Antennal Proteome of the Solenopsis invicta (Hymenoptera: Formicidae): Caste Differences in Olfactory Receptors and Chemosensory Support Proteins.

Little is known about the expression pattern of odorant and pheromone transporters, receptors, and deactivation enzymes in the antennae of ants carrying out different tasks. In order to begin filling in this information gap, we compared the proteomes of the antennae of workers and males of the red fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae). Male ants do not perform any colony work, and their only activity is to leave the nest on a mating flight. Previous studies showed that male ants express fewer types of odorant receptors than workers. Thus, we expected to find large differences between male and worker antennae for expression of receptors, transporters, and deactivators of signaling chemicals. We found that the abundance of receptors was consistent with the expected caste-specific signaling complexity, but the numbers of different antenna-specific transporters and deactivating enzymes in males and workers were similar. It is possible that some of these proteins have antenna-specific functions that are unrelated to chemosensory reception. Alternatively, the similar complexity could be a vestige of ant progenitors that had more behaviorally active males. As the reduced behavior of male ants evolved, the selection process may have favored a complex repertoire of transporters and deactivating enzymes alongside a limited repertoire of odorant receptors.

Surface Polar Lipids Differ in Male and Female Phlebotomus papatasi (Diptera: Psychodidae).

The polar lipids on the surface of the Old World sand fly, Phlebotomus papatasi (Scopoli), were analyzed by high-resolution mass spectrometry. Blood-fed females and nonblood-fed females and males were separately analyzed and compared. The major polar lipids were found to be long-chain diols and fatty acids. Relatively high levels of diacylglycerols were found in blood-fed females and in males. A wide variety of lipids were found at low levels, including esters, sterols, monoacylglycerols, and hydroxy fatty acids. Blood-fed females had several lyso lipids and N-acyl amino acids that were not found on unfed females or males. These substances may be surfactants used in blood feeding. Heneicosenoic acid was found on females at more than twice the level of males, suggesting it could be a component of a female pheromone. Four substances were identified on males at twofold higher levels than on females: tetradienoic acid, methoxyhexadecasphinganine, butyl octadecanoate, and diacylglycerol(14:1/12:0/0:0). These could be short-range pheromones involved in courtship, and they will be further analyzed in future behavioral bioassays.

Surface Polar Lipids Differ in Male and Female Phlebotomus papatasi (Diptera: Psychodidae).

The polar lipids on the surface of the Old World sand fly, Phlebotomus papatasi (Scopoli), were analyzed by high-resolution mass spectrometry. Blood-fed females and nonblood-fed females and males were separately analyzed and compared. The major polar lipids were found to be long-chain diols and fatty acids. Relatively high levels of diacylglycerols were found in blood-fed females and in males. A wide variety of lipids were found at low levels, including esters, sterols, monoacylglycerols, and hydroxy fatty acids. Blood-fed females had several lyso lipids and N-acyl amino acids that were not found on unfed females or males. These substances may be surfactants used in blood feeding. Heneicosenoic acid was found on females at more than twice the level of males, suggesting it could be a component of a female pheromone. Four substances were identified on males at twofold higher levels than on females: tetradienoic acid, methoxyhexadecasphinganine, butyl octadecanoate, and diacylglycerol(14:1/12:0/0:0). These could be short-range pheromones involved in courtship, and they will be further analyzed in future behavioral bioassays.

Arthropod repellent interactions with olfactory receptors and ionotropic receptors analyzed by molecular modeling.

The main insect chemoreceptors are olfactory receptors (ORs), gustatory receptors (GRs) and ionotropic receptors (IRs). The odorant binding sites of many insect ORs appear to be occluded and inaccessible from the surface of the receptor protein, based on the three-dimensional structure of OR5 from the jumping bristletail Machilis hrabei (MhraOR5) and a survey of a sample of vinegar fly (Drosophila melanogaster) OR structures obtained from artificial intellegence (A.I.) modeling. Molecular dynamics simulations revealed that the occluded site can become accessible through tunnels that transiently open and close. The present study extends this analysis to examine seventeen ORs and one GR docking with ligands that have known valence: nine that signal attraction and nine that signal aversion. All but one of the receptors displayed occluded ligand binding sites analogous to MhraOR5, and docking software predicted the known attractant and repellent ligands will bind to the occluded sites. Docking of the repellent DEET was examined, and more than half of the OR ligand sites were predicted to bind DEET, including receptors that signal aversion as well as those that signal attraction. However, DEET may not actually have access to all the attractant binding sites. The larger size and lower flexibility of repellent molecules may restrict their passage through the tunnel bottlenecks, which could act as filters to select access to the ligand binding sites. In contrast to ORs and GRs, the IR ligand binding site is in an extracellular domain known to undergo a large conformational change from an open to a closed state. A.I. models of two D. melanogaster IRs of known valence and two blacklegged tick (Ixodes scapularis) IRs having unknown ligands were computationally tested for attractant and repellent binding. The ligand-binding sites in the closed state appear inaccessible to the protein surface, so attractants and repellents must bind initially at an accessible site in the open state before triggering the conformational change. In some IRs, repellent binding sites were identified at exterior sites adjacent to the ligand-binding site. These may be allosteric sites that, when occupied by repellents, can stabilize the open state of an attractant IR, or stabilize the closed state of an IR in the absence of its activating ligand. The model of D. melanogaster IR64a suggests a possible molecular mechanism for the activation of this IR by H+. The amino acids involved in this proposed mechanism are conserved in IR64a from several Dipteran pest species and disease vectors, potentially offering a route to discovery of new repellents that act via the allosteric site.

Integral membrane protein fragment recombination after transfer from nanolipoprotein particles to bicelles.

A new method for the measurement of membrane protein oligomer association is described. Two engineered fragments of bacteriorhodopsin, which are known to spontaneously associate in bicelles, were expressed in nanolipoprotein particles (NLPs or nanodiscs) using an Escherichia coli S30 cell-free synthesis system. When separately prepared NLPs containing the fragments were mixed, fragment association did not occur, indicating that the apolipoprotein edge blocks transfer between NLPs. However, when bicelles were added to this mixture, fragment association was detected by disulfide cross-linking. The rate of cross-linking was consistent with previously published equilibrium and kinetic parameters. Characterization of the NLP/bicelle mixture by dynamic light scattering and fluorescence spectroscopy indicates that the NLP bilayer transfers to bicelles in a simple reversal of the synthesis of NLPs from bicelles. These experiments validate using cell-free synthesis of membrane proteins in NLPs, followed by treatment with bicelles, as a method for measuring oligomerization of integral membrane protein subunits in a bilayer-like environment.

Tunnel connects lipid bilayer to occluded odorant-binding site of insect olfactory receptor.

MhOR5, an insect olfactory receptor (OR), has an occluded binding site for the odorant eugenol in both the open and closed states of the ion channel. We used atomistic molecular dynamics simulation (MD) and steered molecular dynamics to examine possible tunnels to the odorant binding site from the protein surface. Four high probability tunnels were identified in the MD results. Surprisingly, three of the tunnels connect the ligand binding site to the lipid bilayer. We found sharp 30%-50% increases or decreases in tunnel bottleneck areas over 70 nsec MD trajectories, both in the ligand-bound and unliganded OR structures. Steered MD showed that eugenol follows the tunnels to the protein surface, and the potential of mean force is quantitatively consistent with the known affinity of eugenol for MhOR5. We examined AlphaFold-generated models of 21 other insect ORs, and we found that 19 had odorant binding sites and tunnels in similar positions to MhOR5. The possibility of a tunnel between the odorant binding site and the lipid bilayer in insect ORs suggests new experiments to test molecular mechanisms for insect odorant reception.

Odorant-binding protein from the stable fly (Stomoxys calcitrans) has a high-histidine N-terminal extension that binds transition metals.

The role of odorant- and pheromone-binding proteins (OBPs) in olfactory function is not fully understood. We found an OBP sequence from the stable fly, Stomoxys calcitrans, ScalOBP60, that has a 25 amino acid N-terminal extension with a high content of histidine and acidic amino acids, suggesting a possible metal binding activity. A search of public databases revealed a large number of other fly OBPs with histidine-rich N-terminal extensions, as well as beetle, wasp and ant OBPs with histidine-rich C-terminal extensions. We recombinantly expressed ScalOBP60, as well as a truncated sequence which lacks the histidine-rich N-terminal region, tScalOBP60. Using fluorescence quenching and electrospray quadrupole time-of-flight mass spectrometry (ESI-QTOF), we detected two different types of metal-binding sites. Divalent copper, nickel and zinc bind to the N-terminal histidine-rich region, and divalent copper binds to an internal sequence position. Comparison of the ESI-QTOF spectra of ScalOBP60 and tScalOBP60 showed that the histidine-rich sequence is structurally disordered, but it becomes more ordered in the presence of divalent metal. When copper is bound to the internal site, binding of a hydrophobic ligand to ScalOBP60 is inhibited. The internal and N-terminal metal sites interact allosterically, possibly through a conformational equilibrium, suggesting a mechanism for metal regulation of ligand binding to ScalOBP60. Based on our studies of ScalOBP60, we propose several possible olfactory and non-olfactory functions for this OBP.

Luminescence resonance energy transfer in the cytoplasm of live Escherichia coli cells.

Luminescence resonance energy transfer (LRET) offers many advantages for accurate measurements of distances between specific sites in living cells, but progress in developing a methodology for implementing this technique has been limited. We report here the design, expression, and characterization of a test protein for development of a LRET methodology. The protein, which we call DAL, contains the following domains (from the N-terminus): Escherichia coli dihydrofolate reductase (DHFR), the third and fourth ankyrin repeats of p16(INK4a), a lanthanide-binding tag (LBT), and a hexahistidine tag. LBT binds Tb(3+) with a submicromolar dissociation constant. LRET was measured from the Tb(3+) site on LBT to transition metals bound to the hexa-His tag and to fluorescein methotrexate bound to DHFR. The measured distances were consistent with a molecular model constructed from the known crystal structures of the constituent domains of DAL. The results indicate that the two C-terminal ankyrin domains of p16(INK4a) are stably folded when combined with other protein domains. We found that Tb(3+) binds to DAL in the cytoplasm of live E. coli cells, and thus, DAL is useful as an indicator for studies of metal transport. We also used DAL to measure LRET from Tb(3+) to Cu(2+) in the cytoplasm of live E. coli cells. The rates of Tb(3+) and Cu(2+) transport were not affected by a proton uncoupler or an ATP synthase inhibitor. Reversal of the membrane potential had a small inhibitory effect, and removal of lipopolysaccharide had a small accelerating effect on transport. Changing the external pH from 7 to 5 strongly inhibited the Tb(3+) signal, suggesting that the Tb(3+)-LBT interaction is useful as a cytoplasmic pH indicator in the range of approximately pH 5-6.

Helix insertion into bilayers and the evolution of membrane proteins.

Polytopic alpha-helical membrane proteins cannot spontaneously insert into lipid bilayers without assistance from polytopic alpha-helical membrane proteins that already reside in the membrane. This raises the question of how these proteins evolved. Our current knowledge of the insertion of alpha-helices into natural and model membranes is reviewed with the goal of gaining insight into the evolution of membrane proteins. Topics include: translocon-dependent membrane protein insertion, antibiotic peptides and proteins, in vitro insertion of membrane proteins, chaperone-mediated insertion of transmembrane helices, and C-terminal tail-anchored (TA) proteins. Analysis of the E. coli genome reveals several predicted C-terminal TA proteins that may be descendents of proteins involved in pre-cellular membrane protein insertion. Mechanisms of pre-translocon polytopic alpha-helical membrane protein insertion are discussed.

Surface lipidome of the lone star tick, Amblyomma americanum, provides leads on semiochemicals and lipid metabolism.

Lipids extracted from the surface of the lone star tick, Amblyomma americanum, were analyzed by high resolution mass spectrometry. Prior to lipid extraction, the adult ticks were either unfed or fed on cattle, and the fed ticks were in groups either containing males and females together, or containing only males or females. Cholesteryl esters were found on the surfaces of fed females, and they may provide a more complete description of the composition of the mounting sex pheromone. Dihydrocholesteryl esters were detected on the surfaces of unfed males and females, suggesting a possible role in survival during host-seeking. Dehydrodeoxyecdysone, found on fed females, could be a component of the genital sex pheromone. The most abundant polar surface lipids detected were acylglycerides. High levels of sphingolipids and glycerophospholipids on males fed separately might be derived, in part, from sperm development. A high level of a 20:4 fatty acid, presumably arachidonic acid, was found on the surface of fed females, indicating that it may be a component of the genital sex pheromone. A high level of docosenamide was found on the surface of fed females. Wax esters were found on the surfaces of fed ticks but not on unfed ticks. These esters could be involved in elasticity of the cuticle of engorged females or in wax coating of eggs. N-acylethanolamines were found on the surfaces of male and female ticks fed together, and on male ticks fed separately, but were absent or at low levels on females fed separately and on unfed ticks. This pattern suggests a possible role as a metabolic coordination primer pheromone.

Buried water molecules in helical transmembrane proteins.

Buried water molecules (having no contact with bulk solvent) in 30 helical transmembrane (TM) protein structures were identified. The average amount of buried water in helical TM proteins is about the same as for all water-soluble (WS) proteins, but it is greater than the average for helical WS proteins. Buried waters in TM proteins make more polar contacts, and are more frequently found contacting helices than in WS proteins. The distribution of the buried water binding sites across the membrane profile shows that the sites to some extent reflect protein function. There is also evidence for asymmetry of the sites, with more in the extracellular half of the membrane. Many of the buried water contact sites are conserved across families of proteins, including family members having different functions. This suggests that at least some buried waters play a role in structural stabilization. Disease-causing mutations, which are known to result in misfolded TM proteins, occur at buried water contact sites at a higher than random frequency, which also supports a stabilizing role for buried water molecules.

The chemosensory appendage proteome of Amblyomma americanum (Acari: Ixodidae) reveals putative odorant-binding and other chemoreception-related proteins.

Proteomic analyses were done on 2 chemosensory appendages of the lone star tick, Amblyomma americanum. Proteins in the fore tarsi, which contain the olfactory Haller's organ, and in the palps, that include gustatory sensilla, were compared with proteins in the third tarsi. Also, male and female ticks were compared. Proteins were identified by sequence similarity to known proteins, and by 3-dimensional homology modeling. Proteomic data were also compared with organ-specific transcriptomes from the tick Rhipicephalus microplus. The fore tarsi express a lipocalin not found in the third tarsi or palps. The fore tarsi and palps abundantly express 2 proteins, which are similar to insect odorant-binding proteins (OBPs). Compared with insect OBPs, the tick OBP-like sequences lacked the cysteine absent in C-minus OBPs, and 1 tick OBP-like sequence had additional cysteines that were similar to C-plus OBPs. Four proteins similar to the antibiotic protein microplusin were found: 2 exclusively expressed in the fore tarsi and 1 exclusively expressed in the palps. These proteins lack the microplusin copper-binding site, but they are modeled to have a significant internal cavity, potentially a ligand-binding site. Proteins similar to the dust mite allergens Der p7 and Der f 7 were found differentially expressed in female fore tarsi. A protein exclusively expressed in the fore tarsi has similarities to Neto, which is known to be involved in clustering of ionotropic glutamate receptors. These results constitute the first report of OBP-like protein sequences in ticks and point to several research avenues on tick chemosensory reception.

Neuronal projections from the Haller's organ and palp sensilla to the synganglion of Amblyomma americanum§.

The present study was conducted to elucidate the neuronal pathways between peripheral olfactory and taste sensilla and the synganglion in an Ixodidae tick species. The tarsus of the front legs (olfactory nerves) and the fourth palpal segment (gustatory nerves) of unfed Amblyomma americanum males and females were excised. A neuronal tracer, dextran tetramethylrhodamine, was used for filling of the sensory neurons. The synganglion preparations were examined using a confocal microscope. Neuronal arborizations from the Haller's organ were confined to the olfactory lobes and the first pedal ganglion. The estimated number of olfactory glomeruli ranged from 16 to 22 per olfactory lobe in the females. The number of glomeruli was not counted in males because they were densely packed. Sensory neurons associated with sensilla at the distal end of the palpal organ projected into the palpal ganglion in the synganglion through the palpal nerve. Gustatory sensory neurons associated with palpal sensilla projected into a commissure with several bulges, which are confined in the palpal ganglion. The findings of distinct projection patterns of sensory neurons associated with the Haller's organ and palpal organ in the lone star tick from this study advanced our knowledge on mechanisms of sensory information processing in ticks.

Kinetics of lipid mixing between bicelles and nanolipoprotein particles.

Nanolipoprotein particles (NLPs), also known as nanodiscs, are lipid bilayers bounded by apolipoprotein. Lipids and membrane proteins cannot exchange between NLPs. However, the addition of bicelles opens NLPs and transfers their contents to bicelles, which freely exchange lipids and proteins. NLP-bicelle interactions may provide a new method for studying membrane protein oligomerization. The interaction mechanism was investigated by stopped flow fluorometry. NLPs with lipids having fluorescence resonance energy transfer (FRET) donors and acceptors were mixed with a 200-fold molar excess of dihexanoyl phosphatidylcholine (DHPC)/dimyristoyl phosphatidylcholine (DMPC) bicelles, and the rate of lipid transfer was monitored by the disappearance of FRET. Near or below the DMPC phase transition temperature, the kinetics were sigmoidal. Free DHPC and apolipoprotein were ruled out as participants in autocatalytic mechanisms. The NLP-bicelle mixing rate showed a strong temperature dependence (activation energy = 28 kcal/mol). Models are proposed for the NLP-bicelle mixing, including one involving fusion pores.

Structure and distribution of antennal sensilla of the red imported fire ant.

The morphology of the antenna of the red imported fire ant, Solenopsis invicta, was examined by light microscopy, scanning electron microscopy, and transmission electron microscopy. The antennae are sexually dimorphic: the worker antenna has porous sensilla on the two distal segments (the antennal club), whereas the clubless male antenna has porous sensilla on all segments past the pedicel. The major type of porous sensilla on both male and female is sensilla tricodea curvata. However, the male s. tricodea curvata are rather uniform in size, whereas the female s. tricodea curvata vary considerably in thickness. The number of sensilla on the distal segment of the worker antenna increases with segment length. This suggests a possible mechanism by which task assignments in S. invicta could be determined by the presence or absence of sensilla sensitive to specific task-related odor or pheromone cues. The sensilla basiconica have an invariant spatial pattern on worker and queen antennae.

Proteomic insights into the protective mechanisms of an in vitro oxidative stress model of early stage Parkinson's disease.

Previous studies in Parkinson's disease (PD) models suggest that early events along the path to neurodegeneration involve activation of the ubiquitin-proteasome system (UPS), endoplasmic reticulum-associated degradation (ERAD), and the unfolded protein response (UPR) pathways, in both the sporadic and familial forms of the disease, and thus ER stress may be a common feature. Furthermore, impairments in protein degradation have been linked to oxidative stress as well as pathways associated with ER stress. We hypothesize that oxidative stress is a primary initiator in a multi-factorial cascade driving dopaminergic (DA) neurons towards death in the early stages of the disease. We now report results from proteomic analysis of a rotenone-induced oxidative stress model of PD in the human neuroblastoma cell line, SH-SY5Y. Cells were exposed to sub-micromolar concentrations of rotenone for 48h prior to whole cell protein extraction and shotgun proteomic analysis. Evidence for activation of the UPR comes from our observation of up-regulated binding immunoglobulin protein (BiP), heat shock proteins, and foldases. We also observed up-regulation of proteins that contribute to the degradation of misfolded or unfolded proteins controlled by the UPS and ERAD pathways. Activation of the UPR may allow neurons to maintain protein homeostasis in the cytosol and ER despite an increase in reactive oxygen species due to oxidative stress, and activation of the UPS and ERAD may further augment clean-up and quality control in the cell.

Interaction of a two-transmembrane-helix peptide with lipid bilayers and dodecyl sulfate micelles.

To probe structural changes that occur when a membrane protein is transferred from lipid bilayers to SDS micelles, a fragment of bacteriorhodopsin containing transmembrane helical segments A and B was studied by fluorescence spectroscopy, molecular dynamics (MD) simulation, and stopped flow kinetics. In lipid bilayers, Förster resonance energy transfer (FRET) was observed between tyrosine 57 on helix B and tryptophans 10 and 12 on helix A. FRET efficiency decreased substantially when the peptide was transferred to SDS. MD simulation showed no evidence for significant disruption of helix-helix interactions in SDS micelles. However, a cluster of water molecules was observed to form a hydrogen-bonded network with the phenolic hydroxyl group of tyrosine 57, which probably causes the disappearance of tyrosine-to-tryptophan FRET in SDS. The tryptophan quantum yield decreased in SDS, and the change occurred at nearly the same rate as membrane solubilization. The results provide a clear example of the importance of corroborating distance changes inferred from FRET by using complementary methods.

Neuronal projections from the Haller's organ and palp sensilla to the synganglion of Amblyomma americanum§ / Projeções neuronais do órgão de Haller e sensila palpal até o singânglio de Amblyomma americanum

Abstract The present study was conducted to elucidate the neuronal pathways between peripheral olfactory and taste sensilla and the synganglion in an Ixodidae tick species. The tarsus of the front legs (olfactory nerves) and the fourth palpal segment (gustatory nerves) of unfed Amblyomma americanum males and females were excised. A neuronal tracer, dextran tetramethylrhodamine, was used for filling of the sensory neurons. The synganglion preparations were examined using a confocal microscope. Neuronal arborizations from the Haller’s organ were confined to the olfactory lobes and the first pedal ganglion. The estimated number of olfactory glomeruli ranged from 16 to 22 per olfactory lobe in the females. The number of glomeruli was not counted in males because they were densely packed. Sensory neurons associated with sensilla at the distal end of the palpal organ projected into the palpal ganglion in the synganglion through the palpal nerve. Gustatory sensory neurons associated with palpal sensilla projected into a commissure with several bulges, which are confined in the palpal ganglion. The findings of distinct projection patterns of sensory neurons associated with the Haller’s organ and palpal organ in the lone star tick from this study advanced our knowledge on mechanisms of sensory information processing in ticks.

Resumo O presente estudo foi conduzido para elucidar a trajetória neuronal, entre as sensilas periféricas olfativas e gustativas e o singânglio, em uma espécie de carrapato Ixodidae. O tarso da primeira pata (nervos olfativos) e o quarto segmento palpal (nervos gustativos) de machos e fêmeas não alimentados de Amblyomma americanum foram excisados. Um traçador neuronal, dextran tetrametilrodamina, foi usado para preenchimento dos neurônios sensoriais. Os singânglios foram examinados através de microscopia confocal. Arborizações neuronais do órgão de Haller foram confinadas nos lobos olfativos e primeiro gânglio pedal. O número estimado de glomérulos olfativos variou de 16 a 22 por lobo olfativo nas fêmeas. Em machos, o número de glomérulos não foi contado, pois eles estavam densamente compactados. Os neurônios sensoriais associados com as sensilas, na porção distal do órgão palpal, projetaram-se no gânglio palpal do singânglio através do nervo palpal. Neurônios sensoriais gustativos associados com a sensila palpal projetaram-se numa comissura onde havia vários bulbos. Os resultados obtidos neste estudo de padrões de projeção distintos de neurônios sensoriais associados com os órgãos de Haller e palpal no carrapato A. americanum avançam nosso conhecimento sobre os mecanismos de processamento da informação sensorial em carrapatos.

Occurrence of antennal glands in ants.

A previous report of the discovery of exocrine glands in the antennal club of queens and workers of Solenopsis invicta Buren, 1972 left open the question of the extent to which similar glands occur in the Formicidae family. We wanted to know if these antennal glands are unique to Solenopsis, or they are found in a wider taxonomic group. Using scanning electron microscopy, we examined the antennae of 41 ant species. Presence of the antennal glands was indicated by a characteristic circumferential ring of pores in a distal antennal segment of workers. Pores were found in the 9th antennal segment of all 26 species of Solenopsis examined. Pores were absent in the following: Monomorium minimum, M. pharaonis, Pheidole sp., Crematogaster sp., Linepithema humile, Forelius sp., Dorymyrmex sp., Paratrechina sp., Oecophylla smaragdina, Campanotus sp., Ectatomma ruidum, E. tuberlatum, and Pseudomyrmex ferruginea. However, pores were found in the antennal club of Tetramorium bicarinatum workers and queens. After KOH digestion of T. bicarinatum antennae, internal canals were observed in both workers and queens, and the canals are connected to spherical reservoirs in queens. T. bicarinatum was the only non-Solenopsis species examined, which showed evidence for antennal glands in the distal funiculus.

An unfolding story of helical transmembrane proteins.

Reversible unfolding of helical transmembrane proteins could provide valuable information about the free energy of interaction between transmembrane helices. Thermal unfolding experiments suggest that this process for integral membrane proteins is irreversible. Chemical unfolding has been accomplished with organic acids, but the unfolding or refolding pathways involve irreversible steps. Sodium dodecyl sulfate (SDS) has been used as a perturbant to study reversible unfolding and refolding kinetics. However, the interpretation of these experiments is not straightforward. It is shown that the results could be explained by SDS binding without substantial unfolding. Furthermore, the SDS-perturbed state is unlikely to include all of the entropy terms involved in an unfolding process. Alternative directions for future research are suggested: fluorinated alcohols in homogeneous solvent systems, inverse micelles, and fragment association studies.