Analysis of Integrin αIIb Subunit Dynamics Reveals Long-Range Effects of Missense Mutations on Calf Domains.

Integrin αIIbß3, a glycoprotein complex expressed at the platelet surface, is involved in platelet aggregation and contributes to primary haemostasis. Several integrin αIIbß3 polymorphisms prevent the aggregation that causes haemorrhagic syndromes, such as Glanzmann thrombasthenia (GT). Access to 3D structure allows understanding the structural effects of polymorphisms related to GT. In a previous analysis using Molecular Dynamics (MD) simulations of αIIbCalf-1 domain structure, it was observed that GT associated with single amino acid variation affects distant loops, but not the mutated position. In this study, experiments are extended to Calf-1, Thigh, and Calf-2 domains. Two loops in Calf-2 are unstructured and therefore are modelled expertly using biophysical restraints. Surprisingly, MD revealed the presence of rigid zones in these loops. Detailed analysis with structural alphabet, the Proteins Blocks (PBs), allowed observing local changes in highly flexible regions. The variant P741R located at C-terminal of Calf-1 revealed that the Calf-2 presence did not affect the results obtained with isolated Calf-1 domain. Simulations for Calf-1 + Calf-2, and Thigh + Calf-1 variant systems are designed to comprehend the impact of five single amino acid variations in these domains. Distant conformational changes are observed, thus highlighting the potential role of allostery in the structural basis of GT.

An alternate covalent form of platelet αIIbß3 integrin that resides in focal adhesions and has altered function.

The αIIbß3 integrin receptor coordinates platelet adhesion, activation, and mechanosensing in thrombosis and hemostasis. Using differential cysteine alkylation and mass spectrometry, we have identified a disulfide bond in the αIIb subunit linking cysteines 490 and 545 that is missing in ∼1 in 3 integrin molecules on the resting and activated human platelet surface. This alternate covalent form of αIIbß3 is predetermined as it is also produced by human megakaryoblasts and baby hamster kidney fibroblasts transfected with recombinant integrin. From coimmunoprecipitation experiments, the alternate form selectively partitions into focal adhesions on the activated platelet surface. Its function was evaluated in baby hamster kidney fibroblast cells expressing a mutant integrin with an ablated C490-C545 disulfide bond. The disulfide mutant integrin has functional outside-in signaling but extended residency time in focal adhesions due to a reduced rate of clathrin-mediated integrin internalization and recycling, which is associated with enhanced affinity of the αIIb subunit for clathrin adaptor protein 2. Molecular dynamics simulations indicate that the alternate covalent form of αIIb requires higher forces to transition from bent to open conformational states that is in accordance with reduced affinity for fibrinogen and activation by manganese ions. These findings indicate that the αIIbß3 integrin receptor is produced in various covalent forms that have different cell surface distribution and function. The C490, C545 cysteine pair is conserved across all 18 integrin α subunits, and the disulfide bond in the αV and α2 subunits in cultured cells is similarly missing, suggesting that the alternate integrin form and function are also conserved.

Measurement of Integrin Activation and Conformational Changes on the Cell Surface by Soluble Ligand and Antibody Binding Assays.

Soluble ligand and conformation-dependent antibody binding assay of integrins on the cell surface is an effective approach to evaluate the activation status of integrins in live cells. The ligands or antibodies are usually labeled with biotin or a fluorescent dye and incubated with integrin-expressing cells in suspension. The cell-bound ligands and antibodies are then detected by flow cytometry. Here we describe the detailed protocols of soluble ligand or antibody binding assay for αIIbß3, αVß3, α5ß1, and αLß2 integrins that are transiently or stably expressed in the model cell lines such as HEK293 or CHO-k1 cells.

Vincristine-loaded platelets coated with anti-CD41 mAbs: a new macrophage targeting proposal for the treatment of immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune disorder in which platelet-reactive autoantibodies accelerate the destruction of platelets. Macrophages play an important role in ITP through Fc receptor (FcR)-mediated antigen presenting and platelet clearance. In this study, a novel drug delivery system of vincristine-loaded platelets coated with anti-CD41 mAbs (CD41-VCR-PLT, CD41-VLP) was successfully established. The therapeutic effects and safety of CD41-VLP in vitro and in vivo were evaluated, and the possible mechanism was also explored. The results showed that PLT-CD41 could load VCR with high drug loading (DL) and encapsulation efficiency (EE), which were up to 41.16 ± 1.92% and 60.73 ± 2.79%, respectively, where platelets had no obvious morphological or functional changes. CD41-VLP could facilitate vincristine accumulation in macrophages, where the intracellular VCR concentration was 30.72 ± 3.11% at 72 h, which was significantly increased compared with the other groups (P < 0.01), thus inhibiting macrophage cell viability and inducing apoptosis. The cell viability inhibition rate and total apoptosis rate were 73.06 ± 5.26% and 69.70 ± 4.26%, respectively, both much higher than those of the other groups (P < 0.05). In the ITP mouse model, CD41-VLP increased the platelet count in peripheral blood, which was 720 ± 197.98 × 109 L-1, and significantly improved the platelet count compared with that in the VCR group (P < 0.05); moreover, it reduced the systemic toxicity and peripheral neurotoxicity of vincristine. The possible mechanism was that CD41-VLP could precisely target M1 macrophages in spleen and liver tissues through FcγR, thus reducing the platelet destruction caused by M1 macrophages. Therefore, CD41-VLP provides a new targeted therapy for ITP treatment.

Role of the Helix in Talin F3 Domain (F3 Helix) in Talin-Mediated Integrin Activation.

Increases in ligand binding to cellular integrins (activation) play an important role in platelet and leukocyte function. Talin is necessary in vivo and sufficient in vitro for integrin αIIbß3 activation. The precise mechanisms by which talin activates integrin are still being elucidated. In particular, talin undergoes conformational changes (around the F3 helix) and inserts the F3 helix into lipid bilayer; however, the connection between this lipid-inserting mechanism of talin and talin's capacity to activate integrin has never been explored before. In this work, we used rational mutagenesis, modeled cell systems, and structural modeling to study the potential role of membrane-induced talin conformational changes in talin-mediated integrin activation. Mutations of the residues critical for talin F3 helix to insert into membrane completely abolished talin-mediated integrin activation without affecting the binding of talin to integrins. Furthermore, mutations of the lipid-binding sequences in talin F3 helix significantly reduced the capacity of talin to activate integrin. Our results suggest that the F3 helix may contribute to talin-mediated integrin activation.

Implications of the differing roles of the ß1 and ß3 transmembrane and cytoplasmic domains for integrin function.

Integrins are transmembrane receptors composed of α and ß subunits. Although most integrins contain ß1, canonical activation mechanisms are based on studies of the platelet integrin, αIIbß3. Its inactive conformation is characterized by the association of the αIIb transmembrane and cytosolic domain (TM/CT) with a tilted ß3 TM/CT that leads to activation when disrupted. We show significant structural differences between ß1 and ß3 TM/CT in bicelles. Moreover, the 'snorkeling' lysine at the TM/CT interface of ß subunits, previously proposed to regulate αIIbß3 activation by ion pairing with nearby lipids, plays opposite roles in ß1 and ß3 integrin function and in neither case is responsible for TM tilt. A range of affinities from almost no interaction to the relatively high avidity that characterizes αIIbß3 is seen between various α subunits and ß1 TM/CTs. The αIIbß3-based canonical model for the roles of the TM/CT in integrin activation and function clearly does not extend to all mammalian integrins.

Fluorescence triggering: A general strategy for enumerating and phenotyping extracellular vesicles by flow cytometry.

Plasma contains cell-derived extracellular vesicles (EVs) which participate in various physiopathological processes and have potential biomedical applications. Despite intense research activity, knowledge on EVs is limited mainly due to the difficulty of isolating and characterizing sub-micrometer particles like EVs. We have recently reported that a simple flow cytometry (FCM) approach based on triggering the detection on a fluorescence signal enabled the detection of 50× more Annexin-A5 binding EVs (Anx5+ EVs) in plasma than the conventional FCM approach based on light scattering triggering. Here, we present the application of the fluorescence triggering approach to the enumeration and phenotyping of EVs from platelet free plasma (PFP), focusing on CD41+ and CD235a+ EVs, as well as their sub-populations which bind or do not bind Anx5. Higher EV concentrations were detected by fluorescence triggering as compared to light scattering triggering, namely 40× for Anx5+ EVs, 75× for CD41+ EVs, and 15× for CD235a+ EVs. We found that about 30% of Anx5+ EVs were of platelet origin while only 3% of them were of erythrocyte origin. In addition, a majority of EVs from platelet and erythrocyte origin do not expose PS, in contrast to the classical theory of EV formation. Furthermore, the same PFP samples were analyzed fresh and after freeze-thawing, showing that freeze-thawing processes induce an increase, of about 35%, in the amount of Anx5+ EVs, while the other EV phenotypes remain unchanged. The method of EV detection and phenotyping by fluorescence triggering is simple, sensitive and reliable. We foresee that its application to EV studies will improve our understanding on the formation mechanisms and functions of EVs in health and disease and help the development of EV-based biomarkers.

Natural and artificial mutations in αIIb integrin lead to a structural deformation of a calcium-binding site.

The platelet integrin αIIbß3 is widely accepted as a structural and a functional model of the broad integrin protein family. The four calcium-binding sites in the αIIb subunit contribute to biogenesis and stability of the protein. Mansour et al. (J Thromb Haemost 9:192-200, 2011) showed that the natural Asn2Asp mutation causing Glanzmann thrombasthenia, prevented surface expression of αIIbß3, whereas the artificial Asn2Gln mutation only decreased its level. Molecular dynamics simulations and EDTA chelation assay were used here to explore the mechanism of these structural deformations. We show a considerable expansion of the calcium-binding site 3 in Asn2Asp mutation, whereas the Asn2Gln toggles between normal and expanded conformations. The αIIbß3 surface expression level correlates to the relative spending time in the expanded conformation. By a comparison to other calcium-binding sites of αIIb and of other α integrins we show that the size of a calcium-binding loop is conserved. EDTA chelation assay shows a sensitivity to calcium removal, which correlates with the reduction in αIIbß3 surface expression and with the calcium binding site expansion, thus verifying the simulation data. Here we indicate that Asn2 mutation affects a calcium-binding site 3 of αIIb, which structural deformation is proposed to deprive calcium binding and interfere with an integrin intracellular trafficking and its surface expression.

Integrin αII b tail distal of GFFKR participates in inside-out αII b ß3 activation.

BACKGROUND: Increases in ligand binding to integrins (activation) play critical roles in platelet and leukocyte function. Integrin activation requires talin and kindlin binding to integrin ß cytoplasmic tails. Research has focused on the conserved GFFKR motif in integrin αII b tails, integrin ß cytoplasmic tails and the binding partners of ß tails. However, the roles of αII b tail distal of GFFKR motif are unexplored. OBJECTIVE: To investigate the role of αII b tail distal of GFFKR in talin-mediated inside-out integrin signaling. METHODS: We used model cell systems to examine the role of αII b tail distal of GFFKR in bidirectional αII b ß3 signaling and αII b ß3 -talin interactions. RESULTS: Deletion of amino acid residues after the GFFKR motif in αII b tail moderately decreased ß3 (D723R)-induced activation, abolished talin-induced αII b ß3 activation in model cells, and inhibited agonist-induced αII b ß3 activation in megakaryocytic cells. Furthermore, residues in αII b tail distal of GFFKR did not affect outside-in αII b ß3 signaling or αII b ß3 -talin interaction. Addition of non-homologous or non-specific amino acids to the GFFKR motif restored αII b ß3 activation in model cells and in megakaryocytic cells. Molecular modeling indicates that ß3 -bound talin sterically clashes with the αII b tail in the αII b ß3 complexes, potentially disfavoring the α-ß interactions that keep αII b ß3 inactive. CONCLUSION: The αII b tail sequences distal of GFFKR participate in talin-mediated inside-out αII b ß3 activation through its steric clashes with ß3 -bound talin.

A novel assay for assessing juxtamembrane and transmembrane domain interactions important for receptor heterodimerization.

Understanding the basis of specificity in receptor homodimerization versus heterodimerization is essential in determining the role receptor plays in signal transduction. Specificity in each of the interfaces formed during signal transduction involves cooperative interactions between receptor extracellular, transmembrane (TM), and cytoplasmic domains. While methods exist for studying receptor heterodimerization in cell membranes, they are limited to either TM domains expressed in an inverted orientation or capture only heterodimerization in a single assay. To address this limitation, we have developed an assay (DN-AraTM) that enables simultaneous measurement of homodimerization and heterodimerization of type I receptor domains in their native orientation, including both soluble and TM domains. Using integrin αIIb and RAGE (receptor for advanced glycation end products) as model type I receptor systems, we demonstrate both specificity and sensitivity of our approach, which will provide a novel tool to identify specific domain interactions that are important in regulating signal transduction.

A novel amino acid substitution of integrin αIIb in Glanzmann thrombasthenia confirms that the N-terminal region of the receptor plays a role in maintaining ß-propeller structure.

Mutation screening in Glanzmann thrombasthenia (GT) is now advanced. Despite the large number of genetic defects reported in the ITGA2B gene, few affect the structure of the N-terminal domain of the αIIb subunit. We now report a Catalan family where type I GT is given by compound heterozygosity within ITGA2B with a Gly13Val substitution in αIIb associated with a 13 bp deletion involving the splice site of exon 15. Molecular modelling confirmed that the Gly13Val mutation interfered with the structure of the αIIb ß-propeller and confirms that a fold-back of the N-terminus to interact with residues deep within the propeller is necessary for the normal intracellular processing of the maturing αIIbß3 integrin.

Integrin αIIb-mediated PI3K/Akt activation in platelets.

Integrin αIIbß3 mediated bidirectional signaling plays a critical role in thrombosis and haemostasis. Signaling mediated by the ß3 subunit has been extensively studied, but αIIb mediated signaling has not been characterized. Previously, we reported that platelet granule secretion and TxA2 production induced by αIIb mediated outside-in signaling is negatively regulated by the ß3 cytoplasmic domain residues R(724)KEFAKFEEER(734). In this study, we identified part of the signaling pathway utilized by αIIb mediated outside-in signaling. Platelets from humans and gene deficient mice, and genetically modified CHO cells as well as a variety of kinase inhibitors were used for this work. We found that aggregation of TxA2 production and granule secretion by ß3Δ724 human platelets initiated by αIIb mediated outside-in signaling was inhibited by the Src family kinase inhibitor PP2 and the PI3K inhibitor wortmannin, respectively, but not by the MAPK inhibitor U0126. Also, PP2 and wortmannin, and the palmitoylated ß3 peptide R(724)KEFAKFEEER(734), each inhibited the phosphorylation of Akt residue Ser473 and prevented TxA2 production and storage granule secretion. Similarly, Akt phosphorylation in mouse platelets stimulated by the PAR4 agonist peptide AYPGKF was αIIbß3-dependent, and blocked by PP2, wortmannin and the palmitoylated peptide p-RKEFAKFEEER. Akt was also phosphorylated in response to mAb D3 plus Fg treatment of CHO cells in suspension expressing αIIbß3-Δ724 or αIIbß3E(724)AERKFERKFE(734), but not in cells expressing wild type αIIbß3. In summary, SFK(s) and PI3K/Akt signaling is utilized by αIIb-mediated outside-in signaling to activate platelets even in the absence of all but 8 membrane proximal residues of the ß3 cytoplasmic domain. Our results provide new insight into the signaling pathway used by αIIb-mediated outside-in signaling in platelets.

Platelet-targeting sensor reveals thrombin gradients within blood clots forming in microfluidic assays and in mouse.

BACKGROUND: Thrombin undergoes convective and diffusive transport, making it difficult to visualize during thrombosis. We developed the first sensor capable of revealing inner clot thrombin dynamics. METHODS AND RESULTS: An N-terminal-azido thrombin-sensitive fluorescent peptide (ThS-P) with a thrombin-releasable quencher was linked to anti-CD41 using click chemistry to generate a thrombin-sensitive platelet binding sensor (ThS-Ab). Rapid thrombin cleavage of ThS-P (K(m) = 40.3 µm, k(cat) = 1.5 s(-1) ) allowed thrombin monitoring by ThS-P or ThS-Ab in blood treated with 2-25 pm tissue factor (TF). Individual platelets had > 20-fold more ThS-Ab fluorescence after clotting. In a microfluidic assay of whole blood perfusion over collagen ± linked TF (wall shear rate = 100 s(-1) ), ThS-Ab fluorescence increased between 90 and 450 s for 0.1-1 molecule-TF µm(-2) and co-localized with platelets near fibrin. Without TF, neither thrombin nor fibrin was detected on the platelet deposits by 450 s. Using a microfluidic device to control the pressure drop across a thrombus forming on a porous collagen/TF plug (521 s(-1) ), thrombin and fibrin were detected at the thrombus-collagen interface at a zero pressure drop, whereas 80% less thrombin was detected at 3200 Pa in concert with fibrin polymerizing within the collagen. With anti-mouse CD41 ThS-Ab deployed in a mouse laser injury model, the highest levels of thrombin arose between 40 and 160 s nearest the injury site where fibrin co-localized and where the thrombus was most mechanically stable. CONCLUSION: ThS-Ab reveals thrombin locality, which depends on surface TF, flow and intrathrombus pressure gradients.

Recognition of highly restricted regions in the ß-propeller domain of αIIb by platelet-associated anti-αIIbß3 autoantibodies in primary immune thrombocytopenia.

Platelet-associated (PA) IgG autoantibodies play an essential role in primary immune thrombocytopenia (ITP). However, little is known about the epitopes of these Abs. This study aimed to identify critical binding regions for PA anti-αIIbß3 Abs. Because PA anti-αIIbß3 Abs bound poorly to mouse αIIbß3, we created human-mouse chimera constructs. We first examined 76 platelet eluates obtained from patients with primary ITP. Of these, 26 harbored PA anti-αIIbß3 Abs (34%). Further analysis of 15 patients who provided sufficient materials showed that the epitopes of these Abs were mainly localized in the N-terminal half of the ß-propeller domain in αIIb (L1-W235). We could identify 3 main recognition sites in the region; 2 eluates recognized a conformation formed by the W1:1-2 and W2:3-4 loops, 5 recognized W1:2-3, and 4 recognized W3:4-1. The remaining 4 eluates could not be defined by the binding sites. Within these regions, we identified residues critical for binding, including S29 and R32 in W1:1-2; G44 and P45 in W1:2-3; and P135, E136, and R139 in W2:3-4. Of 11 eluates whose recognition sites were identified, 5 clearly showed restricted κ/λ-chain usage. These results suggested that PA anti-αIIbß3 Abs in primary ITP tended to recognize highly restricted regions of αIIb with clonality.

Use of immuno-magnetic beads for direct capture of nanosized microparticles from plasma.

Increased microparticle tissue factor (TF) activity is not only found in cancer patients, but also in patients with cardiovascular and inflammatory diseases. Methods such as flow cytometry and impedance-based flow cytometry allow the analysis of microparticle subsets but provide no insight on which microparticles carry active TF. Conversely, the microparticle-TF activity itself does not reveal the cellular origin of the microparticles carrying the active TF.For this reason, we developed an immuno-magnetic bead method to capture subsets of microparticles directly from plasma. The method was optimized for capture of platelet-derived microparticles (PMPs) from plasma. Only 100 µl platelet-poor plasma (PPP) was needed in combination with 135 µl (27 µg) of biotinylated antihuman CD41 monoclonal antibody (MoAb) and 200 µl of streptavidin beads to achieve complete separation of PMPs from plasma. As a control, biotinylated mouse IgG1 isotype control MoAb was used instead of the anti-CD41 MoAb. Using biotinylated anti-CD14 MoAb, CD14-positive microparticles were captured from normal plasma spiked with microparticles isolated from the supernatant of lipopolysaccharide-stimulated monocytes (MoMPs). TF activity was found both in the positive (selected) and negative (depleted) fractions indicating that both CD14-positive and negative MoMPs carry active TF. We propose that this method can be used in the future to investigate the source of microparticles carrying active TF in plasma of patients with cancer and other diseases.

A point mutation in the EGF-4 domain of ß(3) integrin is responsible for the formation of the Sec(a) platelet alloantigen and affects receptor function.

Neonatal alloimmune thrombocytopenia (NAIT) is caused by fetomaternal platelet incompatibility with maternal antibodies crossing the placenta and destroying fetal platelets. Antibodies against human platelet antigen-1a (HPA-1a) and HPA-5b are responsible for the majority of NAIT cases. We observed a suspected NAIT in a newborn with a platelet count of 25 G/l and petechial haemorrhages. Serological analysis of maternal serum revealed an immunisation against αIIbß3 on paternal platelets only, indicating the presence of an antibody against a new rare alloantigen (Sec(a)) residing on αIIbß3. The location of Sec(a) on αIIbß3 was confirmed by immunoprecipitation. Nucleotide sequence analysis of paternal ß3 revealed a single nucleotide exchange (G(1818)T) in exon 11 of the ß3 gene (ITGB3), changing Lys(580) (wild-type) to Asn(580) (Sec(a)). Two additional members of the family Sec were typed Sec(a) positive, but none of 300 blood donors. Chinese hamster ovary cells expressing Asn(580), but not Lys(580) αIIbß3, bound anti-Sec(a), which was corroborated by immunoprecipitation. Adhesion of transfected cells onto immobilised fibrinogen showed reduced binding of the Asn(580) variant compared to wild-type αIIbß3. Analysis of transfected cells with anti-LIBS and PAC-1 antibody showed reduced binding when compared to the wild-type. No such effects were observed with Sec(a) positive platelets, which, however, are heterozygous for the Lys(580)Asn mutation. In this study, we describe a NAIT case caused by maternal alloimmunisation against a new antigen on αIIbß3. Analysis with mutant transfected cells showed that the Lys(580)Asn mutation responsible for the formation of the Sec(a) antigenic determinant affects αIIbß3 receptor function.

A helix heterodimer in a lipid bilayer: prediction of the structure of an integrin transmembrane domain via multiscale simulations.

Dimerization of transmembrane (TM) α helices of membrane receptors plays a key role in signaling. We show that molecular dynamics simulations yield models of integrin TM helix heterodimers, which agree well with available NMR structures. We use a multiscale simulation approach, combining coarse-grained and subsequent atomistic simulation, to model the dimerization of wild-type (WT) and mutated sequences of the αIIb and ß3 integrin TM helices. The WT helices formed a stable, right-handed dimer with the same helix-helix interface as in the published NMR structure (PDB: 2K9J). In contrast, the presence of disruptive mutations perturbed the interface between the helices, altering the conformational stability of the dimer. The αIIb/ß3 interface was more flexible than that of, e.g., glycophorin A. This is suggestive of a role for alternative packing modes of the TM helices in transbilayer signaling.

Tyrosine phosphorylation as a conformational switch: a case study of integrin ß3 cytoplasmic tail.

Reversible protein phosphorylation is vital for many fundamental cellular processes. The actual impact of adding and removing phosphate group(s) is 3-fold: changes in the local/global geometry, alterations in the electrostatic potential and, as the result of both, modified protein-target interactions. Here we present a comprehensive structural investigation of the effects of phosphorylation on the conformational as well as functional states of a crucial cell surface receptor, α(IIb)ß(3) integrin. We have analyzed phosphorylated (Tyr(747) and Tyr(759)) ß(3) integrin cytoplasmic tail (CT) primarily by NMR, and our data demonstrate that under both aqueous and membrane-mimetic conditions, phosphorylation causes substantial conformational rearrangements. These changes originate from novel ionic interactions and revised phospholipid binding. Under aqueous conditions, the critical Tyr(747) phosphorylation prevents ß(3)CT from binding to its heterodimer partner α(IIb)CT, thus likely maintaining an activated state of the receptor. This conclusion was tested in vivo and confirmed by integrin-dependent endothelial cells adhesion assay. Under membrane-mimetic conditions, phosphorylation results in a modified membrane embedding characterized by significant changes in the secondary structure pattern and the overall fold of ß(3)CT. Collectively these data provide unique molecular insights into multiple regulatory roles of phosphorylation.

Residue-specific vibrational echoes yield 3D structures of a transmembrane helix dimer.

Two-dimensional (2D) vibrational echo spectroscopy has previously been applied to structural determination of small peptides. Here we extend the technique to a more complex, biologically important system: the homodimeric transmembrane dimer from the α chain of the integrin α(IIb)ß(3). We prepared micelle suspensions of the pair of 30-residue chains that span the membrane in the native structure, with varying levels of heavy ((13)C=(18)O) isotopes substituted in the backbone of the central 10th through 20th positions. The constraints derived from vibrational coupling of the precisely spaced heavy residues led to determination of an optimized structure from a range of model candidates: Glycine residues at the 12th, 15th, and 16th positions form a tertiary contact in parallel right-handed helix dimers with crossing angles of -58° ± 9° and interhelical distances of 7.7 ± 0.5 angstroms. The frequency correlation established the dynamical model used in the analysis, and it indicated the absence of mobile water associated with labeled residues. Delocalization of vibrational excitations between the helices was also quantitatively established.