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Mechanotransduction is the process by which a mechanical force, such as touch, is converted into an electrical signal. Transmembrane channel-like (TMC) proteins are an evolutionarily conserved family of membrane proteins whose function has been linked to a variety of mechanosensory processes, including hearing and balance sensation in vertebrates and locomotion in Drosophila. TMC1 and TMC2 are components of ion channel complexes, but the molecular features that tune these complexes to diverse mechanical stimuli are unknown. Caenorhabditis elegans express two TMC homologs, TMC-1 and TMC-2, both of which are the likely pore-forming subunits of mechanosensitive ion channels but differ in their expression pattern and functional role in the worm. Here, we present the single-particle cryo-electron microscopy structure of the native TMC-2 complex isolated from C. elegans. The complex is composed of two copies of the pore-forming TMC-2 subunit, the calcium and integrin binding protein CALM-1 and the transmembrane inner ear protein TMIE. Comparison of the TMC-2 complex to the recently published cryo-EM structure of the C. elegans TMC-1 complex highlights conserved protein-lipid interactions, as well as a p-helical structural motif in the pore-forming helices, that together suggest a mechanism for TMC-mediated mechanosensory transduction. [ABSTRACT FROM AUTHOR]

Many receptors signal upon phosphorylation of tyrosine-based motifs in their cytosolic tail, with intrinsic disorder as a common feature. Studies on CD3ζ and CD3e tails, which are disordered and polybasic, suggested regulation of phosphorylation through accessibility of tyrosines, governed by electrostatic interactions with membrane anionic lipids. We noticed characteristics of intrinsic disorder and previously unappreciated features in tyrosine-based motif-bearing cytosolic tails of many, especially, inhibitory receptors. They are neutral or acidic polyampholytes, with acidic and basic residues linearly segregated. To explore roles of these electrostatic features, we studied inhibitory killer-cell immunoglobulin-like receptor (KIR). Its cytosolic tail is a disordered neutrally charged polyampholyte, wherein juxtamembrane and membrane distal stretches are basic, and the intervening stretch is acidic. Despite lacking net charge, it interacted electrostatically with the plasma membrane. The juxtamembrane stretch was crucial for overall binding, which sequestered tyrosines in the lipid bilayer and restrained their constitutive phosphorylation. Human leukocyte antigen-C ligand binding to KIR released its tail from the plasma membrane to initiate signaling. Tail release occurred independently of KIR polymerization, clustering, or tyrosine phosphorylation, but required acidic residues of the acidic stretch. Tail interaction with the plasma membrane dictated signaling strength of KIR. These results revealed an electrostatic protein-lipid interaction that is unusual in being governed by segregated clusters of acidic and basic residues in polyampholytic disordered region of protein. In contrast to previously known, segregated distribution of oppositely charged residues made both binding and unbinding modules inherent to receptor tail, which could make the interaction an independent signaling switch. [ABSTRACT FROM AUTHOR]

Caveolae are small plasma membrane invaginations, important for control of membrane tension, signaling cascades, and lipid sorting. The caveola coat protein Cavin1 is essential for shaping such high curvature membrane structures. Yet, a mechanistic understanding of how Cavin1 assembles at the membrane interface is lacking. Here, we used model membranes combined with biophysical dissection and computational modeling to show that Cavin1 inserts into membranes. We establish that initial phosphatidylinositol (4, 5) bisphosphate [PI(4,5)P2]-dependent membrane adsorption of the trimeric helical region 1 (HR1) of Cavin1 mediates the subsequent partial separation and membrane insertion of the individual helices. Insertion kinetics of HR1 is further enhanced by the presence of flanking negatively charged disordered regions, which was found important for the coassembly of Cavin1 with Caveolin1 in living cells. We propose that this intricate mechanism potentiates membrane curvature generation and facilitates dynamic rounds of assembly and disassembly of Cavin1 at the membrane. [ABSTRACT FROM AUTHOR]

Cholesterol decreases the activity of the majority of ion channels while increasing the activity of only a few, yet it remains unclear how. Here, we used the inwardly rectifying potassium channel Kir3.4, which is up-regulated by cholesterol, as a tool to address this question. Employing mutagenesis and electrophysiology, we discovered a molecular switch that controls the impact of cholesterol on the channel. Through a single point mutation at position 182 in the transmembrane domain of Kir3.4, we converted the cholesterol-driven up-regulation of the channel into down-regulation. Microseconds- long coarse-grained and atomistic molecular dynamics simulations revealed that the effect of the point mutation propagated toward the selectivity filter of the channel whose conformation controls the conductance of the channel. Planar lipid bilayer experiments validated these results, showing that although cholesterol up-regulated Kir3.4 by increasing its open probability, cholesterol down-regulated the mutant by decreasing its conductance. Further studies underscored the role of mutation-specific alterations of cholesterol distribution in proximity to the channel in cholesterol's impact on channel activity, highlighting the role of subtle molecular differences in determining how cholesterol distributes around proteins and affects their function. [ABSTRACT FROM AUTHOR]

Sarcoplasmic reticulum (SR) Ca2+-ATPase transports two Ca2+ ions from the cytoplasm to the SR lumen against a large concentration gradient. X-ray crystallography has revealed the atomic structures of the protein before and after the dissociation of Ca2+, while biochemical studies have suggested the existence of intermediate states in the transition between E1P·ADP·2Ca2+ and E2P. Here, we explore the pathway and free energy profile of the transition using atomistic molecular dynamics simulations with the mean-force string method and umbrella sampling. The simulations suggest that a series of structural changes accompany the ordered dissociation of ADP, the A-domain rotation, and the rearrangement of the transmembrane (TM) helices. The luminal gate then opens to release Ca2+ ions toward the SR lumen. Intermediate structures on the pathway are stabilized by transient sidechain interactions between the A- and P-domains. Lipid molecules between TM helices play a key role in the stabilization. Free energy profiles of the transition assuming different protonation states suggest rapid exchanges between Ca2+ ions and protons when the Ca2+ ions are released toward the SR lumen. [ABSTRACT FROM AUTHOR]

Regulation of apoptosis is tightly linked with the targeting of numerous Bcl-2 proteins to the mitochondrial outer membrane (MOM), where their activation or inhibition dictates cell death or survival. According to the traditional view of apoptotic regulation, BH3-effector proteins are indispensable for the cytosol-to-MOM targeting and activation of proapoptotic and antiapoptotic members of the Bcl-2 protein family. This view is challenged by recent studies showing that these processes can occur in cells lacking BH3 effectors by as yet to be determined mechanism(s). Here, we exploit a model membrane system that recapitulates key features of MOM to demonstrate that the proapoptotic Bcl-2 protein BAX and antiapoptotic Bcl-xL have an inherent ability to interact with membranes in the absence of BH3 effectors, but only in the presence of cellular concentrations of Mg2+/Ca2+. Under these conditions, BAX and Bcl-xL are selectively targeted to membranes, refolded, and activated in the presence of anionic lipids especially the mitochondrial-specific lipid cardiolipin. These results provide a mechanistic explanation for the mitochondrial targeting and activation of Bcl-2 proteins in cells lacking BH3 effectors. At cytosolicMg2+ levels, the BH3-independent activation of BAX could provide localized amplification of apoptotic signaling at regions enriched in cardiolipin (e.g., contact sites between MOM and mitochondrial inner membrane). Increases in MOM cardiolipin, as well as cytosolic [Ca2+] during apoptosis could further contribute to its MOM targeting and activity. Meanwhile, the BH3-independent targeting and activation of Bcl-xL to the MOM is expected to counter the action of proapoptotic BAX, thereby preventing premature commitment to apoptosis. [ABSTRACT FROM AUTHOR]

The mechanosensitive channel of small conductance (MscS) protects bacteria against hypoosmotic shock. It can sense the tension in the surrounding membrane and releases solutes if the pressure in the cell is getting too high. The membrane contacts MscS at sensor paddles, but lipids also leave the membrane and move along grooves between the paddles to reside as far as 15 Å away from the membrane in hydrophobic pockets. One sensing model suggests that a higher tension pulls lipids from the grooves back to the membrane, which triggers gating. However, it is still unclear to what degree this model accounts for sensing and what contribution the direct interaction of the membrane with the channel has. Here, we show that MscS opens when it is sufficiently delipidated by incubation with the detergent dodecyl-β-maltoside or the branched detergent lauryl maltose neopentyl glycol. After addition of detergent-solubilized lipids, it closes again. These results support the model that lipid extrusion causes gating: Lipids are slowly removed from the grooves and pockets by the incubation with detergent, which triggers opening. Addition of lipids in micelles allows lipids to migrate back into the pockets, which closes the channel even in the absence of a membrane. Based on the distribution of the aliphatic chains in the open and closed conformation, we propose that during gating, lipids leave the complex on the cytosolic leaflet at the height of highest lateral tension, while on the periplasmic side, lipids flow into gaps, which open between transmembrane helices. [ABSTRACT FROM AUTHOR]

The cytoskeletal protein actin polymerizes into filaments that are essential for the mechanical stability of mammalian cells. In vitro experiments showed that direct interactions between actin filaments and lipid bilayers are possible and that the net charge of the bilayer as well as the presence of divalent ions in the buffer play an important role. In vivo, colocalization of actin filaments and divalent ions are suppressed, and cells rely on linker proteins to connect the plasma membrane to the actin network. Little is known, however, about why this is the case and what microscopic interactions are important. A deeper understanding is highly beneficial, first, to obtain understanding in the biological design of cells and, second, as a possible basis for the building of artificial cortices for the stabilization of synthetic cells. Here, we report the results of coarse-grained molecular dynamics simulations of monomeric and filamentous actin in the vicinity of differently charged lipid bilayers. We observe that charges on the lipid head groups strongly determine the ability of actin to adsorb to the bilayer. The inclusion of divalent ions leads to a reversal of the binding affinity. Our in silico results are validated experimentally by reconstitution assays with actin on lipid bilayer membranes and provide a molecular-level understanding of the actin-membrane interaction. [ABSTRACT FROM AUTHOR]

α-Helical membrane proteins have eluded investigation of their thermodynamic stability in lipid bilayers. Reversible denaturation curves have enabled some headway in determining unfolding free energies. However, these parameters have been limited to detergent micelles or lipid bicelles, which do not possess the same mechanical properties as lipid bilayers that comprise the basis of natural membranes. We establish reversible unfolding of the membrane transporter LeuT in lipid bilayers, enabling the comparison of apparent unfolding free energies in different lipid compositions. LeuT is a bacterial ortholog of neurotransmitter transporters and contains a knot within its 12-transmembrane helical structure. Urea is used as a denaturant for LeuT in proteoliposomes, resulting in the loss of up to 30% helical structure depending upon the lipid bilayer composition. Urea unfolding of LeuT in liposomes is reversible, with refolding in the bilayer recovering the original helical structure and transport activity. A linear dependence of the unfolding free energy on urea concentration enables the free energy to be extrapolated to zero denaturant. Increasing lipid headgroup charge or chain lateral pressure increases the thermodynamic stability of LeuT. The mechanical and charge properties of the bilayer also affect the ability of urea to denature the protein. Thus, we not only gain insight to the long-sought-after thermodynamic stability of an α-helical protein in a lipid bilayer but also provide a basis for studies of the folding of knotted proteins in a membrane environment. [ABSTRACT FROM AUTHOR]

The actin cytoskeleton powers membrane deformation during many cellular processes, such as migration, morphogenesis, and endocytosis. Membrane phosphoinositides, especially phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], regulate the activities of many actinbinding proteins (ABPs), including profilin, cofilin, Dia2, N-WASP, ezrin, and moesin, but the underlying molecular mechanisms have remained elusive. Moreover, because of a lack of available methodology, the dynamics of membrane interactions have not been experimentally determined for any ABP. Here, we applied a combination of biochemical assays, photobleaching/activation approaches, and atomistic molecular dynamics simulations to uncover the molecular principles by which ABPs interact with phosphoinositide-rich membranes. We show that, despite using different domains for lipid binding, these proteins associate with membranes through similar multivalent electrostatic interactions, without specific binding pockets or penetration into the lipid bilayer. Strikingly, our experiments reveal that these proteins display enormous differences in the dynamics of membrane interactions and in the ranges of phosphoinositide densities that they sense. Profilin and cofilin display transient, low-affinity interactions with phosphoinositide-rich membranes, whereas F-actin assembly factors Dia2 and N-WASP reside on phosphoinositide-richmembranes for longer periods to performtheir functions. Ezrin and moesin, which link the actin cytoskeleton to the plasma membrane, bindmembranes with very high affinity and slow dissociation dynamics. Unlike profilin, cofilin, Dia2, and N-WASP, they do not require high "stimulus-responsive" phosphoinositide density for membrane binding. Moreover, ezrin can limit the lateral diffusion of PI(4,5)P2 along the lipid bilayer. Together, these findings demonstrate that membrane-interaction mechanisms of ABPs evolved to precisely fulfill their specific functions in cytoskeletal dynamics. [ABSTRACT FROM AUTHOR]

Relative to the apolipoprotein E (apoE) E3 allele of the APOE gene, apoE4 strongly increases the risk for the development of late-onset Alzheimer's disease. However, apoE4 differs from apoE3 by only a single amino acid at position 112, which is arginine in apoE4 and cysteine in apoE3. It remains unclear why apoE3 and apoE4 are functionally different. Described here is a proposal for understanding the functional differences between these two isoforms with respect to lipid binding. A mechanism is proposed that is based on the full-length monomeric structure of the protein, on hydrogen-deuterium exchange mass spectrometry data, and on the role of intrinsically disordered regions to control protein motions. It is proposed that lipid binds between the N-terminal and C-terminal domains and that separation of the two domains, along with the presence of intrinsically disordered regions, controls this process. The mechanism explains why apoE3 differs from apoE4 with respect to different lipid-binding specificities, why lipid increases the binding of apoE to its receptor, and why specific residues are conserved. [ABSTRACT FROM AUTHOR]

Dimeric tubulin, an abundant water-soluble cytosolic protein known primarily for its role in the cytoskeleton, is routinely found to be associated with mitochondrial outer membranes, although the structure and physiological role of mitochondria-bound tubulin are still unknown. There is also no consensus on whether tubulin is a peripheral membrane protein or is integrated into the outer mitochondrial membrane. Here the results of five independent techniques--surface plasmon resonance, electrochemical impedance spectroscopy, bilayer overtone analysis, neutron reflectometry, and molecular dynamics simulations--suggest that α-tubulin's amphipathic helix H10 is responsible for peripheral binding of dimeric tubulin to biomimetic "mitochondrial" membranes in a manner that differentiates between the two primary lipid headgroups found in mitochondrial membranes, phosphatidylethanolamine and phosphatidylcholine. The identification of the tubulin dimer orientation and membrane-binding domain represents an essential step toward our understanding of the complex mechanisms by which tubulin interacts with integral proteins of the mitochondrial outer membrane and is important for the structure-inspired design of tubulin-targeting agents. [ABSTRACT FROM AUTHOR]

Lipid-mediated signaling events regulate many cellular processes. Investigations of the complex underlying mechanisms are difficult because several different methods need to be used under varying conditions. Here we introduce multifunctional lipid derivatives to study lipid metabolism, lipid−protein interactions, and intracellular lipid localization with a single tool per target lipid. The probes are equipped with two photoreactive groups to allow photoliberation (uncaging) and photo–cross-linking in a sequential manner, as well as a click-handle for subsequent functionalization. We demonstrate the versatility of the design for the signaling lipids sphingosine and diacylglycerol; uncaging of the probe for these two species triggered calcium signaling and intracellular protein translocation events, respectively. We performed proteomic screens to map the lipid-interacting proteome for both lipids. Finally, we visualized a sphingosine transport deficiency in patient-derived Niemann−Pick disease type C fibroblasts by fluorescence as well as correlative light and electron microscopy, pointing toward the diagnostic potential of such tools. We envision that this type of probe will become important for analyzing and ultimately understanding lipid signaling events in a comprehensive manner. [ABSTRACT FROM AUTHOR]

A fundamental objective in membrane biology is to understand and predict how a protein sequence folds and orients in a lipid bilayer. Establishing the principles governing membrane protein folding is central to understanding the molecular basis formembrane proteins that display multiple topologies, the intrinsic dynamic organization of membrane proteins, and membrane protein conformational disorders resulting in disease. We previously established that lactose permease of Escherichia coli displays a mixture of topological conformations and undergoes postassembly bidirectional changes in orientation within the lipid bilayer triggered by a change in membrane phosphatidylethanolamine content, both in vivo and in vitro. However, the physiological implications and mechanism of dynamic structural reorganization of membrane proteins due to changes in lipid environment are limited by the lack of approaches addressing the kinetic parameters of transmembrane protein flipping. In this study, real-time fluorescence spectroscopy was used to determine the rates of protein flipping in the lipid bilayer in both directions and transbilayer flipping of lipids triggered by a change in proteoliposome lipid composition. Our results provide, for the first time to our knowledge, a dynamic picture of these events and demonstrate that membrane protein topological rearrangements in response to lipid modulations occur rapidly following a threshold change in proteoliposome lipid composition. Protein flipping was not accompanied by extensive lipid-dependent unfolding of transmembrane domains. Establishment of lipid bilayer asymmetry was not required but may accelerate the rate of protein flipping. Membrane protein flipping was found to accelerate the rate of transbilayer flipping of lipids. [ABSTRACT FROM AUTHOR]

A cellular feature of Parkinson's disease is cytosolic accumulation and amyloid formation of α-synuclein (α-syn), implicating a misregulation or impairment of protein degradation pathways involving the proteasome and lysosome. Within lysosomes, cathepsin D (CtsD), an aspartyl protease, is suggested to be the main protease for α-syn clearance; however, the protease alone only generates amyloidogenic C terminal-truncated species (e.g., 1-94, 5-94), implying that other proteases and/or environmental factors are needed to facilitate degradation and to avoid α-syn aggregation in vivo. Using liquid chromatography--mass spectrometry, to our knowledge, we report the first peptide cleavage map of the lysosomal degradation process of α-syn. Studies of purified mouse brain and liver lysosomal extracts and individual human cathepsins demonstrate a direct involvement of cysteine cathepsin B (CtsB) and L (CtsL). Both CtsB and CtsL cleave α-syn within its amyloid region and circumvent fibril formation. For CtsD, only in the presence of anionic phospholipids can this protease cleave throughout the α-syn sequence, suggesting that phospholipids are crucial for its activity. Taken together, an interplay exists between α-syn conformation and cathepsin activity with CtsL as the most efficient under the conditions examined. Notably, we discovered that CtsL efficiently degrades α-syn amyloid fibrils, which by definition are resistant to broad spectrum proteases. This work implicates CtsB and CtsL as essential in α-syn lysosomal degradation, establishing groundwork to explore mechanisms to enhance their cellular activity and levels as a potential strategy for clearance of α-syn. [ABSTRACT FROM AUTHOR]

DesK is a bacterial thermosensor protein involved in maintaining membrane fluidity in response to changes in environmental temperature. Most likely, the protein is activated by changes in membrane thickness, but the molecular mechanism of sensing and signaling is still poorly understood. Here we aimed to elucidate the mode of action of DesK by studying the so-called "minimal sensor DesK" (MS-DesK), in which sensing and signaling are captured in a single transmembrane segment. This simplified version of the sensor allows investigation of membrane thickness-dependent protein-lipid interactions simply by using synthetic peptides, corresponding to the membrane-spanning parts of functional and nonfunctional mutants of MS-DesK incorporated in lipid bilayers with varying thicknesses. The lipid-dependent behavior of the peptides was investigated by circular dichroism, tryptophan fluorescence, and molecular modeling. These experiments were complemented with in vivo functional studies on MS-DesK mutants. Based on the results, we constructed a model that suggests a new mechanism for sensing in which the protein is present as a dimer and responds to an increase in bilayer thickness by membrane incorporation of a C-terminal hydrophilic motif. This results in exposure of three serines on the same side of the transmembrane helices of MS-DesK, triggering a switching of the dimerization interface to allow the formation of a serine zipper. The final result is activation of the kinase state of MS-DesK. [ABSTRACT FROM AUTHOR]

Lipid-protein interactions play pivotal roles in biological membranes. Electron crystallography studies of the lens-specific water channel aquaporin-0 (AQP0) revealed atomistic views of such interactions, by providing high-resolution structures of annular lipids surrounding AQPO. It remained unclear, however, whether these lipid structures are representative of the positions of unconstrained lipids surrounding an individual protein, and what molecular determinants define the lipid positions around AQPO. We addressed these questions by using molecular dynamics simulations and crystallographic refinement, and calculated time-averaged densities of dimyristoyl-phosphatidylcholine lipids around AQPO. Our simulations demonstrate that, although the experimentally determined crystallographic lipid positions are constrained by the crystal packing, they appropriately describe the behavior of unconstrained lipids around an individual AQPO tetramer, and thus likely represent physiologically relevant lipid positions. While the acyl chains were well localized, the lipid head groups were not. Furthermore, in silico mutations showed that electrostatic inter actions do not play a major role attracting these phospholipids towards AQPO. Instead, the mobility of the protein crucially modulates the lipid localization and explains the difference in lipid density between extracellular and cytoplasmic leaflets. Moreover, our simulations support a general mechanism in which membrane proteins laterally diffuse accompanied by several layers of localized lipids, with the positions of the annular lipids being influenced the most by the protein surface. We conclude that the acyl chains rather than the head groups define the positions of dimyristoylphosphatidylcholine lipids around AQPO. Lipid localization is largely determined by the mobility of the protein surface, whereas hydrogen bonds play an important but secondary role. [ABSTRACT FROM AUTHOR]

Transferring lipid antigens from membranes into CD1 antigen-presenting proteins represents a major molecular hurdle necessary for T-cell recognition. Saposins facilitate this process, but the mechanisms used are not well understood. We found that saposin B forms soluble saposin protein-lipid complexes detected by native gel electrophoresis that can directly load CD1 proteins. Because saposin B must bind lipids directly to function, we found it could not accommodate long acyl chain containing lipids. In contrast, saposin C facilitates CD1 lipid loading in a different way. It uses a stable, membrane-associated topology and was capable of loading lipid antigens without forming soluble saposin-lipid antigen complexes. These findings reveal how saposins use different strategies to facilitate transfer of structurally diverse lipid antigens. [ABSTRACT FROM AUTHOR]

The saposins are small, membrane-active proteins that exist in both soluble and lipid-bound states. Saposin A has roles in sphingolipid catabolism and transport and is required for the breakdown of galactosylceramide by β-galactosylceramidase. In the absence of lipid, saposin A adopts a closed monomeric apo conformation typical of this family. To study a lipid-bound state of this protein, we determined the crystal structure of saposin A in the presence of detergent to 1.9 Å resolution. The structure reveals two chains of saposin A in an open conformation encapsulating 40 internally bound detergent molecules organized in a highly ordered bilayer-like hydrophobic core. The complex provides a high-resolution view of a discoidal lipoprotein particle in which all of the internalized acyl chains are resolved. Saposin A lipoprotein discs exhibit limited selectivity with respect to the incorporated lipid, and can solubilize phospholipids, sphingolipids, and cholesterol into discrete, monodisperse particles with mass of approximately 27 kDa. These discs may be the smallest possible lipoprotein structures that are stabilized by lipid self-assembly. [ABSTRACT FROM AUTHOR]

The voltage-dependent anion channel (VDAC), located in the outer mitochondrial membrane, acts as a gatekeeper for the entry and exit of mitochondrial metabolites. Here we reveal functional dynamics of isoform one of VDAC (VDAC1) by a combination of solution NMR spectroscopy. Gaussian network model analysis, and molecular dynamics simulation. Micro- to millisecond dynamics are significantly increased for the N-terminal six β-strands of VDAC1 in micellar solution, in agreement with increased B-factors observed in the same region in the bicellar crystal structure of VDAC1. Molecular dynamics simulations reveal that a charge on the membrane-facing glutamic acid 73 (E73) accounts for the elevation of N-terminal protein dynamics as well as a thinning of the nearby membrane. Mutation or chemical modification of E73 strongly reduces the micro- to millisecond dynamics in solution. Because E73 is necessary for hexokinase-l-induced VDAC channel closure and inhibition of apoptosis, our results imply that micro- to millisecond dynamics in the N-terminal part of the barrel are essential for VDAC interaction and gating. [ABSTRACT FROM AUTHOR]

Rhodopsin, the G protein-coupled receptor primarily responsible for sensing light, is found in an environment rich in polyunsaturated lipid chains and cholesterol. Biophysical experiments have shown that lipid unsaturation and cholesterol both have significant effects on rhodopsin's stability and function; ω-3 polyunsaturated chains, such as docosahexaenoic acid (DHA), destabilize rhodopsin and enhance the kinetics of the photocycle, whereas cholesterol has the opposite effect. Here, we use molecular dynamics simulations to investigate the possibility that polyunsaturated chains modulate rhodopsin stability and kinetics via specific direct interactions. By analyzing the results of 26 independent 100-ns simulations of dark-adapted rhodopsin, we found that DHA routinely forms tight associations with the protein in a small number of specific locations qualitatively different from the nonspecific interactions made by saturated chains and cholesterol. Furthermore, the presence of tightly packed DHA molecules tends to weaken the interhelical packing. These results are consistent with recent NMR work, which proposes that rhodopsin binds DHA, and they suggest a molecular rationale for DHA's effects on rhodopsin stability and kinetics. [ABSTRACT FROM AUTHOR]