Understanding the Rhodopsin Worldview Through Atomic Force Microscopy (AFM): Structure, Stability, and Activity Studies.

Rhodopsin is a G protein-coupled receptor (GPCR) present in the rod outer segment (ROS) of photoreceptor cells that initiates the phototransduction cascade required for scotopic vision. Due to the remarkable advancements in technological tools, the chemistry of rhodopsin has begun to unravel especially over the past few decades, but mostly at the ensemble scale. Atomic force microscopy (AFM) is a tool capable of providing critical information from a single-molecule point of view. In this regard, to bolster our understanding of rhodopsin at the nanoscale level, AFM-based imaging, force spectroscopy, and nano-indentation techniques were employed on ROS disc membranes containing rhodopsin, isolated from vertebrate species both in normal and diseased states. These AFM studies on samples from native retinal tissue have provided fundamental insights into the structure and function of rhodopsin under normal and dysfunctional states. We review here the findings from these AFM studies that provide important insights on the supramolecular organization of rhodopsin within the membrane and factors that contribute to this organization, the molecular interactions stabilizing the structure of the receptor and factors that can modify those interactions, and the mechanism underlying constitutive activity in the receptor that can cause disease.

First 3D-Structural Data of Full-Length Guanylyl Cyclase 1 in Rod-Outer-Segment Preparations of Bovine Retina by Cross-Linking/Mass Spectrometry.

The rod-outer-segment guanylyl cyclase 1 (ROS-GC1) is a key transmembrane protein for retinal phototransduction. Mutations of ROS-GC1 correlate with different retinal diseases that often lead to blindness. No structural data are available for ROS-GC1 so far. We performed a 3D-structural analysis of native ROS-GC1 from bovine retina by cross-linking/mass spectrometry (XL-MS) and computational modeling. Absolute quantification and activity measurements of native ROS-GC1 were performed by MS-based assays directly in bovine retina samples. Our data present the first 3D-structural analysis of active, full-length ROS-GC1 derived from bovine retina. We propose a novel domain organization for the intracellular domain ROS-GC1. Our XL-MS data of native ROS-GC1 from rod-outer-segment preparations of bovine retina agree with a dimeric architecture. Our integrated approach can serve as a blueprint for conducting 3D-structural studies of membrane proteins in their native environment.

The GARP Domain of the Rod CNG Channel's ß1-Subunit Contains Distinct Sites for Outer Segment Targeting and Connecting to the Photoreceptor Disk Rim.

Vision begins when light is captured by the outer segment organelle of photoreceptor cells in the retina. Outer segments are modified cilia filled with hundreds of flattened disk-shaped membranes. Disk membranes are separated from the surrounding plasma membrane, and each membrane type has unique protein components. The mechanisms underlying this protein sorting remain entirely unknown. In this study, we investigated the outer segment delivery of the rod cyclic nucleotide-gated (CNG) channel, which is located in the outer segment plasma membrane, where it mediates the electrical response to light. Using Xenopus and mouse models of both sexes, we now show that the targeted delivery of the CNG channel to the outer segment uses the conventional secretory pathway, including protein processing in both ER and Golgi, and requires preassembly of its constituent α1 and ß1 subunits. We further demonstrate that the N-terminal glutamic acid-rich protein (GARP) domain of CNGß1 contains two distinct functional regions. The glutamic acid-rich region encodes specific information targeting the channel to rod outer segments. The adjacent proline-enriched region connects the CNG channel to photoreceptor disk rims, likely through an interaction with peripherin-2. These data reveal fine functional specializations within the structural domains of the CNG channel and suggest that its sequestration to the outer segment plasma membrane requires an interaction with peripherin-2.SIGNIFICANCE STATEMENT Neurons and other differentiated cells have a remarkable ability to deliver and organize signaling proteins at precise subcellular locations. We now report that the CNG channel, mediating the electrical response to light in rod photoreceptors, contains two specialized regions within the N terminus of its ß-subunit: one responsible for delivery of this channel to the ciliary outer segment organelle and another for subsequent channel sequestration into the outer segment plasma membrane. These findings expand our understanding of the molecular specializations used by neurons to populate their critical functional compartments.

Photooxidation mediated by 11-cis and all-trans retinal in single isolated mouse rod photoreceptors.

Retinal, the vitamin A aldehyde, is a potent photosensitizer that plays a major role in light-induced damage to vertebrate photoreceptors. 11-Cis retinal is the light-sensitive chromophore of rhodopsin, the photopigment of vertebrate rod photoreceptors. It is isomerized by light to all-trans, activating rhodopsin and beginning the process of light detection. All-trans retinal is released by activated rhodopsin, allowing its regeneration by fresh 11-cis retinal continually supplied to photoreceptors. The released all-trans retinal is reduced to all-trans retinol in a reaction using NADPH. We have examined the photooxidation mediated by 11-cis and all-trans retinal in single living rod photoreceptors isolated from mouse retinas. Photooxidation was measured with fluorescence imaging from the oxidation of internalized BODIPY C11, a fluorescent dye whose fluorescence changes upon oxidation. We found that photooxidation increased with the concentration of exogenously added 11-cis or all-trans retinal to metabolically compromised rod outer segments that lacked NADPH supply. In dark-adapted metabolically intact rod outer segments with access to NADPH, there was no significant increase in photooxidation following exposure of the cell to light, but there was significant increase following addition of exogenous 11-cis retinal. The results indicate that both 11-cis and all-trans retinal can mediate light-induced damage in rod photoreceptors. In metabolically intact cells, the removal of the all-trans retinal generated by light through its reduction to retinol minimizes all-trans retinal-mediated photooxidation. However, because the enzymatic machinery of the rod outer segment cannot remove 11-cis retinal, 11-cis-retinal-mediated photooxidation may play a significant role in light-induced damage to photoreceptor cells.

Multistep peripherin-2/rds self-assembly drives membrane curvature for outer segment disk architecture and photoreceptor viability.

Rod and cone photoreceptor outer segment (OS) structural integrity is essential for normal vision; disruptions contribute to a broad variety of retinal ciliopathies. OSs possess many hundreds of stacked membranous disks, which capture photons and scaffold the phototransduction cascade. Although the molecular basis of OS structure remains unresolved, recent studies suggest that the photoreceptor-specific tetraspanin, peripherin-2/rds (P/rds), may contribute to the highly curved rim domains at disk edges. Here, we demonstrate that tetrameric P/rds self-assembly is required for generating high-curvature membranes in cellulo, implicating the noncovalent tetramer as a minimal unit of function. P/rds activity was promoted by disulfide-mediated tetramer polymerization, which transformed localized regions of curvature into high-curvature tubules of extended lengths. Transmission electron microscopy visualization of P/rds purified from OS membranes revealed disulfide-linked tetramer chains up to 100 nm long, suggesting that chains maintain membrane curvature continuity over extended distances. We tested this idea in Xenopus laevis photoreceptors, and found that transgenic expression of nonchain-forming P/rds generated abundant high-curvature OS membranes, which were improperly but specifically organized as ectopic incisures and disk rims. These striking phenotypes demonstrate the importance of P/rds tetramer chain formation for the continuity of rim formation during disk morphogenesis. Overall, this study advances understanding of the normal structure and function of P/rds for OS architecture and biogenesis, and clarifies how pathogenic loss-of-function mutations in P/rds cause photoreceptor structural defects to trigger progressive retinal degenerations. It also introduces the possibility that other tetraspanins may generate or sense membrane curvature in support of diverse biological functions.

Investigating the Nanodomain Organization of Rhodopsin in Native Membranes by Atomic Force Microscopy.

Membrane proteins play an integral role in cellular communication. They are often organized within the crowded cell membrane into nanoscale domains (i.e., nanodomains), which facilitates their function in cell signaling processes. The visualization of membrane proteins and nanodomains within biological membranes under physiological conditions presents a challenge and is not possible using conventional microscopy methods. Atomic force microscopy (AFM) provides an opportunity to study the organization of membrane proteins within biological membranes with sub-nanometer resolution. An example of a membrane protein organized into nanodomains is rhodopsin. Rhodopsin is expressed in photoreceptor cells of the retina and upon photoactivation initiates a series of biochemical reactions called phototransduction, which represents the first steps of vision. AFM has provided an opportunity to directly visualize the packing of rhodopsin in native retinal membranes and the quantitative analysis of AFM images is beginning to reveal insights about the nanodomain organization of rhodopsin in the membrane. In this report, we outline procedures for imaging rhodopsin nanodomains by AFM and the quantitative analysis of those AFM images.

Palmitoylation is a prerequisite for dimerization-dependent raftophilicity of rhodopsin.

The visual photopigment rhodopsin (Rh) is a prototypical G protein-coupled receptor (GPCR) responsible for initiation of the phototransduction cascade in rod photoreceptors. Similar to other GPCRs, Rh can form dimers or even higher oligomers and tends to have a supramolecular organization that is likely important in the dim light response. Rh also exhibits high affinity for lipid rafts (i.e. raftophilicity) upon light-dependent binding with the cognate G protein transducin (Gt), suggesting the presence of lipid raft-like domains in the retinal disk membrane and their importance in phototransduction. However, the relationship between Rh oligomerization and lipid rafts in the disk membrane remains to be explored. Given previous findings that Gt binds to dimeric Rh and that Rh is posttranslationally modified with two highly raftophilic palmitoyl moieties, we hypothesized that Rh becomes raftophilic upon dimerization. Here, using biochemical assays, we found that Rh*-Gt complexes in the detergent-resistant membrane are partially resistant to cholesterol depletion by methyl-ß-cyclodextrin and that the Rh-to-Gt stoichiometry in this methyl-ß-cyclodextrin-resistant complex is 2:1. Next, we found that IgG-mediated Rh-Rh cross-linking renders Rh highly raftophilic, supporting the premise that Rh becomes raftophilic upon dimerization. Rh depalmitoylation via reduction of thioester linkages blocked the translocation of IgG-cross-linked Rh to the detergent-resistant membrane, highlighting that the two palmitoyl moieties are important for the dimerization-dependent raftophilicity of Rh. These results indicate that palmitoylated GPCRs such as Rh can acquire raftophilicity upon G protein-stabilized dimerization and thereby organize receptor-cluster rafts by recruiting raftophilic lipids.

Rhodopsin Forms Nanodomains in Rod Outer Segment Disc Membranes of the Cold-Blooded Xenopus laevis.

Rhodopsin forms nanoscale domains (i.e., nanodomains) in rod outer segment disc membranes from mammalian species. It is unclear whether rhodopsin arranges in a similar manner in amphibian species, which are often used as a model system to investigate the function of rhodopsin and the structure of photoreceptor cells. Moreover, since samples are routinely prepared at low temperatures, it is unclear whether lipid phase separation effects in the membrane promote the observed nanodomain organization of rhodopsin from mammalian species. Rod outer segment disc membranes prepared from the cold-blooded frog Xenopus laevis were investigated by atomic force microscopy to visualize the organization of rhodopsin in the absence of lipid phase separation effects. Atomic force microscopy revealed that rhodopsin nanodomains form similarly as that observed previously in mammalian membranes. Formation of nanodomains in ROS disc membranes is independent of lipid phase separation and conserved among vertebrates.

Crystallization of proteins from crude bovine rod outer segments.

Obtaining protein crystals suitable for X-ray diffraction studies comprises the greatest challenge in the determination of protein crystal structures, especially for membrane proteins and protein complexes. Although high purity has been broadly accepted as one of the most significant requirements for protein crystallization, a recent study of the Escherichia coli proteome showed that many proteins have an inherent propensity to crystallize and do not require a highly homogeneous sample (Totir et al., 2012). As exemplified by RPE65 (Kiser, Golczak, Lodowski, Chance, & Palczewski, 2009), there also are cases of mammalian proteins crystallized from less purified samples. To test whether this phenomenon can be applied more broadly to the study of proteins from higher organisms, we investigated the protein crystallization profile of bovine rod outer segment (ROS) crude extracts. Interestingly, multiple protein crystals readily formed from such extracts, some of them diffracting to high resolution that allowed structural determination. A total of seven proteins were crystallized, one of which was a membrane protein. Successful crystallization of proteins from heterogeneous ROS extracts demonstrates that many mammalian proteins also have an intrinsic propensity to crystallize from complex biological mixtures. By providing an alternative approach to heterologous expression to achieve crystallization, this strategy could be useful for proteins and complexes that are difficult to purify or obtain by recombinant techniques.

Crystallization of G protein-coupled receptors.

Oligomerization is one of several mechanisms that can regulate the activity of G protein-coupled receptors (GPCRs), but little is known about the structure of GPCR oligomers. Crystallography and NMR are the only methods able to reveal the details of receptor-receptor interactions at an atomic level, and several GPCR homodimers already have been described from crystal structures. Two clusters of symmetric interfaces have been identified from these structures that concur with biochemical data, one involving helices I, II, and VIII and the other formed mainly by helices V and VI. In this chapter, we describe the protocols used in our laboratory for the crystallization of rhodopsin and the ß2-adrenergic receptor (ß2-AR). For bovine rhodopsin, we developed a new purification strategy including a (NH4)2SO4-induced phase separation that proved essential to obtain crystals of photoactivated rhodopsin containing parallel dimers. Crystallization of native bovine rhodopsin was achieved by the classic vapor-diffusion technique. For ß2-AR, we developed a purification strategy based on previously published protocols employing a lipidic cubic phase to obtain diffracting crystals of a ß2-AR/T4-lysozyme chimera bound to the antagonist carazolol.

Kinetic, energetic, and mechanical differences between dark-state rhodopsin and opsin.

Rhodopsin, the photoreceptor pigment of the retina, initiates vision upon photon capture by its covalently linked chromophore 11-cis-retinal. In the absence of light, the chromophore serves as an inverse agonist locking the receptor in the inactive dark state. In the absence of chromophore, the apoprotein opsin shows low-level constitutive activity. Toward revealing insight into receptor properties controlled by the chromophore, we applied dynamic single-molecule force spectroscopy to quantify the kinetic, energetic, and mechanical differences between dark-state rhodopsin and opsin in native membranes from the retina of mice. Both rhodopsin and opsin are stabilized by ten structural segments. Compared to dark-state rhodopsin, the structural segments stabilizing opsin showed higher interaction strengths and mechanical rigidities and lower conformational variabilities, lifetimes, and free energies. These changes outline a common mechanism toward activating G-protein-coupled receptors. Additionally, we detected that opsin was more pliable and frequently stabilized alternate structural intermediates.

Divalent cations modulate membrane binding and pore formation of a potent antibiotic peptide analog of alamethicin.

The Ca(2+) modulation of pore formation (and disaggregation) kinetics of a synthetic analog of alamethicin F50/5 ([l-Glu(OMe)(7,18,19)]), a potent antibiotic peptide, was investigated in situ and in vitro. The in situ experiments consisted in whole-cell recording from isolated retinal rod outer segments (OS), because once blocking the only OS endogenous conductance with saturating light, the current flows entirely through the (exogenous) channels formed by the peptide. The kinetics of current change induced by peptide application and removal (in ∼50ms) on the OS extracellular side was measured in the presence of divalent cations at different concentrations. The in vitro experiments consisted on the divalent cations modulation of [l-Glu(OMe)(7,18,19)] binding to a mimetic OS membrane immobilized on a sensor chip surface, employing surface plasmon resonance spectroscopy (SPR). The presence of even low mM Ca(2+) or Mg(2+) sufficed to increase the [l-Glu(OMe)(7,18,19)] apparent affinity for the mimetic OS membrane up to ∼4-fold, which accelerated the activation of the peptide-induced current in OS by ∼10-fold with respect to low Ca(2+). In situ and in vitro experiments indicate that high concentrations of divalent cations increased also membrane rigidity, contrasting their effect on increasing the pore formation rate.

Three-dimensional architecture of the rod sensory cilium and its disruption in retinal neurodegeneration.

Defects in primary cilia lead to devastating disease because of their roles in sensation and developmental signaling but much is unknown about ciliary structure and mechanisms of their formation and maintenance. We used cryo-electron tomography to obtain 3D maps of the connecting cilium and adjacent cellular structures of a modified primary cilium, the rod outer segment, from wild-type and genetically defective mice. The results reveal the molecular architecture of the cilium and provide insights into protein functions. They suggest that the ciliary rootlet is involved in cellular transport and stabilizes the axoneme. A defect in the BBSome membrane coat caused defects in vesicle targeting near the base of the cilium. Loss of the proteins encoded by the Cngb1 gene disrupted links between the disk and plasma membranes. The structures of the outer segment membranes support a model for disk morphogenesis in which basal disks are enveloped by the plasma membrane.

Quantifying the differential effects of DHA and DPA on the early events in visual signal transduction.

A range of evidence from animal, clinical and epidemiological studies indicates that highly polyunsaturated acyl chains play important roles in development, cognition, vision and other aspects of neurological function. In a number of these studies n3 polyunsaturated fatty acids (PUFAs) appear to be more efficacious than n6 PUFAs. In a previous study of retinal rod outer segments obtained from rats raised on either an n3 adequate or deficient diet, we demonstrated that the replacement of 22:6n3 by 22:5n6 in the n3 deficient rats led to functional deficits in each step in the visual signaling process (Niu et al., 2004). In this study, we examined rhodopsin and phosphodiesterase function and acyl chain packing properties in membranes consisting of phosphatidylcholines with sn-1=18:0, and sn-2=22:6n3, 22:5n6, or 22:5n3 in order to determine if differences in function are due to the loss of one double bond or due to differences in double bond location. At 37 °C the n6 lipid shifted the equilibrium between the active metarhodopsin II (MII) state and inactive metarhodopsin I (MI) state towards MI. In addition, 22:5n6 reduced the rates of MII formation and MII-transducin complex formation by 2- and 6-fold, respectively. At a physiologically relevant level of rhodopsin light stimulation, the activity of phosphodiesterase was reduced by 50% in the 22:5n6 membrane, relative to either of the n3 membranes. Activity levels in the two n3 membranes were essentially identical. Ensemble acyl chain order was assessed with time-resolved fluorescence measurements of the membrane probe diphenylhexatriene (DPH). Analysis in terms of the orientational distribution of DPH showed that acyl chain packing in the two n3 membranes is quite similar, while in the 22:5n6 membrane there was considerably less packing disorder in the bilayer midplane. These results demonstrate that the n3 bond configuration uniquely optimizes the early steps in signaling via a mechanism which may involve acyl chain packing deep in the bilayer.

Role of membrane integrity on G protein-coupled receptors: Rhodopsin stability and function.

Rhodopsin is a prototypical G protein-coupled receptor (GPCR) - a member of the superfamily that shares a similar structural architecture consisting of seven-transmembrane helices and propagates various signals across biological membranes. Rhodopsin is embedded in the lipid bilayer of specialized disk membranes in the outer segments of retinal rod photoreceptor cells where it transmits a light-stimulated signal. Photoactivated rhodopsin then activates a visual signaling cascade through its cognate G protein, transducin or Gt, that results in a neuronal response in the brain. Interestingly, the lipid composition of ROS membranes not only differs from that of the photoreceptor plasma membrane but is critical for visual transduction. Specifically, lipids can modulate structural changes in rhodopsin that occur after photoactivation and influence binding of transducin. Thus, altering the lipid organization of ROS membranes can result in visual dysfunction and blindness.

Binding of retinoids to ABCA4, the photoreceptor ABC transporter associated with Stargardt macular degeneration.

ABCA4 is a member of the superfamily of ATP-binding cassette (ABC) transporters, which has been implicated in the clearance of all-trans retinal derivatives from rod and cone photoreceptor cells following photoexcitation as part of the visual cycle. Mutations in ABCA4 are known to cause Stargardt macular degeneration and related disorders, associated with a severe loss in vision. Recently, a solid-phase binding assay has been developed to identify retinoids that likely serve as substrates for this transporter. In this procedure, monoclonal antibodies directed either against an epitope within ABCA4 (Rim 3F4 antibody) or against the 9 amino acid 1D4 epitope tag engineered onto the C-terminus of expressed ABCA4 (Rho 1D4 antibody) are covalently bound to a Sepharose matrix. This immunoaffinity matrix is then used to isolate ABCA4 from photoreceptor outer segments or transfected cells. All-trans retinal is added to immobilized ABCA4 in the presence of a phospholipid mixture containing phosphatidylethanolamine. The bound retinoid is then analyzed directly by spectrophotometry or identified by HPLC and/or mass spectrometry following extraction with organic solvents. Using this procedure, it has been shown that unprotonated N-retinylidene-phosphatidylethanolamine binds with high affinity to ABCA4 and is released by the addition of ATP. These procedures and related radiometric assays using titrated retinal have been used to study the binding of N-retinylidene-PE to wild-type and mutant ABCA4 in the absence and presence of nucleotides for structure-function studies.

Immunochemical or fluorescent labeling of vesicular subcellular fractions for microscopy imaging.

We describe a procedure for the labeling of membranous vesicular purified subcellular fractions, to image them, typically by confocal laser scanning microscopy. Being intracellular organelles, these fractions, once purified cannot be attached to glass slides as for cells. Fractions are labeled "in batch" without prior embedding or freezing. Each labeling step performed by passages of resuspension/centrifugation is followed by washings. Then samples are dispersed on the glass slides. Mammalian retinal rod outer segment disks, intact brain stem myelin vesicles, and brain synaptosomes were chosen, as these subcellular fractions can be purified by well established procedures. These fractions were immunolabeled with specific antibodies. Moreover, by the earlier procedure, we show that the mitochondrial vital membrane potential probe MitoTracker Deep Red 633 stains myelin vesicles and rod disks before fixation, consistently with our previous reports of a respiring capacity of these membranes. Therefore, the technique seems adequate to become an instrument to study the structure and the function of these and other subcellular fractions.

[Purification of transducin betagamma-subunit complex from isotonic extracts of bovine retinal rod outer segments].

A method for obtaining a free complex of transducin betagamma-subunits from bovine retinal rod outer segments in a highly purified state has been suggested.

RPGRIP1 is essential for normal rod photoreceptor outer segment elaboration and morphogenesis.

The function of the retinitis pigmentosa GTPase regulator interacting protein 1 (RPGRIP1) gene is currently not known. However, mutations within the gene lead to Leber Congenital Amaurosis and autosomal recessive retinitis pigmentosa in human patients. In a previously described knockout mouse model of the long splice variant of Rpgrip1, herein referred to as Rpgrip1(tm1Tili) mice, mislocalization of key outer segment proteins and dysmorphogenesis of outer segment discs preceded subsequent photoreceptor degeneration. In this report, we describe a new mouse model carrying a splice acceptor site mutation in Rpgrip1, herein referred to as Rpgrip1(nmf247) that is phenotypically distinct from Rpgrip1(tm1Tili) mice. Photoreceptor degeneration in homozygous Rpgrip1(nmf247) mice is earlier in onset and more severe when compared with Rpgrip1(tm1Tili) mice. Also, ultrastructural studies reveal that whereas Rpgrip1(nmf247) mutants have a normal structure and number of connecting cilia, unlike Rpgrip1(tm1Tili) mice, they do not elaborate rod outer segments (OS). Therefore, in addition to its role in OS disc morphogenesis, RPGRIP1 is essential for rod OS formation. Our study indicates the absence of multiple Rpgrip1 isoforms in Rpgrip1(nmf247) mice, suggesting different isoforms may play different roles in photoreceptors and underscores the importance of considering splice variants when generating targeted null mutations.

Aggregation of frog rhodopsin to oligomers and their dissociation to monomer: application of BN- and SDS-PAGE.

After solubilization of frog rod outer segments (ROS) with mild detergents (digitonin, n-dodecyl-beta-D-maltoside, Chaps, Triton X-100) and subsequent one-dimensional blue native polyacrylamide gel electrophoresis (1D BN-PAGE), the position of rhodopsin (Rh) on the gradient gel does not match the monomer with molecular weight of 40 kDa but appears self-associated into aggregate of Rh (RhA) with molecular mass varying in different detergents from 85 to 125 kDa. Short-term treatment (~2 h) of the excised BN-PAGE strip containing RhA by denaturing detergent mixture (10% SDS + 1 mM dithiothreitol (DTT)) followed by 2D SDS-PAGE revealed dissociation of the RhA into opsin monomer and unidentified proteins. Long-term treatment (approximately 2 days) of RhA that included extraction, denaturation, concentration, and electrophoresis induced, along with dissociation of RhA into opsin monomer + unidentified proteins, also formation of opsin dimers, trimers, and higher oligomers owing to a secondary aggregation of opsin. Direct solubilization of the ROS by harsh SDS + DTT detergent mixture followed by 1D SDS-PAGE revealed only opsin monomer that upon heating disappeared, transforming into higher oligomers owing to secondary aggregation. The data show that degree of Rh oligomerization depends on specific conditions in which it stays. In the native state in the photoreceptor membrane as well as in mild detergents frog Rh exists mainly as dimers or higher oligomers. After solubilization with denaturing detergents, RhA can dissociate into monomers that then spontaneously self-associate into higher oligomers under the influence of various factors (for example, heating).