Self-association Mechanism Research Articles

The intrinsically disordered N-terminal domain of galectin-3 dynamically mediates multisite self-association of the protein through fuzzy interactions

Galectins are a family of lectins that bind β-galactosides through their conserved carbohydrate recognition domain (CRD) and can induce aggregation with glycoproteins or glycolipids on the cell surface and thereby regulate cell activation, migration, adhesion, and signaling. Galectin-3 has an intrinsically disordered N-terminal domain and a canonical CRD. Unlike the other 14 known galectins in mammalian cells, which have dimeric or tandem-repeated CRDs enabling multivalency for various functions, galectin-3 is monomeric, and its functional multivalency therefore is somewhat of a mystery. Here, we used NMR spectroscopy, mutagenesis, small-angle X-ray scattering, and computational modeling to study the self-association-related multivalency of galectin-3 at the residue-specific level. We show that the disordered N-terminal domain (residues ∼20-100) interacts with itself and with a part of the CRD not involved in carbohydrate recognition (β-strands 7-9; residues ∼200-220), forming a fuzzy complex via inter- and intramolecular interactions, mainly through hydrophobicity. These fuzzy interactions are characteristic of intrinsically disordered proteins to achieve liquid-liquid phase separation, and we demonstrated that galectin-3 can also undergo liquid-liquid phase separation. We propose that galectin-3 may achieve multivalency through this multisite self-association mechanism facilitated by fuzzy interactions.

Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity.

TRIM E3 ubiquitin ligases regulate a wide variety of cellular processes and are particularly important during innate immune signalling events. They are characterized by a conserved tripartite motif in their N‐terminal portion which comprises a canonical RING domain, one or two B‐box domains and a coiled‐coil region that mediates ligase dimerization. Self‐association via the coiled‐coil has been suggested to be crucial for catalytic activity of TRIMs; however, the precise molecular mechanism underlying this observation remains elusive. Here, we provide a detailed characterization of the TRIM ligases TRIM25 and TRIM32 and show how their oligomeric state is linked to catalytic activity. The crystal structure of a complex between the TRIM25 RING domain and an ubiquitin‐loaded E2 identifies the structural and mechanistic features that promote a closed E2~Ub conformation to activate the thioester for ubiquitin transfer allowing us to propose a model for the regulation of activity in the full‐length protein. Our data reveal an unexpected diversity in the self‐association mechanism of TRIMs that might be crucial for their biological function.

Hierarchical looping of zigzag nucleosome chains in metaphase chromosomes

The architecture of higher-order chromatin in eukaryotic cell nuclei is largely unknown. Here, we use electron microscopy-assisted nucleosome interaction capture (EMANIC) cross-linking experiments in combination with mesoscale chromatin modeling of 96-nucleosome arrays to investigate the internal organization of condensed chromatin in interphase cell nuclei and metaphase chromosomes at nucleosomal resolution. The combined data suggest a novel hierarchical looping model for chromatin higher-order folding, similar to rope flaking used in mountain climbing and rappelling. Not only does such packing help to avoid tangling and self-crossing, it also facilitates rope unraveling. Hierarchical looping is characterized by an increased frequency of higher-order internucleosome contacts for metaphase chromosomes compared with chromatin fibers in vitro and interphase chromatin, with preservation of a dominant two-start zigzag organization associated with the 30-nm fiber. Moreover, the strong dependence of looping on linker histone concentration suggests a hierarchical self-association mechanism of relaxed nucleosome zigzag chains rather than longitudinal compaction as seen in 30-nm fibers. Specifically, concentrations lower than one linker histone per nucleosome promote self-associations and formation of these looped networks of zigzag fibers. The combined experimental and modeling evidence for condensed metaphase chromatin as hierarchical loops and bundles of relaxed zigzag nucleosomal chains rather than randomly coiled threads or straight and stiff helical fibers reconciles aspects of other models for higher-order chromatin structure; it constitutes not only an efficient storage form for the genomic material, consistent with other genome-wide chromosome conformation studies that emphasize looping, but also a convenient organization for local DNA unraveling and genome access.

Nuclear protein kinase CLK1 uses a non-traditional docking mechanism to select physiological substrates.

Phosphorylation-dependent cell communication requires enzymes that specifically recognize key proteins in a sea of similar, competing substrates. The protein kinases achieve this goal by utilizing docking grooves in the kinase domain or heterologous protein adaptors to reduce 'off pathway' targeting. We now provide evidence that the nuclear protein kinase CLK1 (cell division cycle2-like kinase 1) important for splicing regulation departs from these classic paradigms by using a novel self-association mechanism. The disordered N-terminus of CLK1 induces oligomerization, a necessary event for targeting its physiological substrates the SR protein (splicing factor containing a C-terminal RS domain) family of splicing factors. Increasing the CLK1 concentration enhances phosphorylation of the splicing regulator SRSF1 (SR protein splicing factor 1) compared with the general substrate myelin basic protein (MBP). In contrast, removal of the N-terminus or dilution of CLK1 induces monomer formation and reverses this specificity. CLK1 self-association also occurs in the nucleus, is induced by the N-terminus and is important for localization of the kinase in sub-nuclear compartments known as speckles. These findings present a new picture of substrate recognition for a protein kinase in which an intrinsically disordered domain is used to capture physiological targets with similar disordered domains in a large oligomeric complex while discriminating against non-physiological targets.

Mechanism of Reversible Self-Association of a Monoclonal Antibody: Role of Electrostatic and Hydrophobic Interactions

Reversible self-association of protein therapeutics, the phenomenon of formation of native reversible oligomeric species as a result of noncovalent intermolecular interactions, can add additional manufacturing, stability, delivery, and safety complexities in biopharmaceutical development. Its early detection, characterization, and mitigation can therefore contribute to the success of drug development. A variety of structural and environmental factors can contribute to the modulation of self-association with mechanisms still elusive in some cases due to the inherent structural complexity of proteins. By combining the capabilities of dynamic and static light scattering techniques, the modulatory effects of a variety of solution conditions on a model IgG1's (mAbA) intermolecular interactions have been utilized to derive mechanism of its self-association at relatively low-protein concentration. The analysis of the effect of pH, buffer type, Hofmeister salts, and aromatic amino acids utilizing light scattering supported a combined role of hydrophobic and electrostatic interactions in mAbA self-association. Fitting of the data into the equilibrium models obtained from the multiangle static light scattering provided the enthalpic and entropic contributions of self-association, highlighting the more dominant effect of electrostatic interactions. In addition, studies of the Fab and Fc fragments of mAbA suggested the key role of the former in observed self-association.

De novo design and experimental characterization of ultrashort self-associating peptides.

Self-association is a common phenomenon in biology and one that can have positive and negative impacts, from the construction of the architectural cytoskeleton of cells to the formation of fibrils in amyloid diseases. Understanding the nature and mechanisms of self-association is important for modulating these systems and in creating biologically-inspired materials. Here, we present a two-stage de novo peptide design framework that can generate novel self-associating peptide systems. The first stage uses a simulated multimeric template structure as input into the optimization-based Sequence Selection to generate low potential energy sequences. The second stage is a computational validation procedure that calculates Fold Specificity and/or Approximate Association Affinity (K*association) based on metrics that we have devised for multimeric systems. This framework was applied to the design of self-associating tripeptides using the known self-associating tripeptide, Ac-IVD, as a structural template. Six computationally predicted tripeptides (Ac-LVE, Ac-YYD, Ac-LLE, Ac-YLD, Ac-MYD, Ac-VIE) were chosen for experimental validation in order to illustrate the self-association outcomes predicted by the three metrics. Self-association and electron microscopy studies revealed that Ac-LLE formed bead-like microstructures, Ac-LVE and Ac-YYD formed fibrillar aggregates, Ac-VIE and Ac-MYD formed hydrogels, and Ac-YLD crystallized under ambient conditions. An X-ray crystallographic study was carried out on a single crystal of Ac-YLD, which revealed that each molecule adopts a β-strand conformation that stack together to form parallel β-sheets. As an additional validation of the approach, the hydrogel-forming sequences of Ac-MYD and Ac-VIE were shuffled. The shuffled sequences were computationally predicted to have lower K*association values and were experimentally verified to not form hydrogels. This illustrates the robustness of the framework in predicting self-associating tripeptides. We expect that this enhanced multimeric de novo peptide design framework will find future application in creating novel self-associating peptides based on unnatural amino acids, and inhibitor peptides of detrimental self-aggregating biological proteins.

Small Angle X-ray Scattering-Based Elucidation of the Self-Association Mechanism of Human Insulin Analogue LysB29(Nεω-carboxyheptadecanoyl) des(B30)

Lys(B29)(N(ε)ω-carboxyheptadecanoyl) des(B30) human insulin is an insulin analogue belonging to a class of analogues designed to form soluble depots in subcutis by self-association, aiming at a protracted action. On the basis of small angle X-ray scattering (SAXS) supplemented by a range of biophysical and structural methods (field flow fractionation, dynamic and multiangle light scattering, circular dichroism, size exclusion chromatography, and crystallography), we propose a mechanism for the self-association expected to occur upon subcutaneous injection of this insulin analogue. SAXS data provide evidence of the in solution structure of the self-associated oligomer, which is a long straight rod composed of "tense" state insulin hexamers (T(6)-hexamers) as the smallest repeating unit. The smallest oligomer building block in the process is a T(6)T(6)-dihexamer. This tense dihexamer is formed by the allosteric change of the initial equilibrium between a proposed "relaxed" state R(6)-hexamer and an R(3)T(3)T(3)R(3)-dihexamer. The allosteric change from relaxed to tense is triggered by removal of phenol, mimicking subcutaneous injection. The data hence provide the first unequivocal evidence of the mechanism of self-association for this type of insulin analogue.

Influence of the Valine Zipper Region on the Structure and Aggregation of the Basic Leucine Zipper (bZIP) Domain of Activating Transcription Factor 5 (ATF5)

Protein aggregation is a major problem for biopharmaceuticals. While the control of aggregation is critically important for the future of protein pharmaceuticals, mechanisms of aggregate assembly, particularly the role that structure plays, are still poorly understood. Increasing evidence indicates that partially folded intermediates critically influence the aggregation pathway. We have previously reported the use of the basic leucine zipper (bZIP) domain of activating transcription factor 5 (ATF5) as a partially folded model system to investigate protein aggregation. This domain contains three regions with differing structural propensity: a N-terminal polybasic region, a central helical leucine zipper region, and a C-terminal extended valine zipper region. Additionally, a centrally positioned cysteine residue readily forms an intermolecular disulfide bond that reduces aggregation. Computational analysis of ATF5 predicts that the valine zipper region facilitates self-association. Here we test this hypothesis using a truncated mutant lacking the C-terminal valine zipper region. We compare the structure and aggregation of this mutant to the wild-type (WT) form under both reducing and nonreducing conditions. Our data indicate that removal of this region results in a loss of α-helical structure in the leucine zipper and a change in the mechanism of self-association. The mutant form displays increased association at low temperature but improved resistance to thermally induced aggregation.

Mechanisms of self-association of a human monoclonal antibody CNTO607

Some antibodies have a tendency to self-associate leading to precipitation at relatively low concentrations. CNTO607, a monoclonal antibody, precipitates irreversibly in phosphate-buffered saline at concentrations above 13 mg/ml. Previous mutagenesis work based on the Fab crystal structure pinpointed a three residue fragment in the heavy chain CDR-3, (99)FHW(100a), as an aggregation epitope that is anchored by two salt bridges. Biophysical characterization of variants reveals that F99 and W100a, but not H100, contribute to the intermolecular interaction. A K210T/K215T mutant designed to disrupt the charge interactions in the aggregation model yielded an antibody that does not precipitate but forms reversible aggregates. An isotype change from IgG1 to IgG4 prevents the antibody from precipitating at low concentration yet the solution viscosity is elevated. To further understand the nature of the antibody self-association, studies on the Fab fragment found high solubility but significant self- and cross-interactions remain. Dynamic light scattering data provides evidence for higher order Fab structure at increased concentrations. Our results provide direct support for the aggregation model that CNTO607 precipitation results primarily from the specific interaction of the Fab arms of neighboring antibodies followed by the development of an extensive network of antibodies inducing large-scale aggregation and precipitation.

The Structure of Dimeric Apolipoprotein A-IV and Its Mechanism of Self-Association

Apolipoproteins are key structural elements of lipoproteins and critical mediators of lipid metabolism. Their detergent-like properties allow them to emulsify lipid or exist in a soluble lipid-free form in various states of self-association. Unfortunately, these traits have hampered high-resolution structural studies needed to understand the biogenesis of cardioprotective high-density lipoproteins (HDLs). We derived a crystal structure of the core domain of human apolipoprotein (apo)A-IV, an HDL component and important mediator of lipid absorption. The structure at 2.4 Å depicts two linearly connected 4-helix bundles participating in a helix swapping arrangement that offers a clear explanation for how the protein self-associates as well as clues to the structure of its monomeric form. This also provides a logical basis for antiparallel arrangements recently described for lipid-containing particles. Furthermore, we propose a "swinging door" model for apoA-IV lipid association.

Solution Structure Analysis of the HPV16 E6 Oncoprotein Reveals a Self-Association Mechanism Required for E6-Mediated Degradation of p53

The viral oncoprotein E6 is an essential factor for cervical cancers induced by "high-risk" mucosal HPV. Among other oncogenic activities, E6 recruits the ubiquitin ligase E6AP to promote the ubiquitination and subsequent proteasomal degradation of p53. E6 is prone to self-association, which long precluded its structural analysis. Here we found that E6 specifically dimerizes through its N-terminal domain and that disruption of the dimer interface strongly increases E6 solubility. This allowed us to raise structural data covering the entire HPV16 E6 protein, including the high-resolution NMR structures of the two zinc-binding domains of E6 and a robust data-driven model structure of the N-terminal domain homodimer. Interestingly, homodimer interface mutations that disrupt E6 self-association also inactivate E6-mediated p53 degradation. These data suggest that E6 needs to self-associate via its N-terminal domain to promote the polyubiquitination of p53 by E6AP.

Structure of dimeric apoA‐IV: basis for HDL model

A major contributing factor to cardiovascular disease is atherosclerosis and its progression is dramatically affected by HDL and LDL levels. HDL and LDL are lipoprotein complexes consisting of lipids and proteins from the apolipoprotein family. Apolipoprotein members have a unique detergent‐like property, which enable them to bind lipids and form large complexes such as chylomicrons, VLDL, LDL and HDL. Another key property of apolipoprotein is self‐association and elucidating this mechanism is key to understanding the functions of cardio‐protective lipoprotein complexes like HDL. The few solved structures of apolipoproteins have failed to provide insight into self‐association. We have determined the first dimeric structure of apoA‐IV, one of the largest of the apolipoproteins at 43kDa. From the structure, we observed a novel dimerization mechanism that buries over 11,000 Å2, forming an incredibly stable dimer that requires over 48 hours at 37°C to reach monomer:dimer equilibrium. This unique mechanism also maintains a four‐helical bundle upon dimerization and creats a hydrophobic core that stretches over 150Å and providing an ideal environment for sequestering lipids. Using this structure in conjunction with biochemical data, we have generated a model of apoA‐IV transitioning from a monomer to a HDL in three steps. The apoA‐IV HDL model is supported by previous observations and rectifies some conflicting biochemical data in the field. This structure not only reveals the mechanism for apolipoprotein self‐association, but also provides a clear picture that demonstrates the apoA‐IV structure as the basis for its biological function. We believe this transition model is a general mechanism and can extend to apoA‐I and apoA‐V, which also form HDL.

Molecular Mechanism of Alpha-CaMKII Self-Association

Calcium/calmodulin-dependent protein kinase II, CaMKII, is a calcium-dependent serine/threonine kinase associated with ischemia and other neurodegenerative diseases. Biochemical studies have shown that CaMKII undergoes an activity-dependent aggregation, termed self-association, when activated in an ischemic-like environment - reduced pH, reduced ATP availability. To date, the molecular mechanisms differentiating CaMKII self-association from targeting are not known, as mutants such as Thr286Ala which disrupt self-association also disrupt targeting. Interestingly, self-association is only seen with αCaMKII and not βCaMKII. While these isoforms are highly conserved, sequence alignment of the catalytic and autoregulatory domains highlight several amino acids that vary between αCaMKII and βCaMKII, including residues which may serve as pH sensors (βH38, αH84, αH273) or potential crosslink sites (βC35, βC148, βC272). Thus, we constructed αCaMKII/βCaMKII chimeras to identify mutations in αCaMKII that would prevent self-association. Unlike wild-type αCaMKII, GFP-α/βCaMKII chimeras failed to self-associate in HEK293 cells subjected to ionomycin/calcium at pH 6.5 - a cell-based model where self-association is monitored in real-time using fluorescent microscopy. Recombinant αCaMKII, βCaMKII, and α/βCaMKII chimeras were expressed, purified, and their enzymatic properties and ability to self-associate were measured in controlled conditions in vitro. No differences were observed for any of the wild-type or mutant enzymes to bind or phosphorylate substrates, suggesting that the α/βCaMKII chimeras are enzymatically identical to wild-type proteins. Using Rayleigh light-scattering to monitor αCaMKII self-association in vitro, the α/βCaMKII chimera, like βCaMKII, did not undergo self-association. These data suggest that key residues in the catalytic and autoregulatory domain account for either the ability of αCaMKII to self-associate or βCaMKII to be unable to self-associate. Identification of these mutations that disrupt self-association and still allow targeting affords the opportunity to definitively determine the role that self-association plays in the subcellular localization of CaMKII during physiological and pathological conditions.

Atomic structure of a nanobody-trapped domain-swapped dimer of an amyloidogenic β2-microglobulin variant

Atomic-level structural investigation of the key conformational intermediates of amyloidogenesis remains a challenge. Here we demonstrate the utility of nanobodies to trap and characterize intermediates of β2-microglobulin (β2m) amyloidogenesis by X-ray crystallography. For this purpose, we selected five single domain antibodies that block the fibrillogenesis of a proteolytic amyloidogenic fragment of β2m (ΔN6β2m). The crystal structure of ΔN6β2m in complex with one of these nanobodies (Nb24) identifies domain swapping as a plausible mechanism of self-association of this amyloidogenic protein. In the swapped dimer, two extended hinge loops--corresponding to the heptapetide NHVTLSQ that forms amyloid in isolation--are unmasked and fold into a new two-stranded antiparallel β-sheet. The β-strands of this sheet are prone to self-associate and stack perpendicular to the direction of the strands to build large intermolecular β-sheets that run parallel to the axis of growing oligomers, providing an elongation mechanism by self-templated growth.

The structural basis for autonomous dimerization of the pre-T-cell antigen receptor

The pre-T-cell antigen receptor (pre-TCR), expressed by immature thymocytes, has a pivotal role in early T-cell development, including TCR β-selection, survival and proliferation of CD4(-)CD8(-) double-negative thymocytes, and subsequent αβ T-cell lineage differentiation. Whereas αβTCR ligation by the peptide-loaded major histocompatibility complex initiates T-cell signalling, pre-TCR-induced signalling occurs by means of a ligand-independent dimerization event. The pre-TCR comprises an invariant α-chain (pre-Tα) that pairs with any TCR β-chain (TCRβ) following successful TCR β-gene rearrangement. Here we provide the basis of pre-Tα-TCRβ assembly and pre-TCR dimerization. The pre-Tα chain comprised a single immunoglobulin-like domain that is structurally distinct from the constant (C) domain of the TCR α-chain; nevertheless, the mode of association between pre-Tα and TCRβ mirrored that mediated by the Cα-Cβ domains of the αβTCR. The pre-TCR had a propensity to dimerize in solution, and the molecular envelope of the pre-TCR dimer correlated well with the observed head-to-tail pre-TCR dimer. This mode of pre-TCR dimerization enabled the pre-Tα domain to interact with the variable (V) β domain through residues that are highly conserved across the Vβ and joining (J) β gene families, thus mimicking the interactions at the core of the αβTCR's Vα-Vβ interface. Disruption of this pre-Tα-Vβ dimer interface abrogated pre-TCR dimerization in solution and impaired pre-TCR expression on the cell surface. Accordingly, we provide a mechanism of pre-TCR self-association that allows the pre-Tα chain to simultaneously 'sample' the correct folding of both the V and C domains of any TCR β-chain, regardless of its ultimate specificity, which represents a critical checkpoint in T-cell development. This unusual dual-chaperone-like sensing function of pre-Tα represents a unique mechanism in nature whereby developmental quality control regulates the expression and signalling of an integral membrane receptor complex.

Differences between reversible (self-association) and irreversible aggregation of r HuG-CSF in carbohydrate and polyol formulations

Severe immunogenic and other debilitating human disorders potentially induced by protein aggregates have brought this phenomenon into the focus of biopharmaceutical science over the past decade. Depending on its driving forces, the process induced in the model protein r HuG-CSF may be either reversible or irreversible, resulting in the assembly of self-associated protein species or irreversible aggregates of various final morphologies. The aim of our work was to investigate the correlation between irreversible and reversible aggregation and the protective effect of non-specific formulation stabilisers, selected from the group of carbohydrates and polyols including trehalose, xylitol, cellobiitol, turanose, cellobiose, leucrose, lactitol, lyxose, and sorbitol, against both irreversible protein aggregation and reversible self-association processes of the r HuG-CSF. The formation of irreversible aggregates was thermally induced and evaluated using differential scanning calorimetry and size-exclusion chromatography. As opposed to the irreversible aggregation process, the process of self-association was induced by the agitation experiment by directly augmenting the protein solution contact surfaces. Absence of statistical connectivity between different stabilisers’ ability to inhibit self-association or aggregation reactions indicates that these are two distinct physicochemical processes with different formulation stabilizing outcomes. Reaction mechanism of thermally induced aggregation observed in the study was in line with published literature data, while the reaction mechanism for self-association process was postulated. The postulate has been verified experimentally by isothermal calorimetry and agitation set of experiments conducted after size-exclusion chromatography and asymmetrical flow field-flow fractionation separation of monomeric, dimeric, trimeric, oligomeric, and large self-associated forms detected on multi-angle light scattering, fluorescence, UV, and refractive index detectors. Besides defining the mechanism and kinetic of self-association in stabilized rHuG-CSF formulations, special attention was also paid to the shifts and ranks of the free energy of the aggregation or self-association transition states.

Collagen–DNA Complex

Previously presented models of collagen-DNA (7) and collagen-siRNA (8) complexes point to a general description of delivery systems and indicate to what specific topology that system should be equipped with to effectively deliver the gene into the living body via in vivo and in vitro injection. We focused our interest on the nature of collagen-DNA complex structure and the molecular and environmental determinants of the self-association processes of collagen with the presence of DNA. In this aspect, the self-association of collagen-DNA complex offers an opportunity to characterize a unique system, which may be related to the general mechanisms of self-association of fiber macromolecules by water bridges. For characterizing the collagen-DNA interaction, we used FTIR-ATR, NMR, and AFM experiments done on a separate collagen film, DNA film, and on the peptide-DNA aqueous solution. We demonstrate that collagen-DNA spontaneously forms self-assembling complex systems in aqueous solution. Such self-association of the complex could be induced by electrostatic interactions between neutral collagen cylinders, having strong dipole moment, and negatively charged DNA cylinders. A final complex could be formed by hydrogen bonds between specified donor groups of collagen and phosphate acceptor groups of DNA. According to FTIR measurements, a collagen triple helix should not change global conformation during collagen-DNA complex formation.

Fundamental Studies on Membrane Protein Folding Using Model Transmembrane Helices

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Fundamental Studies on Membrane Protein Folding Using Model Transmembrane Helices

To understand the folding process of alpha-helical membrane proteins in a lipid bilayer environment, the mechanisms of membrane partitioning and self-association of the helix should be elucidated. Considering the inhomogeneity of biological membranes composed of various lipids, not only the amino acid sequence of the transmembrane helices but also the composition of the lipid bilayers are determinants for folding and intramembrane distribution of membrane proteins thorough the balance between helix-helix, helix-lipid, and lipid-lipid interactions. Thermodynamic study using model transmembrane helices is fascinating to measure these complex interactions experimentally. The effects of lipid composition on membrane partitioning and self-association of an inert model transmembrane helix, (AALALAA)3, were examined. Partitioning of the helix into phosphoethanolamine-containing bilayers, gel-phase bilayers, or liquid ordered-phase bilayers significantly decreased, presumably by decreasing the fluidity in the hydrophobic region of the bilayer. It was found that the difference in the length of the hydrophobic regions between helix and lipid bilayers is energetically unfavorable, and partitioning into thicker and thinner membranes were weakened by increasing enthalpic and entropic terms, respectively. In contrast, stronger helix associations driven by the decrease in enthalpy were observed with increasing membrane thickness. These results demonstrate that the surrounding lipids are also important factors determining the behavior of transmembrane helices.

Self-Association Process of a Peptide in Solution: From β-Sheet Filaments to Large Embedded Nanotubes

Lanreotide is a synthetic octapeptide used in the therapy against acromegaly. When mixed with pure water at 10% (w/w), Lanreotide (acetate salt) forms liquid crystalline and monodisperse nanotubes with a radius of 120 Å. The molecular and supramolecular organization of these structures has been determined in a previous work as relying on the lateral association of 26 β-sheet filaments made of peptide noncovalent dimers, the basic building blocks. The work presented here has been devoted to the corresponding self-association mechanisms, through the characterization of the Lanreotide structures formed in water, as a function of peptide (acetate salt) concentration (from 2% to 70% (w/w)) and temperature (from 15°C to 70°C). The corresponding states of water were also identified and quantified from the thermal behavior of water in the Lanreotide mixtures. At room temperature and below 3% (w/w) Lanreotide acetate in water, soluble aggregates were detected. From 3% to 20% (w/w) long individual and monodisperse nanotubes crystallized in a hexagonal lattice were evidenced. Their molecular and supramolecular organizations are identical to the ones characterized for the 10% (w/w) sample. Heating induces the dissolution of the nanotubes into soluble aggregates of the same structural characteristics as the room temperature ones. The solubilization temperature increases from 20°C to 70°C with the peptide concentration and reaches a plateau between 15% and 25% (w/w) in peptide. These aggregates are proposed to be the β-sheet filaments that self-associate to build the walls of the nanotubes. Above 20% (w/w) of Lanreotide acetate in water, polydisperse embedded nanotubes are formed and the hexagonal lattice is lost. These embedded nanotubes exhibit the same molecular and supramolecular organizations as the individual monodisperse nanotubes formed at lower peptide concentration. The embedded nanotubes do not melt in the range of temperature studied indicating a higher thermodynamic stability than individual nanotubes. In parallel, the thermal behaviors of water in mixtures containing 2–80% (w/w) in peptide have been studied by differential scanning calorimetry, and three different types of water were characterized: 1), bulk water melting at 0°C, 2), nonfreezing water, and 3), interfacial water melting below 0°C. The domains of existence and coexistence of these different water states are related to the different Lanreotide supramolecular structures. All these results were compiled into a binary Lanreotide-water phase diagram and allowed to propose a self-association mechanism of Lanreotide filaments into monodisperse individual nanotubes and embedded nanotubes.