Studies on analogs of a peptide derived from alpha-fetoprotein having antigrowth properties.

A 34-amino acid portion of the third domain of alpha-fetoprotein possesses antigrowth and anticancer activities. Three analogs of this sequence were chemically synthesized, in which the two cysteines of the original sequence were replaced by alanines, glycines or serines. The original cysteine and alanine peptides formed trimers at 0.20 g/L in pH 7.4 phosphate buffer, and the glycine and serine peptides formed dimers. Trimer preparations were more potent in inhibiting estrogen-induced growth in the mouse uterine assays than the two dimeric oligomers. Of salient importance is that the alanine peptide retained its trimeric form in solution much longer than the cysteine peptide. Antigrowth assays were performed starting with stock solutions at a peptide concentration of 0.20 g/L, because at very high peptide concentration (8.0 g/L) the peptides aggregated extensively. All the peptides, although differing in biological activity, had almost identical secondary structures. Unlike alpha-fetoprotein, the three peptides have low amounts of alpha-helix. Trifluoroethanol has the ability to convert peptides into a helical conformation when they have a propensity for that structure. At trifluoroethanol concentrations of 20% and higher, the alanine and glycine peptides were changed into highly helical structures.     

Characterization and isolation of intermediates in beta-lactoglobulin heat aggregation at high pH

The early stages of heat induced aggregation at 67.5 degrees C of beta-lactoglobulin were studied by combined static light scattering and size exclusion chromatography. At all conditions studied (pH 8.7 without salt and pH 6.7 with or without 60 mM NaCl) we observe metastable heat-modified dimers, trimers, and tetramers. These oligomers reach a maximum in concentration at about the time when large aggregates (1000-4000 kg/mol) appear, after which they decline in concentration. By isolating the oligomers it was demonstrated that they rapidly form aggregates upon heating in the absence of monomeric protein, showing that these species are central to the aggregation process. To our knowledge this is the first time that intermediates in protein aggregation have been isolated. At all stages of aggregation the dominant oligomer was the heat-modified dimer. Whereas the heat-modified oligomers are formed at a higher rate at pH 8.7 than at pH 6.7, the opposite is the case for the formation of aggregates from the metastable oligomers indicating cross-linking via disulfide bridges for the oligomers and noncovalent interaction in the formation of the aggregates. The data suggest that an aggregate nucleus is formed from four oligomers. For protein concentrations of 10 or 20 g/l a heat-modified monomer can be observed until about the time when the maximum in concentration appears of the heat-modified dimer. The disappearance of this heat-modified monomer correlates to the formation of dimers (trimers and tetramers).     

Effect of structural parameters of peptides on dimer formation and highly oxidized side products in the oxidation of thiols of linear analogues of human β‐defensin 3 by DMSO

The purpose of this study was to examine the effects of structural parameters of peptides on their oxidation by DMSO, including location of cysteine, effect of adjunct group participation, molecular hydrophobicity, steric hindrance or the accessibility of thiol group and peptide conformation, on oxidation rates, dimer formation and associated side products. We designed and synthesized two series of linear cysteine‐containing analogues of human β‐defensin 3 (the C1‐peptides with cysteine at the N‐terminus residue 1, the C29‐peptides with cysteine located at residue 29 in the centre of peptide), which were used for preparation of disulphide‐linked homodimers. HPLC–ESI–MS was used to monitor the oxidation process and to characterize the molecular weights of dimers and side products of high oxidation. The formations of dimers and side products were dependent on the position of cysteines. Hydrophobicity generally rendered the thiol groups less accessible and hence exposed them to slow oxidation to form dimers (or even fail to form dimers during the timescale of observation). Molecular dynamics simulations showed that the exposure of cysteines (and sulphurs) of the C1‐peptides was much larger than for the C29‐peptides. The larger hydrophobic side chains tended to enable clustering of the side chains that sequester cysteine, particularly in the C29‐peptides, which provided a molecular explanation for the observed trends in oxidation rates. Together with molecular modelling, we propose a reaction mechanism to elucidate the oxidation results of these peptides. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

The assembly of amyloidogenic yeast sup35 as assessed by scanning (atomic) force microscopy: an analogy to linear colloidal aggregation?

Amyloidosis is a class of diseases caused by protein aggregation and deposition in various tissues and organs. In this paper, a yeast amyloid-forming protein Sup35 was used as a model for understanding amyloid fiber formation. The dynamics of amyloid formation by Sup35 were studied with scanning force microscopy. We found that: 1) the assembly of Sup35 fibers begins with individual NM peptides that aggregate to form large beads or nucleation units which, in turn, form dimers, trimers, tetramers and longer linear assemblies appearing as a string of beads; 2) the morphology of the linear assemblies differ; and 3) fiber assembly suggests an analogy to the aggregation of colloidal particles. A dipole assembly model is proposed based on this analogy that will allow further experimental testing.     

Study of the aggregation mechanism of polyglutamine peptides using replica exchange molecular dynamics simulations

Polyglutamine (polyQ, a peptide) with an abnormal repeat length is the causative agent of polyQ diseases, such as Huntington’s disease. Although glutamine is a polar residue, polyQ peptides form insoluble aggregates in water, and the mechanism for this aggregation is still unclear. To elucidate the detailed mechanism for the nucleation and aggregation of polyQ peptides, replica exchange molecular dynamics simulations were performed for monomers and dimers of polyQ peptides with several chain lengths. Furthermore, to determine how the aggregation mechanism of polyQ differs from those of other peptides, we compared the results for polyQ with those of polyasparagine and polyleucine. The energy barrier between the monomeric and dimeric states of polyQ was found to be relatively low, and it was observed that polyQ dimers strongly favor the formation of antiparallel β-sheet structures. We also found a characteristic behavior of the monomeric polyQ peptide: a turn at the eighth residue is always present, even when the chain length is varied. We previously showed that a structure including more than two sets of β-turns is stable, so a long monomeric polyQ chain can act as an aggregation nucleus by forming several pairs of antiparallel β-sheet structures within a single chain. Since the aggregation of polyQ peptides has some features in common with an amyloid fibril, our results shed light on the mechanism for the aggregation of polyQ peptides as well as the mechanism for the formation of general amyloid fibrils, which cause the onset of amyloid diseases.     

Aggregation kinetics of interrupted polyglutamine peptides

Abnormally expanded polyglutamine domains are associated with at least nine neurodegenerative diseases, including Huntington's disease. Expansion of the glutamine region facilitates aggregation of the impacted protein, and aggregation has been linked to neurotoxicity. Studies of synthetic peptides have contributed substantially to our understanding of the mechanism of aggregation because the underlying biophysics of polyglutamine-mediated association can be probed independent of their context within a larger protein. In this report, interrupting residues were inserted into polyglutamine peptides (Q20), and the impact on conformational and aggregation properties was examined. A peptide with two alanine residues formed laterally aligned fibrillar aggregates that were similar to the uninterrupted Q20 peptide. Insertion of two proline residues resulted in soluble, nonfibrillar aggregates, which did not mature into insoluble aggregates. In contrast, insertion of a β-turn template _DPG rapidly accelerated aggregation and resulted in a fibrillar aggregate morphology with little lateral alignment between fibrils. These results are interpreted to indicate that (a) long-range nonspecific interactions lead to the formation of soluble oligomers, while maturation of oligomers into fibrils requires conformational conversion and (b) that soluble oligomers dynamically interact with each other, while insoluble aggregates are relatively inert. Kinetic analysis revealed that the increase in aggregation caused by the _DPG insert is inconsistent with the nucleation-elongation mechanism of aggregation featuring a monomeric β-sheet nucleus. Rather, the data support a mechanism of polyglutamine aggregation by which monomers associate into soluble oligomers, which then undergo slow structural rearrangement to form sedimentable aggregates.     

Lysozyme inactivation and aggregation in stirred-reactor

Mechanisms of enzyme inactivation and aggregation are still poorly understood. In this work, we are considering the characterisation of both inactivation and aggregation in stirred tank reactor, with lysozyme as the model enzyme.

The inactivation kinetics are first order. For stirring speeds in the range of 0–700 rpm, the kinetic constant is found to be proportional to the power brought by the impeller. It suggests that inactivation depends on collisions between enzyme molecules. Efficient collisions between native and inactive molecules induce native molecules to turn into inactive molecules and lead to lysozyme aggregation.

During inactivation, enzymes are found to aggregate as shown by light scattering measurements. The structure of aggregates was studied on samples treated for chemical denaturation and reduction. The aggregates are supramolecular edifices, mainly made up of inactivated enzymes linked by weak forces. But aggregates are also made up of dimers and trimers of lysozyme, linked by disulfide bridges. Dimers and trimers are 18% and 5%, respectively, of the total amount of lysozyme aggregates.

Whatever the stage of aggregate formation and the initial enzyme concentration are, these aggregates are irreversibly inactivated. Enzyme activity is definitely lost even if stirring is stopped and/or temperature decreased.

This study points out the importance of hydrodynamics in bioreactors and highlights the nature of the aggregates resulting from the interactions between native and inactive enzymes.     

Molecular resolution visualization of a pore formed by trichogin,an antimicrobial peptide,in a phospholipid matrix

Electrochemical scanning tunneling microscopy (EC-STM) was employed to study the aggregation of trichogin OMe (TCG), an antimicrobial peptide, incorporated into a lipid monolayer. High-resolution EC-STM images show that trichogin molecules aggregate to form channels in the lipid monolayer. Two types of aggregates were observed in the images. The first consisted of a bundle of six TCG molecules surrounding a central pore. The structure and dimensions of this channel are similar to aggregates that in bilayers are described by the barrel-stave model. The EC-STM images also reveal that channels aggregate further to form a hexagonal lattice of a two dimensional (2D) nanocrystal. The model of 2D lattice was built from trimers of TCG molecules that alternatingly are oriented with either hydrophilic or hydrophobic faces to each other. In this way each TCG molecule is oriented partially with its hydrophilic face towards the hexameric pore allowing the formation of the column of water inside this pore.     

Mapping the conformational dynamics and pathways of spontaneous steric zipper Peptide oligomerization

The process of protein misfolding and self-assembly into various, polymorphic aggregates is associated with a number of important neurodegenerative diseases. Only recently, crystal structures of several short peptides have provided detailed structural insights into -sheet rich aggregates, known as amyloid fibrils. Knowledge about early events of the formation and interconversion of small oligomeric states, an inevitable step in the cascade of peptide self-assembly, however, remains still limited. We employ molecular dynamics simulations in explicit solvent to study the spontaneous aggregation process of steric zipper peptide segments from the tau protein and insulin in atomistic detail. Starting from separated chains with random conformations, we find a rapid formation of structurally heterogeneous, -sheet rich oligomers, emerging from multiple bimolecular association steps and diverse assembly pathways. Furthermore, our study provides evidence that aggregate intermediates as small as dimers can be kinetically trapped and thus affect the structural evolution of larger oligomers. Alternative aggregate structures are found for both peptide sequences in the different independent simulations, some of which feature characteristics of the known steric zipper conformation (e.g., -sheet bilayers with a dry interface). The final aggregates interconvert with topologically distinct oligomeric states exclusively via internal rearrangements. The peptide oligomerization was analyzed through the perspective of a minimal oligomer, i.e., the dimer. Thereby all observed multimeric aggregates can be consistently mapped onto a space of reduced dimensionality. This novel method of conformational mapping reveals heterogeneous association and reorganization dynamics that are governed by the characteristics of peptide sequence and oligomer size.     

Genetic analysis of the leucine heptad repeats of Lac repressor: evidence for a 4-helical bundle.

Gel-filtration experiments indicate that a peptide (P2) composed of the basic region of GCN4 fused to the leucine heptad repeats of Lac repressor forms tetrameric aggregates. Gel-shift experiments were performed to determine the orientation of the helices in the tetrameric P2 aggregate. Sandwich-complex formation of peptide P2 with two DNA fragments containing two symmetrical CRE binding sites (5'-ATGACGTCAT-3') at a distance of 21 bp suggests antiparallel aggregation of the Lac leucine heptad repeats. Thus, we conclude that the leucine heptad repeats of Lac repressor have the ability to form homomeric 4-helical bundles with an antiparallel arrangement of the helices. This topology enables the two DNA fragments in the sandwich complexes to be held together by two tetramers of peptide P2. Replacement of the uncharged amino acids of the helical g and e positions of peptide P2 by the corresponding charged residues of GCN4 (peptide P4) results in a dimeric and parallel aggregation of the leucine heptad repeats, and consequently abolishes the potential to form sandwich structures. Similarly, a hybrid Lac repressor in which the GCN4 leucine zipper replaces the natural Lac leucine heptad repeats forms dimers only. It regains the ability to form tetramers when the charged amino acids in helical positions g and e are replaced by uncharged alanines.     

A potent new class of reductively activated peptide gene delivery agents

A new class of peptide gene delivery agents were developed by inserting multiple cysteine residues into short (dp 20) synthetic peptides. Substitution of one to four cysteine residues for lysine residues in Cys-Trp-Lys(18) resulted in low molecular weight DNA condensing peptides that spontaneously oxidize after binding to plasmid DNA to form interpeptide disulfide bonds. The stability of cross-linked peptide DNA condensates increased in proportion to the number of cysteines incorporated into the peptide. Disulfide bond formation led to a decrease in particle size relative to control peptide DNA condensates and prevented dissociation of peptide DNA condensates in concentrated sodium chloride. Cross-linked peptide DNA condensates were 5-60-fold more potent at mediating gene expression in HepG2 and COS 7 cells relative to uncross-linked peptide DNA condensates. The enhanced gene expression was dependent on the number of cysteine residues incorporated, with a peptide containing two cysteines mediating maximal gene expression. Cross-linking peptides caused elevated gene expression without increasing DNA uptake by cells, suggesting a mechanism involving intracellular release of DNA triggered by disulfide bond reduction. The results establish cross-linking peptides as a novel class of potent gene delivery agents that enhance gene expression through a new mechanism of action.     

A limited role for disulfide cross-linking in the aggregation of mutant SOD1 linked to familial amyotrophic lateral sclerosis

One of the mechanisms by which mutations in superoxide dismutase 1 (SOD1) cause familial amyotrophic lateral sclerosis (fALS) is proposed to involve the accumulation of detergent-insoluble, disulfide-cross-linked, mutant protein. Recent studies have implicated cysteine residues at positions 6 and 111 as critical in mediating disulfide cross-linking and promoting aggregation. In the present study, we used a panel of experimental and disease-linked mutations at cysteine residues of SOD1 (positions 6, 57, 111, and 146) in cell culture assays for aggregation to demonstrate that extensive disulfide cross-linking is not required for the formation of mutant SOD1 aggregates. Experimental mutants possessing only a single cysteine residue or lacking cysteine entirely were found to retain high potential to aggregate. Furthermore we demonstrate that aggregate structures in symptomatic SOD1-G93A mice can be dissociated such that they no longer sediment upon ultracentrifugation (i.e. appear soluble) under relatively mild conditions that leave disulfide bonds intact. Similar to other recent work, we found that cysteines 6 and 111, particularly the latter, play interesting roles in modulating the aggregation of human SOD1. However, we did not find that extensive disulfide cross-linking via these residues, or any other cysteine, is critical to aggregate structure. Instead we suggest that these residues participate in other features of the protein that, in some manner, modulate aggregation.     

Both the environment and somatic mutations govern the aggregation pathway of pathogenic immunoglobulin light chain.

Deposition of monoclonal immunoglobulin light chain (LC) aggregates in tissues is the hallmark of a class of fatal diseases with no effective treatment. In the most prevalent diseases two different types of LC aggregates are observed: fibrillar deposits in LC amyloidosis (AL) and granular aggregates in LC deposition disease (LCDD). The mechanisms by which a given LC forms either type of aggregate are not understood. Although some LCs are more aggregation-prone than others, this does not appear to be due to specific sequence determinants, but more likely from global properties that can be introduced by multiple somatic mutations. Moreover, a single LC isotype can sometimes form both fibrillar and granular aggregates within the same patient. To better understand how the different aggregation pathways arise, we developed a series of in vitro assays to analyze the formation of distinct aggregate types. The recombinant kappa IV LC (SMA) assembles into fibrils when agitated. We now show that SMA can also form granular aggregates upon exposure to copper, and that this aggregation can occur not only in vitro, but also in cells. A constellation of somatic mutations, consisting of His89/His94/Gln96, is sufficient to confer sensitivity to copper on wild-type kappa IV proteins. The formation of both types of aggregates is inhibited by synthetic peptides derived from the LC variable domain. However, the peptide that inhibits fibrillar aggregation is different from the peptide that inhibits copper-induced aggregation. Thus, distinct molecular surfaces of the LC underly each type of aggregate. We conclude that both the intrinsic properties of the sequence and extrinsic conditions govern the aggregation pathway of a LC.     

Thermodynamic analysis of the aggregation propensity of oxidized Alzheimer's beta-amyloid variants

We have determined the critical concentrations of a set of 18 variants of Alzheimer's Abeta(1-40) peptide, each carrying a different residue at position 18. We find that the critical concentrations depend on the hydrophobicity and beta-sheet propensity of residue 18, and therefore on properties that we identified previously to affect also the kinetics by which these peptides aggregate. Since the critical concentrations can be related to the Gibbs free energy of aggregation (DeltaG), these data imply a link between the thermodynamics and the kinetics of aggregation in that sequences that form very stable aggregates are also those that form such aggregates very rapidly.     

Structural properties of trimers and tetramers of ribonuclease A

Ribonuclease A aggregates (dimers, trimers, tetramers, pentamers) can be obtained by lyophilization from 40% acetic acid solutions. Each aggregate forms two conformational isomers distinguishable by different basic net charge. The crystal structure of the two dimers has recently been determined; the structure of the higher oligomers is unknown. The results of the study of the two trimeric and tetrameric conformers can be summarized as follows: (1) RNase A trimers and tetramers form by a 3D domain-swapping mechanism. N-terminal and C-terminal types of domain swapping could coexist; (2) the secondary structures of the trimeric and tetrameric conformers do not show significant differences if compared with the secondary structure of monomeric RNase A or its two dimers; (3) a different exposure of tyrosine residues indicates that in the aggregates they have different microenvironments; (4) the two trimeric and tetrameric conformers show different susceptibility to digestion by subtilisin; (5) dimers, trimers, and tetramers of RNase A show unwinding activity on double-helical poly(dA-dT) x poly(dA-dT), that increases as a function of the size of the oligomers; (6) the less basic conformers are more stable than the more basic ones, and a low concentration in solution of trimers and tetramers favors their stability, which is definitely increased by the interaction of the aggregates with poly(dA-dT) x poly(dA-dT); (7) the products of thermal dissociation of the two trimers indicate that their structures could be remarkably different. The dissociation products of the two tetramers allow the proposal of two models for their putative structures.     

Self-assembling and membrane modifying properties of a lipopeptaibol studied by CW-ESR and PELDOR spectroscopies.

Trichogin GA IV is a short lipopeptaibol antibiotic that is capable of enhancing the transport of small cations through the phospholipid double layer of the membrane. The antibiotic activity of the undecapeptide is thought to be based on either its self-assembling or membrane-modifying property. The chemical equilibrium between self-aggregated and non-aggregated molecular states was studied by CW-ESR spectroscopy using solutions of TOAC nitroxide spin-labelled trichogin analogues in an apolar solvent to mimic the membrane bound state. At room temperature the two different sets of signals observed in the spectrum were attributed to the presence of both monomers and aggregates in the sample. The ESR spectra of the monomeric and aggregated forms were separated and the dependence of the fraction of monomeric peptide molecules on concentration was obtained over the range 5 x 10(-6) to 7 x 10(-4) M. A two-step aggregation mechanism is proposed: dimerization of peptide molecules followed by aggregation of dimers to assemblies of four peptide molecules per aggregate. The equilibrium constants were estimated for both steps. In addition, the lower lifetime limit was determined for dimers and tetramers. It is shown that when the peptide concentration exceeds 10(-5) M. the major part of the peptide molecules in solution has the form of tetrameric aggregates. Independently, the PELDOR technique was used to investigate the concentration dependence of the parameters of the dipole-dipole interaction between spin labels in frozen (77 K] glassy solutions of aggregates of mono-labelled TOAC analogues. The number of molecules in aggregates as well as the frequency and amplitude of PELDOR signal oscillations were found to be concentration independent in the range 5 x 10(-4) to 8 x 10(-3) M. In the frozen glassy solution state, the number of peptide molecules per aggregate was determined to be close to four, which is in agreement with the value obtained for spin-labelled trichogin at room temperature. The present data provide experimental evidence in favour of a self-assembling rather than a membrane-modifying ion conduction mechanism.     

Surface aggregation and membrane penetration by peptides: relation to pore formation and fusion

The peptide GALA undergoes a conformational change to an amphipathic alpha -helix when the pH is reduced, inducing leakage of contents from vesicles. Leakage from neutral or negativelycharged vesicles at pH 5.0 was similar and could be adequately explained by a mathematical model which assumed that GALA becomes incorporated into the vesicle bilayer and irreversibly aggregates to form a pore consisting of M =10+/-2 peptides. Increasing cholesterol content in the membranes resulted in reduced leakage, and increased reversibility of surface aggregation of the peptide. Employing fluorescently labelled peptides confirmed that the degree of reversibility of surface aggregation of GALA was significantly larger in cholesterol containing liposomes. Orientation of the peptide GALA in bilayers was determined by a bodipy-avidin/ biotin binding assay. The peptide was labelled by biotin at the N- or Cterminus and bodipy-avidin molecules were added externally or were preencapsulated in the vesicles. The peptides are arranged in the pore perpendicularly to the membrane, such that 3/4 of the N-termini are on the internal side of the membrane. The pores are stable and persist for at least 10 min. When the peptides form an aggregate of size smaller than M, the orientation of the peptide is mostly parallel to the surface and the biotinylated peptide does not translocate. When a critical size of the aggregate is attained, a rearrangement of the peptide occurs, which amounts to rapid penetration and formation of a pore structure. Induction of fusion by peptides may be antagonistic to pore formation, the outcome being dependent on vesicle aggregation.     

A recipe for designing water-soluble, beta-sheet-forming peptides.

Based on observations of solubility and folding properties of peptide 33-mers derived from the beta-sheet domains of platelet factor-4 (PF4), interleukin-8 (IL-8), and growth related protein (Gro-alpha), as well as other beta-sheet-forming peptides, general guidelines have been developed to aid in the design of water soluble, self-association-induced beta-sheet-forming peptides. CD, 1H-NMR, and pulsed field gradient NMR self-diffusion measurements have been used to assess the degree of folding and state of aggregation. PF4 peptide forms native-like beta-sheet tetramers and is sparingly soluble above pH 6. IL-8 peptide is insoluble between pH 4.5 and pH 7.5, yet forms stable, native-like beta-sheet dimers at higher pH. Gro-alpha peptide is soluble at all pH values, yet displays no discernable beta-sheet structure even when diffusion data indicate dimer-tetramer aggregation. A recipe used in the de novo design of water-soluble beta-sheet-forming peptides calls for the peptide to contain 40-50% hydrophobic residues, usually aliphatic ones (I, L, V, A, M) (appropriately paired and mostly but not always alternating with polar residues in the sheet sequence), a positively charged (K, R) to negatively charged (E, D) residue ratio between 4/2 and 6/2, and a noncharged polar residue (N, Q, T, S) composition of about 20% or less. Results on four de novo designed, 33-residue peptides are presented supporting this approach. Under near physiologic conditions, all four peptides are soluble, form beta-sheet structures to varying degrees, and self-associate. One peptide folds as a stable, compact beta-sheet tetramer, whereas the others are transient beta-sheet-containing aggregates.     

Development of a synthetic cyclized peptide derived from alpha-fetoprotein that prevents the growth of human breast cancer.

The peptide, EMTPVNPG, derived from alpha-fetoprotein, inhibits estrogen-stimulated growth of immature mouse uterus and estrogen-dependent proliferation of human breast cancer cells. However, the biological activities of the peptide diminish over time in storage, even when in the lyophilized state, probably because of peptide aggregation through hydrophobic interaction among monomers. Two analogs of EMTPVNPG were designed with the intent of minimizing aggregation and retaining biological activity during prolonged storage. EMTOVNOG, where O is 4-hydroxyproline, is a linear peptide generated by substituting 4-hydroxyproline for the two prolines, thereby increasing peptide hydrophilicity. This analog exhibited a dose-dependent inhibition of estrogen-stimulated growth of immature mouse uterus similar to that of EMTPVNPG (maximal activity at 1 microg/mouse). A second analog, cyclo-(EMTOVNOGQ), a hydrophilic, cyclic analog with increased conformational constraint, was as potent as the other peptides in its inhibition of estrogen-dependent growth of immature mouse uterus, and had an expanded effective dose range. Both linear and cyclized hydroxyproline-substituted analogs exhibited indefinite shelf-life. Furthermore, both analogs inhibited the estrogen-dependent growth of MCF-7 human breast cancer growing as a xenograft in SCID mice. These analogs may become significant, novel agents for the treatment of breast cancer.     

Synergistic interactions between aqueous and membrane domains of a designed protein determine its fold and stability

Membrane-spanning proteins contain both aqueous and membrane-spanning regions, both of which contribute to folding and stability. To explore the interplay between these two domains we have designed and studied the assembly of coiled-coil peptides that span from the membrane into the aqueous phase. The membrane-spanning segment is based on MS1, a transmembrane coiled coil that contains a single Asn at a buried a position of a central heptad in its sequence. This Asn has been shown to drive assembly of the monomeric peptide in a membrane environment to a mixture of dimers and trimers. The coiled coil has now been extended into the aqueous phase by addition of water-soluble helical extensions. Although too short to fold in isolation, these helical extensions were expected to interact synergistically with the transmembrane domain and modulate its stability as well as its conformational specificity for forming dimers versus trimers. One design contains Asn at a position of the aqueous helical extension, which was expected to specify a dimeric state; a second peptide, which contains Val at this position, was expected to form trimers. The thermodynamics of assembly of the hybrid peptides were studied in micelles by sedimentation equilibrium ultracentrifugation. The aqueous helical extensions indeed conferred additional stability and conformational specificity to MS1 in the expected manner. These studies highlight the delicate interplay between membrane-spanning and water-soluble regions of proteins, and demonstrate how these different environments define the thermodynamics of a given specific interaction. In this case, an Asn in the transmembrane domain provided a strong driving force for folding but failed to specify a unique oligomerization state, while an Asn in the water-soluble domain was able to define specificity for a specific aggregation state as well as modulate stability.