Flexible docking of an amyloid-forming peptide from beta(2)-microglobulin

Using an all-atom, molecular dynamics-based, flexible docking method, the tertiary and quaternary structures of protofilaments of the "K3" fragment from beta(2)-microglobulin (residues Ser20-Lys41) were predicted at low pH in a continuous mixture of water and 2,2,2-trifluoroethanol (TFE). Tetramers with energies very close to the global minimum were produced with C(alpha) root-mean square deviation values under 4A over 88 residues compared to a recently solved SSNMR structure. The most accurate model distinguishes itself from other low-energy solutions in that it shows high structural similarity to another known fold, the parallel beta-helix, in agreement with models proposed previously by several other groups. The method achieves efficiency without loss of generality or atomic detail by enforcing local symmetry on the individual peptides, rewarding intermolecular contacts, and iteratively building up the protofilaments by successively doubling the number of chains. Solvent effects were included in the model by treating the dielectric constant and surface tension as functions of the TFE concentration. In order to understand the physical basis for the stabilizing effects of TFE, the TFE concentration was varied from 0% to 50% (v/v) and a peak in stability was observed at 16%, where the polar and hydrophobic terms cancel out and close to the experimentally determined value of 20%.     

Interaction of discoidin domain receptor 1 with collagen type 1

Discoidin domain receptor 1 (DDR1) is a widely expressed tyrosine kinase receptor which binds to and gets activated by collagens including collagen type 1. Little is understood about the interaction of DDR1 with collagen and its possible functional implications. Here, we elucidate the binding pattern of the DDR1 extracellular domain (ECD) to collagen type 1 and its impact on collagen fibrillogenesis. Our in vitro assays utilized DDR1-Fc fusion proteins, which contain only the ECD of DDR1. Using surface plasmon resonance, we confirmed that further oligomerization of DDR1-Fc (by means of anti-Fc antibody) greatly enhances its binding to immobilized collagen type 1. Single-molecule imaging by means of atomic force microscopy revealed that DDR1 oligomers bound at overlapping or adjacent collagen molecules and were nearly absent on isolated collagen molecules. Interaction of DDR1 oligomers with collagen was found to modulate collagen fibrillogenesis both in vitro and in cell-based assays. Collagen fibers formed in the presence of DDR1 had a larger average diameter, were more cross-linked and lacked the native banded structure. The presence of DDR1 ECD resulted in "locking" of collagen molecules in an incomplete fibrillar state both in vitro and on surfaces of cells overexpressing DDR1. Our results signify an important functional role of the DDR1 ECD, which occurs naturally in kinase-dead isoforms of DDR1 and as a shedded soluble protein. The modulation of collagen fibrillogenesis by the DDR1 ECD elucidates a novel mechanism of collagen regulation by DDR1.     

Structural analysis and dynamics of retinal chromophore in dark and meta I states of rhodopsin from 2H NMR of aligned membranes

Rhodopsin is a prototype for G protein-coupled receptors (GPCRs) that are implicated in many biological responses in humans. A site-directed (2)H NMR approach was used for structural analysis of retinal within its binding cavity in the dark and pre-activated meta I states. Retinal was labeled with (2)H at the C5, C9, or C13 methyl groups by total synthesis, and was used to regenerate the opsin apoprotein. Solid-state (2)H NMR spectra were acquired for aligned membranes in the low-temperature lipid gel phase versus the tilt angle to the magnetic field. Data reduction assumed a static uniaxial distribution, and gave the retinylidene methyl bond orientations plus the alignment disorder (mosaic spread). The dark-state (2)H NMR structure of 11-cis-retinal shows torsional twisting of the polyene chain and the beta-ionone ring. The ligand undergoes restricted motion, as evinced by order parameters of approximately 0.9 for the spinning C-C(2)H(3) groups, with off-axial fluctuations of approximately 15 degrees . Retinal is accommodated within the rhodopsin binding pocket with a negative pre-twist about the C11=C12 double bond that explains its rapid photochemistry and the trajectory of 11-cis to trans isomerization. In the cryo-trapped meta I state, the (2)H NMR structure shows a reduction of the polyene strain, while torsional twisting of the beta-ionone ring is maintained. Distortion of the retinal conformation is interpreted through substituent control of receptor activation. Steric hindrance between trans retinal and Trp265 can trigger formation of the subsequent activated meta II state. Our results are pertinent to quantum and molecular mechanics simulations of ligands bound to GPCRs, and illustrate how (2)H NMR can be applied to study their biological mechanisms of action.     

Macromolecular properties of cepacian in water and in dimethylsulfoxide

Cepacian is the exopolysaccharide produced by the majority of the so far investigated clinical strains of the Burkholderia cepacia complex. This is a group of nine closely related bacterial species that might cause serious lung infections in cystic fibrosis patients, in some cases leading to death. In this paper the aggregation ability and the conformational properties of cepacian chain were investigated to understand its role in biofilm formation. Viscosity and atomic force microscopy studies in water and in mixed (dimethylsulfoxide/water) solvent indicated the formation of double stranded molecular structures in aqueous solutions. Inter-residue short distances along cepacian chain were investigated by NOE NMR, which showed that two side chains of cepacian were not conformationally free due to strong interactions with the polymer backbone. These interactions were attributed to hydrogen bonding and contributed to structure rigidity.     

S100 proteins interact with the N-terminal domain of MDM2

S100 proteins interact with the transactivation domain and the C-terminus of p53. Further, S100B has been shown to interact with MDM2, a central negative regulator of p53. Here, we show that S100B bound directly to the folded N-terminal domain of MDM2 (residues 2-125) by size exclusion chromatography and surface plasmon resonance experiments. This interaction with MDM2 (2-125) is a general feature of S100 proteins; S100A1, S100A2, S100A4 and S100A6 also interact with MDM2 (2-125). These interactions with S100 proteins do not result in a ternary complex with MDM2 (2-125) and p53. Instead, we observe the ability of a subset of S100 proteins to disrupt the extent of MDM2-mediated p53 ubiquitylation in vitro.

Structured summary

MINT-7905256: MDM2 (uniprotkb:Q00987) binds (MI:0407) to s100A6 (uniprotkb:P06703) by surface plasmon resonance (MI:0107)MINT-7905063: MDM2 (uniprotkb:Q00987) and s100A1 (uniprotkb:P23297) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905376: s100A4 (uniprotkb:P26447) and MDM2 (uniprotkb:Q00987) physically interact (MI:0915) by competition binding (MI:0405)MINT-7905130: s100A6 (uniprotkb:P06703) and MDM2 (uniprotkb:Q00987) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905207: s100A6 (uniprotkb:P06703) and p53 (uniprotkb:P04637) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905043: s100B (uniprotkb:P04271) and MDM2 (uniprotkb:Q00987) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905196: p53 (uniprotkb:P04637) and s100A4 (uniprotkb:P26447) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905358: p53 (uniprotkb:P04637) and s100A4 (uniprotkb:P26447) physically interact (MI:0915) by fluorescence polarization spectroscopy (MI:0053)MINT-7905220: MDM2 (uniprotkb:Q00987) binds (MI:0407) to s100B (uniprotkb:P04271) by surface plasmon resonance (MI:0107)MINT-7905104: s100A4 (uniprotkb:P26447) and MDM2 (uniprotkb:Q00987) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905229: MDM2 (uniprotkb:Q00987) binds (MI:0407) to s100A1 (uniprotkb:P23297) by surface plasmon resonance (MI:0107)MINT-7905317, MINT-7905162: s100B (uniprotkb:P04271) and p53 (uniprotkb:P04637) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905238: MDM2 (uniprotkb:Q00987) binds (MI:0407) to s100A2 (uniprotkb:P29034) by surface plasmon resonance (MI:0107)MINT-7905174, MINT-7905308: s100A1 (uniprotkb:P23297) and p53 (uniprotkb:P04637) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905247: MDM2 (uniprotkb:Q00987) binds (MI:0407) to s100A4 (uniprotkb:P26447) by surface plasmon resonance (MI:0107)MINT-7905090: s100A2 (uniprotkb:P29034) and MDM2 (uniprotkb:Q00987) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905142, MINT-7905326: MDM2 (uniprotkb:Q00987) and p53 (uniprotkb:P04637) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905185, MINT-7905347: s100A2 (uniprotkb:P29034) and p53 (uniprotkb:P04637) bind (MI:0407) by molecular sieving (MI:0071)     

A switch in the mechanism of communication between the two DNA-binding sites in the SfiI restriction endonuclease

While many Type II restriction enzymes are dimers with a single DNA-binding cleft between the subunits, SfiI is a tetramer of identical subunits. Two of its subunits (a dimeric unit) create one DNA-binding cleft, and the other two create a second cleft on the opposite side of the protein. The two clefts bind specific DNA cooperatively to give a complex of SfiI with two recognition sites. This complex is responsible for essentially all of the DNA-cleavage reactions by SfiI: virtually none is due to the complex with one site. The communication between the DNA-binding clefts was examined by disrupting one of the very few polar interactions in the otherwise hydrophobic interface between the dimeric units: a tyrosine hydroxyl was removed by mutation to phenylalanine. The mutant protein remained tetrameric in solution and could bind two DNA sites. But instead of being activated by binding two sites, like wild-type SfiI, it showed maximal activity when bound to a single site and had a lower activity when bound to two sites. This interaction across the dimer interface thus enforces in wild-type SfiI a cooperative transition between inactive and active states in both dimers, but without this interaction as in the mutant protein, a single dimer can undergo the transition to give a stable intermediate with one inactive dimer and one active dimer.     

Fine structural properties of natural and synthetic glycogens

Glycogen, highly branched (1→4)(1→6)-linked α-d-glucan, can be extracted from natural sources such as animal tissues or shellfish (natural source glycogen, NSG). Glycogen can also be synthesized in vitro from glucose-1-phosphate using the cooperative action of α-glucan phosphorylase (GP, EC 2.4.1.1) and branching enzyme (BE, EC 2.4.1.18), or from short-chain amylose by the cooperative action of BE and amylomaltase (AM, EC 2.4.1.25). It has been shown that enzymatically synthesized glycogen (ESG) has structural and physicochemical properties similar to those of NSG. In this study, the fine structures of ESG and NSG were analyzed using isoamylase and α-amylase. Isoamylase completely hydrolyzed the α-1,6 linkages of ESG and NSG. The unit-chain distribution (distribution of degrees of polymerization (DP) of α-1,4 linked chains) of ESG was slightly narrower than that of NSG. α-Amylase treatment revealed that initial profiles of hydrolyses of ESG and NSG were almost the same: both glycogens were digested slowly, compared with starch. The final products from NSG by α-amylase hydrolysis were glucose, maltose, maltotriose, branched oligosaccharides with DP ? 4, and highly branched macrodextrin molecules with molecular weights of up to 10,000. When ESG was digested with excess amounts of α-amylase, much larger macrodextrins (molecular weight > 10⁶) were detected. In contrast, oligosaccharides with DP 4-7 could not be detected from ESG. These results suggest that the α-1,6 linkages in ESG molecules are more regularly distributed than those in NSG molecules.     

The unfolded state of the villin headpiece helical subdomain: computational studies of the role of locally stabilized structure

The 36 residue villin headpiece helical subdomain (HP36) is one of the fastest cooperatively folding proteins, folding on the microsecond timescale. HP36's simple three helix topology, fast folding and small size have made it an attractive model system for computational and experimental studies of protein folding. Recent experimental studies have explored the denatured state of HP36 using fragment analysis coupled with relatively low-resolution spectroscopic techniques. These studies have shown that there is apparently only a small tendency to form locally stabilized secondary structure. Here, we complement the experimental studies by using replica exchange molecular dynamics with explicit solvent to investigate the structural features of these peptide models of unfolded HP36. To ensure convergence, two sets of simulations for each fragment were performed with different initial structures, and simulations were continued until these generated very similar final ensembles. These simulations reveal low populations of native-like structure and early folding events that cannot be resolved by experiment. For each fragment, calculated J-coupling constants and helical propensities are in good agreement with experimental trends. HP-1, corresponding to residues 41 to 53 and including the first alpha-helix, contains the highest helical population. HP-3, corresponding to residues 62 through 75 and including the third alpha-helix, contains a small population of helical turn residing at the N terminus while HP-2, corresponding to residues 52 through 61 and including the second alpha-helix, formed little to no structure in isolation. Overall, HP-1 was the only fragment to adopt a native-like conformation, but the low population suggests that formation of significant structure only occurs after formation of specific tertiary interactions.     

Nonantibiotic Properties of Tetracyclines: Structural Basis for Inhibition of Secretory Phospholipase A2

Secretory phospholipase A₂ is involved in inflammatory processes and was previously shown to be inhibited by lipophilic tetracyclines such as minocycline (minoTc) and doxycycline. Lipophilic tetracyclines might be a new lead compound for the design of specific inhibitors of secretory phospholipase A₂, which play a crucial role in inflammatory processes. Our X-ray crystal structure analysis at 1.65 Å resolution of the minoTc complex of phospholipase A₂ (PLA₂) of the Indian cobra (Naja naja naja) is the first example of nonantibiotic tetracycline interactions with a protein. MinoTc interferes with the conformation of the active-site Ca²⁺-binding loop, preventing Ca²⁺ binding, and shields the active site from substrate entrance, resulting in inhibition of the enzyme. MinoTc binding to PLA₂ is dominated by hydrophobic interactions quite different from antibiotic recognition of tetracyclines by proteins or the ribosome. The affinity of minoTc for PLA₂ was determined by surface plasmon resonance, resulting in a dissociation constant K_d = 1.8 × 10^− 4 M.     

Structural and thermodynamic signatures of DNA recognition by Mycobacterium tuberculosis DnaA

An essential protein, DnaA, binds to 9-bp DNA sites within the origin of replication oriC. These binding events are prerequisite to forming an enigmatic nucleoprotein scaffold that initiates replication. The number, sequences, positions, and orientations of these short DNA sites, or DnaA boxes, within the oriCs of different bacteria vary considerably. To investigate features of DnaA boxes that are important for binding Mycobacterium tuberculosis DnaA (MtDnaA), we have determined the crystal structures of the DNA binding domain (DBD) of MtDnaA bound to a cognate MtDnaA-box (at 2.0 Å resolution) and to a consensus Escherichia coli DnaA-box (at 2.3 Å). These structures, complemented by calorimetric equilibrium binding studies of MtDnaA DBD in a series of DnaA-box variants, reveal the main determinants of DNA recognition and establish the [T/C][T/A][G/A]TCCACA sequence as a high-affinity MtDnaA-box. Bioinformatic and calorimetric analyses indicate that DnaA-box sequences in mycobacterial oriCs generally differ from the optimal binding sequence. This sequence variation occurs commonly at the first 2 bp, making an in vivo mycobacterial DnaA-box effectively a 7-mer and not a 9-mer. We demonstrate that the decrease in the affinity of these MtDnaA-box variants for MtDnaA DBD relative to that of the highest-affinity box TTGTCCACA is less than 10-fold. The understanding of DnaA-box recognition by MtDnaA and E. coli DnaA enables one to map DnaA-box sequences in the genomes of M. tuberculosis and other eubacteria.     

The alpha-helix of the second chromodomain of the 43 kDa subunit of the chloroplast signal recognition particle facilitates binding to the 54 kDa subunit

Chloroplasts of higher plants contain a unique signal recognition particle (cpSRP) that consists of two proteins, cpSRP54 and cpSRP43. CpSRP43 is composed of a four ankyrin repeat domain and three functionally distinct chromodomains (CDs). In this report we confirm previously published data that the second chromodomain (CD2) provides the primary binding site for cpSRP54. However, quantitative binding analysis demonstrates that cpSRP54 binds to CD2 significantly less efficiently than it binds to full-length cpSRP43. Further analysis of the binding interface of cpSRP by mutagenesis studies and a pepscan approach demonstrates that the C-terminal alpha-helix of CD2 facilitates binding to cpSRP54.     

Evolution of Stability in a Cold-Active Enzyme Elicits Specificity Relaxation and Highlights Substrate-Related Effects on Temperature Adaptation

Molecular aspects of thermal adaptation of proteins were studied by following the co-evolution of temperature dependence, conformational stability, and substrate specificity in a cold-active lipase modified via directed evolution. We found that the evolution of kinetic stability was accompanied by a relaxation in substrate specificity. Moreover, temperature dependence and selectivity turned out to be mutually dependent. While the wild-type protein was strictly specific for short-chain triglycerides (C4) in the temperature range 10-50 °C and displayed highest activity in the cold, its stabilized variant was able to accept C8 and C12 molecules and its selectivity was temperature dependent. We could not detect any improvement in the overall structural robustness of the mutant when the structure was challenged by temperature or chemical denaturants. There is, however, strong evidence for local stabilization effects in the active-site region provided by two independent approaches. Differential scanning fluorimetry revealed that the exposure of hydrophobic patches (as the active site is) precedes denaturation, and molecular dynamics simulations confirmed that stability was obtained by restriction of the mobility of the lid, a flexible structure that regulates the access to the enzyme active site and influences its stability. This reduction of lid movements is suggested to be accompanied by a concomitant increase in the mobility of other protein regions, thus accounting for the observed broadening of substrate specificity.     

Crystal structure of the Marasmius oreades mushroom lectin in complex with a xenotransplantation epitope

MOA, a lectin from the mushroom Marasmius oreades, is one of the few reagents that specifically agglutinate blood group B erythrocytes. Further, it is the only lectin known to have exclusive specificity for Galalpha(1,3)Gal-containing sugar epitopes, which are antigens that pose a severe barrier to animal-to-human organ transplantation. We describe here the structure of MOA at 2.4 A resolution, in complex with the linear trisaccharide Galalpha(1,3)Galbeta(1,4)GlcNAc. The structure is dimeric, with two distinct domains per protomer: the N-terminal lectin module adopts a ricinB/beta-trefoil fold and contains three putative carbohydrate-binding sites, while the C-terminal domain serves as a dimerization interface. This latter domain, which has an unknown function, reveals a novel fold with intriguing conservation of an active site cleft. A number of indications suggest that MOA may have an enzymatic function in addition to the sugar-binding properties.     

A simple model of backbone flexibility improves modeling of side-chain conformational variability

The considerable flexibility of side-chains in folded proteins is important for protein stability and function, and may have a role in mediating allosteric interactions. While sampling side-chain degrees of freedom has been an integral part of several successful computational protein design methods, the predictions of these approaches have not been directly compared to experimental measurements of side-chain motional amplitudes. In addition, protein design methods frequently keep the backbone fixed, an approximation that may substantially limit the ability to accurately model side-chain flexibility. Here, we describe a Monte Carlo approach to modeling side-chain conformational variability and validate our method against a large dataset of methyl relaxation order parameters derived from nuclear magnetic resonance (NMR) experiments (17 proteins and a total of 530 data points). We also evaluate a model of backbone flexibility based on Backrub motions, a type of conformational change frequently observed in ultra-high-resolution X-ray structures that accounts for correlated side-chain backbone movements. The fixed-backbone model performs reasonably well with an overall rmsd between computed and predicted side-chain order parameters of 0.26. Notably, including backbone flexibility leads to significant improvements in modeling side-chain order parameters for ten of the 17 proteins in the set. Greater accuracy of the flexible backbone model results from both increases and decreases in side-chain flexibility relative to the fixed-backbone model. This simple flexible-backbone model should be useful for a variety of protein design applications, including improved modeling of protein-protein interactions, design of proteins with desired flexibility or rigidity, and prediction of correlated motions within proteins.     

Functional characterization and conformational analysis of the Herpesvirus saimiri Tip-C484 protein

Tyrosine kinase interacting protein (Tip) of Herpesvirus saimiri (HVS) activates the lymphoid-specific member of the Src family kinase Lck. The Tip:Lck interaction is essential for transformation and oncogenesis in HVS-infected cells. As there are no structural data for Tip, hydrogen-exchange mass spectrometry was used to investigate the conformation of a nearly full-length form (residues 1-187) of Tip from HVS strain C484. Disorder predictions suggested that Tip would be mostly unstructured, so great care was taken to ascertain whether recombinant Tip was functional. Circular dichroism and gel-filtration analysis indicated an extended, unstructured protein. In vitro and in vivo binding and kinase assays confirmed that purified, recombinant Tip interacted with Lck, was capable of activating Lck kinase activity strongly and was multiply phosphorylated by Lck. Hydrogen-exchange mass spectrometry of Tip then showed that the majority of backbone amide hydrogen atoms became deuterated after only 10 s of labeling. Such a result suggested that Tip was almost totally unstructured in solution. Digestion of deuterium-labeled Tip revealed some regions with minor protection from exchange. Overall, it was found that, although recombinant Tip is still functional and capable of binding and activating its target Lck, it is largely unstructured.     

Splicing of intron 3 of human BACE requires the flanking introns 2 and 4

Regulation of proteolytic cleavage of the amyloid precursor protein by the aspartic protease BACE may occur by alternative splicing and the generation of enzymatically inactive forms. In fact, the presence of exonic donor and acceptor sites for intron 3 generates the two deficient variants BACE457 and BACE476. In HEK293 cells, when introns are inserted separately in the BACE cDNA, we found that whilst introns 2 and 4 are efficiently spliced out, intron 3 is not removed. On the other hand, splicing to wild-type BACE is restored when intron 3 is flanked by the two other introns. The presence of all three introns also leads to alternative splicing of intron 3 and the generation of BACE476. In contrast, BACE457 expression takes place only after mutating the donor splice site of intron 3, indicating that additional regulatory elements are necessary for the use of the splicing site within exon 4. Overall, our data demonstrate that a complex splicing of intron 3 regulates the maturation of the BACE mRNA. This appears orchestrated by domains present in the exons and introns flanking intron 3. Excessive BACE activity is a risk factor for Alzheimer’s disease, therefore this complex regulation might guarantee low neuronal BACE activity and disease prevention.     

X-ray crystallography and QM/MM investigation on the oligosaccharide synthesis mechanism of rice BGlu1 glycosynthases

Nucleophile mutants of retaining β-glycosidase can act as glycosynthases to efficiently catalyze the synthesis of oligosaccharides. Previous studies proved that rice BGlu1 mutants E386G, E386S and E386A catalyze the oligosaccharide synthesis with different rates. The E386G mutant gave the fastest transglucosylation rate, which was approximately 3- and 19-fold faster than those of E386S and E386A. To account for the differences of their activities, in this paper, the X-ray crystal structures of BGlu1 mutants E386S and E386A were solved and compared with that of E386G mutant. However, they show quite similar active sites, which implies that their activities cannot be elucidated from the crystal structures alone. Therefore, a combined quantum mechanical/molecular mechanical (QM/MM) calculations were further performed. Our calculations reveal that the catalytic reaction follows a single-step mechanism, i.e., the extraction of proton by the acid/base, E176, and the formation of glycosidic bond are concerted. The energy barriers are calculated to be 19.9, 21.5 and 21.9 kcal/mol for the mutants of E386G, E386S and E386A, respectively, which is consistent with the order of their experimental relative activities. But based on the calculated activation energies, 1.1 kcal/mol energy difference may translate to nearly 100 fold rate difference. Although the rate limiting step in these mutants has not been established, considering the size of the product and the nature of the active site, it is likely that the product release, rather than chemistry, is rate limiting in these oligosaccharides synthesis catalyzed by BGlu1 mutants.     

The crystal structure of CHIR-AB1: a primordial avian classical Fc receptor

CHIR-AB1 is a newly identified avian immunoglobulin (Ig) receptor that includes both activating and inhibitory motifs and was therefore classified as a potentially bifunctional receptor. Recently, CHIR-AB1 was shown to bind the Fc region of chicken IgY and to induce calcium mobilization via association with the common γ-chain, a subunit that transmits signals upon ligation of many different immunoreceptors. Here we describe the 1.8-Å-resolution crystal structure of the CHIR-AB1 ectodomain. The receptor ectodomain consists of a single C2-type Ig domain resembling the Ig-like domains found in mammalian Fc receptors such as FcγRs and FcαRI. Unlike these receptors and other monomeric Ig superfamily members, CHIR-AB1 crystallized as a 2-fold symmetrical homodimer that bears no resemblance to variable or constant region dimers in an antibody. Analytical ultracentrifugation demonstrated that CHIR-AB1 exists as a mixture of monomers and dimers in solution, and equilibrium gel filtration revealed a 2:1 receptor/ligand binding stoichiometry. Measurement of the 1:1 CHIR-AB1/IgY interaction affinity indicates a relatively low affinity complex, but a 2:1 CHIR-AB1/IgY interaction allows an increase in apparent affinity due to avidity effects when the receptor is tethered to a surface. Taken together, these results add to the structural understanding of Fc receptors and their functional mechanisms.     

Solid-State H NMR spectroscopy of retinal proteins in aligned membranes

Solid-state ²H NMR spectroscopy gives a powerful avenue to investigating the structures of ligands and cofactors bound to integral membrane proteins. For bacteriorhodopsin (bR) and rhodopsin, retinal was site-specifically labeled by deuteration of the methyl groups followed by regeneration of the apoprotein. ²H NMR studies of aligned membrane samples were conducted under conditions where rotational and translational diffusion of the protein were absent on the NMR time scale. The theoretical lineshape treatment involved a static axial distribution of rotating C-C²H₃ groups about the local membrane frame, together with the static axial distribution of the local normal relative to the average normal. Simulation of solid-state ²H NMR lineshapes gave both the methyl group orientations and the alignment disorder (mosaic spread) of the membrane stack. The methyl bond orientations provided the angular restraints for structural analysis. In the case of bR the retinal chromophore is nearly planar in the dark- and all-trans light-adapted states, as well upon isomerization to 13-cis in the M state. The C13-methyl group at the “business end” of the chromophore changes its orientation to the membrane upon photon absorption, moving towards W182 and thus driving the proton pump in energy conservation. Moreover, rhodopsin was studied as a prototype for G protein-coupled receptors (GPCRs) implicated in many biological responses in humans. In contrast to bR, the retinal chromophore of rhodopsin has an 11-cis conformation and is highly twisted in the dark state. Three sites of interaction affect the torsional deformation of retinal, viz. the protonated Schiff base with its carboxylate counterion; the C9-methyl group of the polyene; and the β-ionone ring within its hydrophobic pocket. For rhodopsin, the strain energy and dynamics of retinal as established by ²H NMR are implicated in substituent control of activation. Retinal is locked in a conformation that is twisted in the direction of the photoisomerization, which explains the dark stability of rhodopsin and allows for ultra-fast isomerization upon absorption of a photon. Torsional strain is relaxed in the meta I state that precedes subsequent receptor activation. Comparison of the two retinal proteins using solid-state ²H NMR is thus illuminating in terms of their different biological functions.