaneously repressed by the osmoregulatory EnvZ/OmpR signal transduction pathway. The entericidin locus encodes tandem paralogous genes (ecnAB) and directs the synthesis of two small cell-envelope lipoproteins. An attenuator precedes ecnA and an ompR-sensitive sigmaS promoter governs expression of ecnB. The entericidin A lipoprotein is an antidote to the bacteriolytic lipoprotein entericidin B. The entericidins are predicted to adopt amphipathic alpha-helical structures and to reciprocally modulate membrane stability. The entericidin locus is not present on any known plasmids, but is conserved in the homologous region of the Citrobacter freundii chromosome. Although the cloned C. freundii entericidin locus is expressed in E. coli independently of ompR, it carries an additional ompR-like gene called ecnR. The organization of the entericidin locus as a chromosomal antidote/toxin gene pair, which is regulated by both positive and negative osmotic signals during starvation, is consistent with an emerging paradigm of programmed bacterial cell death.

Amino Acid Sequence↗

PubMed1998

Binding of an oligopeptide to a specific plane of ice.

The alpha-helical antifreeze protein (AFP) from winter flounder inhibits ice growth by binding to a specific set of pyramidal surface planes that are not otherwise macroscopically expressed. The 37-residue AFP contains three 11-amino acid repeats that make a stereo-specific fit to the ice lattice along the <01-12> direction of the (20-21) and equivalent binding planes. When the AFP was shortened to delete two of the three 11-amino acid ice-binding repeats, the resulting 15-residue peptide and its variants were less helical and showed no antifreeze activity. However, when the helicity of the peptide was reinforced by an internal lactam bridge between Glu-7 and Lys-11, the minimized AFP was able to stably express the pyramidal plane (20-21) on the surface of growing ice crystals. This dynamic shaping of the ice surface by a single ice-binding repeat provides evidence that AFP adsorption to the ice lattice is not an "all-or-nothing" interaction. Instead, a partial interaction can help develop the binding site on ice to which the remainder of the AFP (or other AFP molecules) can orient and bind.

Antifreeze Proteins↗

PubMed1998

Liquid chromatographic-high-resolution mass spectrometric and tandem mass spectrometric identification of synthetic peptides using electrospray ionization.

Liquid chromatography-high-resolution electrospray mass spectrometry (LC-ESI-MS) was investigated for the identification of known and unknown synthetic peptides in a research effort designed to evaluate the applicability of this and complementary MS techniques for peptide characterization and identification. The monoisotopic molecular masses of five related peptides with molecular masses between 2000 and 2500 u were acquired with a resolution of 3000 (10% valley). Under narrow and wide mass range magnetic sector scanning conditions monoisotopic molecular mass errors were typically in the 10-20 and 30-40 ppm range, respectively. Tryptic maps were generated for each peptide following LC-ESI-MS analysis and collisionally activated dissociation (CAD) in the ESI interface resulted in the production of characteristic product ions that enabled amino acid sequencing of the tryptic fragments. Unknown identification was demonstrated during analysis of an incomplete synthetic peptide reaction mixture. The synthesis of an 18 amino acid peptide, LTTAVKKVLTTGLPALIS, was not successful. In its place were six unknown peptides that were identified on the basis of monoisotopic molecular mass and amino acid sequence data. The monoisotopic molecular masses of these unknowns were determined to within 10-20 ppm with a resolution of 3500 (10% valley). Amino acid sequences for the six peptides were generated during ESI-MS-MS analysis. Finally two synthetic peptides differing only by the incorporation of a 13C at leucine were analysed with a resolution of 6000 (10% valley) to confirm that the isotopic distributions were consistent with theoretical expectations.

Amino Acid Sequence↗

PubMed1998

Molecular organization and dynamics of 1-palmitoyl-2-oleoylphosphatidylcholine bilayers containing a transmembrane alpha-helical peptide.

The molecular organization and dynamics have been investigated in membranes consisting of 1-palmitoyl-2-oleoyl-l-alpha-phosphatidylcholine (POPC) and various ratios of a transmembrane alpha-helical peptide, Ac-K2L24K2-amide (L24), in order to gain insights into how the transmembrane portions of membrane proteins are mixed with phospholipids and organized in biological membranes. Particular attention was paid to membranes with high peptide concentrations. The molecular organization and dynamics were studied in the ps-to-micros regime using various spin-labeling techniques. Conventional ESR spectra as well as saturation-recovery curves measured in both the presence and the absence of molecular oxygen showed that phosphatidylcholine spin-labels detect the existence of a single homogeneous environment, indicating that both L24 and POPC are likely to be undergoing fast translational diffusion in L24-POPC membranes of up to 9 mol % peptide. Since 16-18 molecules of phosphatidylcholine are required to surround a transmembrane alpha-helical peptide [Morrow, M. R., Huschilt, J. C., and Davis, J. H. (1985) Biochemistry 24, 5396-5406], L24 must form L24-rich regions at a P/L ratio of 1/10 instantaneously. However, these results suggest that the lipid exchange rates among the bulk, boundary, and L24-rich regions are fast, and that the L24-rich regions must be forming and dispersing rapidly in a time scale shorter than 0.1 micros, the conventional ESR spin-label time scale and the electron spin-lattice relaxation time scale in the presence of molecular oxygen. Although this does not exclude the possibility of the formation of small, stable oligomers of L24, it is unlikely because L24 lacks features that would favor their formation. L24 (9 mol %) increases the hydrophobicity of the central part of the POPC membrane from the level of 1-decanol to that of pure hexane and also increases the hydrophobicity near the membrane surface from the level of 2-propanol to that of 1-decanol. The effect of 9 mol % L24 on the order parameter profile is similar to that of decreasing the temperature by approximately 8 degrees C between 10 and 55 degrees C. It is concluded that L24 is highly miscible in POPC membranes even at high concentrations in the membrane.

Binding Sites↗

PubMed1998

Breakdown and release of myofilament proteins during ischemia and ischemia/reperfusion in rat hearts: identification of degradation products and effects on the pCa-force relation.

Our objective in experiments reported here was to identify myofilament proteins of rat hearts either lost or degraded by cardiac ischemia (15- or 60-minute duration) with and without 45 minutes of reperfusion. We correlated these changes with alterations in myofilament sensitivity to Ca2+ and maximum force generation. Protein degradation and loss were assessed by high-performance liquid chromatography, SDS-PAGE, Western blotting analysis, and amino acid sequencing. Compared with nonischemic control hearts, bundles of skinned fibers from hearts subjected to ischemia alone demonstrated a decrease in maximum force generation and an increase in sensitivity to Ca2+. These changes in function were increased with the duration of the ischemia and with reperfusion. With increasing duration of ischemia, there was an increased loss and degradation of myofibrillar alpha-actinin and troponin I (TnI) at its C-terminus. Alpha-actinin and TnI were most susceptible to ischemia, but with 60 minutes of ischemia/reperfusion, there was also degradation of myosin light chain-1 (MLC1) involving a clip of residues 1 to 19. The MLC1 degradation product was detected in the reperfusion effluent (along with troponin T, tropomyosin, and alpha-actinin) but not in the tissue with 60 minutes of ischemia with no reperfusion. Moreover, with ischemia the following proteins became associated with the myofibrils: GAPDH and proteins of the mitochondrial ATP synthase complex. Our results provide new evidence regarding the mechanism by which ischemia/reperfusion causes myocardial injury and support the hypothesis that an important element in the injury is altered activity and structure of the myofilaments.

Actin Cytoskeleton↗

PubMed1998

Structural cassette mutagenesis in a de novo designed protein: proof of a novel concept for examining protein folding and stability.

The solution to the protein folding problem lies in defining the relative energetic contributions of short-range and long-range interactions. In other words, the tendency of a stretch of amino acids to adopt a final secondary structural fold is context dependent. Our approach to this problem is to address whether an amino acid sequence, a "cassette," with a defined secondary structure in the three-dimensional structure of a native protein, can adopt a different conformation when placed into a different protein environment. Thus, we designed de novo a disulfide-bridged two-stranded alpha-helical parallel coiled coil, where each polypeptide chain consisted of 39 residues, as a "cassette holder." The 11-residue cassette would be inserted into the center of each polypeptide chain between the two nucleating alpha-helices to replace the control sequence. This Structural Cassette Mutagenesis model permits the analysis of short-range interactions within the inserted cassette as well as long-range interactions between the nucleating helices and the cassette region. The cassette holder, with a control sequence as the cassette, had a GdnHCl transition midpoint during denaturation of 5.6M. To demonstrate the feasibility of our model, an 11-residue beta-strand cassette from an immunoglobulin fold was inserted. The cassette was fully induced into the alpha-helical conformation with a [GdnHCl]1/2 value of 3.2M. To demonstrate the importance of short-range interactions (beta-sheet/alpha-helical propensities of amino acid side chains) in modulating structure and stability, a series of 1-5 threonine residues (highest beta-sheet propensity) were substituted into the solvent-exposed portions of the cassette in the alpha-helical conformation. Each successive substitution systematically decreased the stability of the coiled coil with peptide T4b (4 Thr residues) having a [GdnHCl]1/2 value of 2.2M. The single substitution of Ile in the hydrophobic core of the cassette with Ala or Thr had the most dramatic effect on protein stability (peptide 120T, [GdnHCl]1/2 value of 1.4M). Though these substitutions were able to modulate stability, they were not able to disrupt the alpha-helical conformation of the cassette, showing the importance of the nucleating alpha-helices on either side of the cassette in controlling conformation of the cassette. We have demonstrated the feasibility of our model protein to accept a beta-strand cassette. The effect of cassettes containing other beta-strands, beta-turns, loops, regions of undefined structure, and helical segments on conformation and stability of our model protein will also be determined.

Amino Acid Sequence↗

PubMed1998

Unusual beta-sheet periodicity in small cyclic peptides.

Cyclic peptide homologs of gramicidin S containing 6, 8, 10, 12, 14 and 16 residues were synthesized and characterized using circular dichroism (CD) and 1H NMR spectroscopy. Based on the three-dimensional structures generated from these data we have found strong evidence of a periodic sequence-length dependence on beta-sheet content. In particular, peptides of length 6, 10 and 14 residues exhibit a high beta-sheet content, while peptides of 8, 12 and 16 residues appear to exist as random coils. This unusual beta-sheet periodicity may have important implications in our understanding of beta-sheet formation and in the design of constrained beta-sheet and beta-hairpin mimics.

Amino Acid Sequence↗