Docosahexaenoyl Chains Isomerize on the Sub-Nanosecond Time Scale

The dynamics of docosahexaenoyl acyl chains (DHA) in 18:0-22:6n3-PC bilayers were studied by 13C MAS NMR relaxation measurements. A Lipari−Szabo-type analysis yielded site-specific correlation times of DHA chain isomerization and C−H bond order parameters. It is concluded that DHA chains perform rapid isomerization with correlation times from 80 ps near the carbonyl group to 8 ps near the terminal methyl group. Spin−lattice relaxation rates remained unaltered after rhodopsin incorporation into the bilayers, indicating that the majority of lipids maintain their rapid chain isomerization in the presence of the protein. However, spin−spin relaxation rates revealed that rhodopsin increased motional correlation times of slow collective DHA motions

Lipid−Rhodopsin Hydrophobic Mismatch Alters Rhodopsin Helical Content

The ability of photoactivated rhodopsin to achieve the enzymatically active metarhodopsin II conformation is exquisitely sensitive to bilayer hydrophobic thickness. The sensitivity of rhodopsin to the lipid matrix has been explained by the hydrophobic matching theory, which predicts that lipid bilayers adjust elastically to the hydrophobic length of transmembrane helices. Here, we examined if bilayer thickness adjusts to the length of the protein or if the protein alters its conformation to adapt to the bilayer. Purified bovine rhodopsin was reconstituted into a series of mono-unsaturated phosphatidylcholines with 14−20 carbons per hydrocarbon chain. Changes of hydrocarbon chain length were measured by 2H NMR, and protein helical content was quantified by synchrotron radiation circular dichroism and conventional circular dichroism. Experiments were conducted on dark-adapted rhodopsin, the photo-intermediates metarhodopsin I/II/III, and opsin. Changes of bilayer thickness upon rhodopsin incorporation and photoactivation were mostly absent. In contrast, the helical content of rhodopsin increased with membrane hydrophobic thickness. Helical content did not change measurably upon photoactivation. The increases of bilayer thickness and helicity of rhodopsin are accompanied by higher metarhodopsin II/metarhodopsin I ratios, faster rates of metarhodopsin II formation, an increase of tryptophan fluorescence, and higher temperatures of rhodopsin denaturation. The data suggest a surprising adaptability of this G protein-coupled membrane receptor to properties of the lipid matrix

Functional Expression and Characterization of Human Myristoylated-Arf1 in Nanodisc Membrane Mimetics

Lipidated small GTP-binding proteins of the Arf family interact with multiple cellular partners and with membranes to regulate intracellular traffic and organelle structure. Here, we focus on the ADP-ribosylation factor 1 (Arf1), which interacts with numerous proteins in the Arf pathway, such as the ArfGAP ASAP1 that is highly expressed and activated in several cancer cell lines and associated with enhanced migration, invasiveness, and poor prognosis. Understanding the molecular and mechanistic details of Arf1 regulation at the membrane via structural and biophysical studies requires large quantities of fully functional protein bound to lipid bilayers. Here, we report on the production of a functional human Arf1 membrane platform on nanodiscs for biophysical studies. Large scale bacterial production of highly pure, N-myristoylated human Arf1 has been achieved, including complex isotopic labeling for nuclear magnetic resonance (NMR) studies, and the myr-Arf1 can be readily assembled in small nanoscale lipid bilayers (nanodiscs, NDs). It is determined that myr-Arf1 requires a minimum binding surface in the NDs of ∼20 lipids. Fluorescence and NMR were used to establish nucleotide exchange and ArfGAP-stimulated GTP hydrolysis at the membrane, indicating that phophoinositide stimulation of the activity of the ArfGAP ASAP1 is ≥2000-fold. Differences in nonhydrolyzable GTP analogues are observed, and GMPPCP is found to be the most stable. Combined, these observations establish a functional environment for biophysical studies of Arf1 effectors and interactions at the membrane

Myr-Arf1 conformational flexibility at the membrane surface: insights for ArfGAP ASAP1 interactions

Experimental and computational data used in an integrated structural biology study of membrane surface complexe

Arf GAP activity using the indicated Arf GAP enzymes and myrArf1•GTP as substrate.

(A) ASAP1 GAP activity using the indicated myrArf1•GTP as substrate. For these assays, the His-tagged ASAP1PZA construct (see top for comparison with full-length [FL] construct) was titrated into a reaction with myrArf1 constructs bound to [α32P]GTP on an LUV surface. After a fixed period of time, the ratio of [α32P]GDP and [α32P]GTP bound to myrArf1 was measured. Data shown are a representative example from multiple experiments. BAR, Bin/Amphiphysin/Rvs domain; PH, Pleckstrin Homology domain; Arf GAP, Arf GAP catalytic domain; Ank, Ankyrin repeats; Pro, Proline rich region; E/DLPPKP, E/DLPPKP repeat region; SH3, Src Homology 3 domain. (B) Summary of ASAP1 GAP activity assays using the indicated myrArf1•GTP as substrate. C50 values (the concentration of ASAP1PZA required to achieve 50% of maximum GTP hydrolysis) from each independent experiment are shown. Error bars represent standard deviation. ns, not significant; ****, p 10 transformed data in order to satisfy the assumption of equal variances, and the significance using transformed data is displayed. (C) Summary of GAP activity assays of His-tagged proteins ASAP1PZA, full-length (FL) AGAP1, and ARAP1PPZA using WT or [F13A]myrArf1•GTP as substrates. Assays were conducted as described in (A). Error bars represent standard deviation. ****, p 10 transformed data in order to satisfy the assumption of equal variances, and the significance using transformed data is displayed. GLD, GTP-binding protein-like domain; SAM, sterile-α motif; Rho GAP, Rho GAP catalytic domain; RA, Ras-associating domain. All other protein regions are as described in (A).</p

Theoretical mechanistic models for nucleotide and membrane cycling of myrArf proteins.

(A) Mechanistic model for Arf GEF-mediated exchange of GDP for GTP. In step 1, the Arf GEF first binds to myrArf•GDP. At this stage, the N-terminal extension is disordered, and the myristate moiety is localized within the G domain hydrophobic cavity. In step 2, GDP dissociates from the G domain, which is now stabilized by the Arf GEF. Movement of the interswitch region causes the hydrophobic cavity to shrink, thereby ejecting the N-terminal extension and myristate and allowing them to associate with the membrane. In step 3, the N-terminal extension folds into an alpha helix and GTP associates with myrArf, causing the Arf GEF to dissociate. At the end of the reaction, myrArf•GTP is bound to the membrane via the N-terminal extension hydrophobic amino acids and the myristate. (B) Mechanistic model for Arf GAP-mediated degradation of GTP to GDP and inorganic phosphate. In step 1, the Arf GAP first binds to myrArf•GTP. At this stage, the N-terminal extension is associated with the membrane and myristate and is folded into an alpha helix. In step 2, interactions between the Arf GAP (e.g., through a PH domain) and the myristoylated N-terminal extension likely help to stabilize the removal of the N-terminus from the membrane. The Arf GAP also catalyzes the degradation of GTP to GDP and inorganic phosphate (the latter shown as a yellow circle with the letter "P"). Inorganic phosphate dissociates from the myrArf•GDP:Arf GAP complex. In step 3, movement of the interswitch region causes the hydrophobic cavity in the G domain to reopen, allowing the N-terminal extension to become disordered and for the myristate to enter the cavity. Finally, myrArf•GDP dissociates from the Arf GAP and the membrane.</p

S1 File -

The ADP-ribosylation factors (Arfs) constitute a family of small GTPases within the Ras superfamily, with a distinguishing structural feature of a hypervariable N-terminal extension of the G domain modified with myristate. Arf proteins, including Arf1, have roles in membrane trafficking and cytoskeletal dynamics. While screening for Arf1:small molecule co-crystals, we serendipitously solved the crystal structure of the non-myristoylated engineered mutation [L8K]Arf1 in complex with a GDP analogue. Like wild-type (WT) non-myristoylated Arf1•GDP, we observed that [L8K]Arf1 exhibited an N-terminal helix that occludes the hydrophobic cavity that is occupied by the myristoyl group in the GDP-bound state of the native protein. However, the helices were offset from one another due to the L8K mutation, with a significant change in position of the hinge region connecting the N-terminus to the G domain. Hypothesizing that the observed effects on behavior of the N-terminus affects interaction with regulatory proteins, we mutated two hydrophobic residues to examine the role of the N-terminal extension for interaction with guanine nucleotide exchange factors (GEFs) and GTPase Activating Proteins (GAPs. Different than previous studies, all mutations were examined in the context of myristoylated Arf. Mutations had little or no effect on spontaneous or GEF-catalyzed guanine nucleotide exchange but did affect interaction with GAPs. [F13A]myrArf1 was less than 1/2500, 1/1500, and 1/200 efficient as substrate for the GAPs ASAP1, ARAP1 and AGAP1; however, [L8A/F13A]myrArf1 was similar to WT myrArf1. Using molecular dynamics simulations, the effect of the mutations on forming alpha helices adjacent to a membrane surface was examined, yet no differences were detected. The results indicate that lipid modifications of GTPases and consequent anchoring to a membrane influences protein function beyond simple membrane localization. Hypothetical mechanisms are discussed.</div

Statistics of molecular dynamics simulations of myrArf1 N-terminal extensions.

Statistics of molecular dynamics simulations of myrArf1 N-terminal extensions.</p

Positioning and conservation of residues L8 and F13 in Arf N-terminal extensions.

(A) NMR structure of the N-terminal extension of S. cerevisiae WT myrArf1•GTP bound to bicelles (PDB: 2KSQ [17]). Note that all NMR states of the structure are depicted. The rough positioning of the outer layer of the phospholipid membrane is shown as a dashed line, and the myristate moiety, L8, and F13 are labeled and shown as colored sticks (key on top). (B) Carbon-carbon distances between the indicated carbons from the myristoyl moiety and L8 in yeast WT myrArf1•GTP. Distances were obtained from each of the 20 states within the NMR structure. (C) Alignments of the N-terminal extension primary sequences of S. cerevisiae Arf1 as well as human Arf1, Arf3, Arf4, Arf5, and Arf6. Amino acids that are identical are marked with an asterisk, whereas those with low and high similarity are marked with a period or a colon. Colors of amino acids that align with yeast L8 and F13 are the same as in (A). The N-terminal extensions are represented by the horizontal line on top. Swiss-Prot identifiers are shown on the left, and amino acid positions on the right.</p

Spontaneous guanine nucleotide exchange of myrArf1 constructs.

For these assays, 0.5 μM myrArf1 constructs was added to a reaction with low (1–10 μM) Mg2+ to promote exchange, as well as [35S]GTPγS and LUVs. After the indicated period of time, the fraction of myrArf1 bound to [35S]GTPγS was measured. Data shown are a representative example from multiple experiments. (TIF)</p