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PubMed2003

Relation between the secondary structure of carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) and the fluorescence of the protein.

We studied in this work the relation that exists between the secondary structure of the glycans of alpha(1)-acid glycoprotein and the fluorescence of the Trp residues of the protein. We calculated for that the efficiency of quenching and the radiative and non-radiative constants. Our results indicate that the glycans display a spatial structure that is modified upon asialylation. The asialylated conformation is closer to the protein matrix than the sialylated form, inducing by that a decrease in the fluorescence parameters of the Trp residues. In fact, the mean quantum yield of Trp residues in sialylated and asialylated alpha(1)-acid glycoprotein are 0.0645 and 0.0385, respectively. Analysis of the fluorescence emission of alpha(1)-acid glycoprotein as the result of two contributions (surface and hydrophobic domains) indicates that quantum yields of both classes of Trp residues are lower when the protein is in the asialylated form. Also, the mean fluorescence lifetime of Trp residues decreases from 2.285 ns in the sialylated protein to 1.948 ns in the asialylated one. The radiative rate constant k(r) of the Trp residues in the sialylated alpha(1)-acid glycoprotein is higher than that in the asialylated protein. Thus, the carbohydrate residues are closer to the Trp residues in the absence of sialic acid. The modification of the spatial conformation of the glycans upon asialylation is confirmed by the decrease of the fluorescence lifetimes of Calcofluor, a fluorophore that binds to the carbohydrate residues. Finally, thermal intensity quenching of Calcofluor bound to alpha(1)-acid glycoprotein shows that the carbohydrate residues have slower residual motions in the absence of sialic acid residues.

Carbohydrates↗

PubMed2025

Sweeps in Space: Leveraging Geographic Data to Identify Beneficial Alleles in Anopheles gambiae.

As organisms adapt to environmental changes, natural selection modifies the frequency of nonneutral alleles. For beneficial mutations, the outcome of this process may be a selective sweep, in which an allele rapidly increases in frequency and perhaps reaches fixation within a population. Selective sweeps have well-studied effects on patterns of local genetic variation in panmictic populations, but much less is known about the dynamics of sweeps in continuous space. In particular, because limited movement across a landscape leads to unique patterns of population structure, spatial dynamics may influence the trajectory of selected mutations. Here, we use forward-in-time, individual-based simulations in continuous space to study the impact of space on beneficial mutations as they sweep through a population. In particular, we show that selection changes the joint distribution of allele frequency and geographic range occupied by a focal allele and demonstrate that this signal can be used to identify selective sweeps. We then leverage this signal to identify in-progress selective sweeps within the malaria vector Anopheles gambiae, a species under strong selection pressure from vector control measures. By considering space, we identify multiple previously undescribed variants with potential phenotypic consequences, including mutations impacting known IR-associated genes and altering protein structure and properties. Our results demonstrate a novel signal for detecting selection in spatial population genetic data that may have implications for genomic surveillance and understanding geographic patterns of genetic variation.

Animals↗

PubMed2025

Sweeps in space: leveraging geographic data to identify beneficial alleles in Anopheles gambiae.

As organisms adapt to environmental changes, natural selection modifies the frequency of non-neutral alleles. For beneficial mutations, the outcome of this process may be a selective sweep, in which an allele rapidly increases in frequency and perhaps reaches fixation within a population. Selective sweeps have well-studied effects on patterns of local genetic variation in panmictic populations, but much less is known about the dynamics of sweeps in continuous space. In particular, because limited movement across a landscape leads to unique patterns of population structure, spatial dynamics may influence the trajectory of selected mutations. Here, we use forward-in-time, individual-based simulations in continuous space to study the impact of space on beneficial mutations as they sweep through a population. In particular, we show that selection changes the joint distribution of allele frequency and geographic range occupied by a focal allele and demonstrate that this signal can be used to identify selective sweeps. We then leverage this signal to identify in-progress selective sweeps within the malaria vector Anopheles gambiae , a species under strong selection pressure from vector control measures. By considering space, we identify multiple previously undescribed variants with potential phenotypic consequences, including mutations impacting known IR-associated genes and altering protein structure and properties. Our results demonstrate a novel signal for detecting selection in spatial population genetic data that may have implications for genomic surveillance and understanding geographic patterns of genetic variation.

Journal Article↗

PubMed1992

The refined 1.9-A X-ray crystal structure of D-Phe-Pro-Arg chloromethylketone-inhibited human alpha-thrombin: structure analysis, overall structure, electrostatic properties, detailed active-site geometry, and structure-function relationships.

Thrombin is a multifunctional serine proteinase that plays a key role in coagulation while exhibiting several other key cellular bioregulatory functions. The X-ray crystal structure of human alpha-thrombin was determined in its complex with the specific thrombin inhibitor D-Phe-Pro-Arg chloromethylketone (PPACK) using Patterson search methods and a search model derived from trypsinlike proteinases of known spatial structure (Bode, W., Mayr, I., Baumann, U., Huber, R., Stone, S.R., & Hofsteenge, J., 1989, EMBO J. 8, 3467-3475). The crystallographic refinement of the PPACK-thrombin model has now been completed at an R value of 0.156 (8 to 1.92 A); in particular, the amino- and the carboxy-termini of the thrombin A-chain are now defined and all side-chain atoms localized; only proline 37 was found to be in a cis-peptidyl conformation. The thrombin B-chain exhibits the characteristic polypeptide fold of trypsinlike serine proteinases; 195 residues occupy topologically equivalent positions with residues in bovine trypsin and 190 with those in bovine chymotrypsin with a root-mean-square (r.m.s.) deviation of 0.8 A for their alpha-carbon atoms. Most of the inserted residues constitute novel surface loops. A chymotrypsinogen numbering is suggested for thrombin based on the topological equivalences. The thrombin A-chain is arranged in a boomeranglike shape against the B-chain globule opposite to the active site; it resembles somewhat the propeptide of chymotrypsin(ogen) and is similarly not involved in substrate and inhibitor binding. Thrombin possesses an exceptionally large proportion of charged residues. The negatively and positively charged residues are not distributed uniformly over the whole molecule, but are clustered to form a sandwichlike electrostatic potential; in particular, two extended patches of mainly positively charged residues occur close to the carboxy-terminal B-chain helix (forming the presumed heparin-binding site) and on the surface of loop segment 70-80 (the fibrin[ogen] secondary binding exosite), respectively; the negatively charged residues are more clustered in the ringlike region between both poles, particularly around the active site. Several of the charged residues are involved in salt bridges; most are on the surface, but 10 charged protein groups form completely buried salt bridges and clusters. These electrostatic interactions play a particularly important role in the intrachain stabilization of the A-chain, in the coherence between the A- and the B-chain, and in the surface structure of the fibrin(ogen) secondary binding exosite (loop segment 67-80).(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Chloromethyl Ketones↗

PubMed2005

Spatial distribution of suppressive signals outside the classical receptive field in lateral geniculate nucleus.

A suppressive surround modulates the responsiveness of cells in the lateral geniculate nucleus (LGN), but we know nothing of its spatial structure or the way in which it combines signals arising from different locations. It is generally assumed that suppressive signals are either uniformly distributed or balanced in opposing regions outside the receptive field. Here, we examine the spatial distribution and summation of suppressive signals outside the receptive field in extracellular recordings from 46 LGN cells in anesthetized marmosets. The receptive field of each cell was stimulated with a drifting sinusoidal grating of the preferred size and spatial and temporal frequency; we probed different positions in the suppressive surround with either a large half-annular grating or a small circular grating patch of the preferred spatial and temporal frequency. In many of the cells with a strong suppressive surround (29/46), the spatial distribution of suppression showed clear deviation from circular symmetry. In the majority of these of cells, suppressive signals were spatially asymmetrical or balanced in opposing areas outside the receptive field. A suppressive area was larger than the classical receptive field itself and spatial summation within and between these areas was nonlinear. There was no bias for suppression to arise from foveal or nasal retina where cone density is higher and no other sign of a systematic spatial organization to the suppressive surround. We conclude that nonclassical suppressive signals in LGN deviate from circular symmetry and are nonlinearly combined.

Action Potentials↗

PubMed2003

Microbial composition and structure of a rotating biological contactor biofilm treating ammonium-rich wastewater without organic carbon.

High nitrogen losses were observed in a rotating biological contactor (RBC) treating ammonium-rich (up to 500 mg NH4(+)-N/L) but organic-carbon-poor leachate from a hazardous waste landfill in Kölliken, Switzerland. The composition and spatial structure of the microbial community in the biofilm on the RBC was analyzed with specific attention for the presence of aerobic ammonium and nitrite oxidizing bacteria and anaerobic ammonium oxidizers. Anaerobic ammonium oxidation (anammox) involves the oxidation of ammonium with nitrite to N2. First the diversity of the biofilm community was determined from sequencing cloned PCR-amplified 16S rDNA fragments. This revealed the presence of a number of very unusual 16S rDNA sequences, but very few sequences related to known ammonium or nitrite oxidizing bacteria. From analysis of biofilm samples by fluorescence in situ hybridization with known phylogenetic probes and by dot-blot hybridization of the same probes to total RNA purified from biofilm samples, the main groups of microorganisms constituting the biofilm were found to be ammonium-oxidizing bacteria from the Nitrosomonas europaea/eutropha group, anaerobic ammonium-oxidizing bacteria of the "Candidatus Kuenenia stuttgartiensis" type, filamentous bacteria from the phylum Bacteroidetes, and nitrite-oxidizing bacteria from the genus Nitrospira. Aerobic and anaerobic ammonium-oxidizing bacteria were present in similar amounts of around 20 to 30% of the biomass, whereas members of the CFB phylum were present at around 7%. Nitrite oxidizing bacteria were only present in relatively low amounts (less than 5% determined with fluorescence in situ hybridization). Data from 16S rRNA dot-blot and in situ hybridization were not in all cases congruent. FISH analysis of thin-sliced and fixed biofilm samples clearly showed that the aerobic nitrifiers were located at the top of the biofilm in an extremely high density and in alternating clusters. Anammox bacteria were exclusively present in the lower half of the biofilm, whereas CFB-type filamentous bacteria were present throughout the biofilm. The structure and composition of these biofilms correlated very nicely with the proposed physiological functional separations in ammonium conversion.

Bacteria, Aerobic↗

PubMed1980

Synthesis and structure--activity relationship of a pyrimido[4,5-d]pyrimidine derivative with antidepressant activity.

The synthesis and antidepressant properties of a new pyrimido[4,5-d]pyrimidine are described. Spectral data determined in solution and in the solid state allowed establishment of the relationship between the activity and the conformation of the molecule. The spatial structure seems to be in accordance with a possible binding at the presynaptic alpha-receptor sites.

Antidepressive Agents↗

PubMed1992

Three-dimensional structure of proteolytic fragment 163-231 of bacterioopsin determined from nuclear magnetic resonance data in solution.

546 NOESY cross-peak volumes were measured in the two-dimensional NOESY spectrum of proteolytic fragment 163-231 of bacterioopsin in organic solution. These data and 42 detected hydrogen bonds were applied for determining the peptide spatial structure. The fold of the polypeptide chain was determined by local structure analysis, a distance geometry approach and systematic search for energetically allowed side-chain rotamers which are consistent with experimental NOESY cross-peak volumes. The effective rotational correlation time of 6 ns for the molecule was evaluated from optimization of the local structure to meet NOE data and from the dependence on mixing time of the NiH/Ci alpha H cross-peak volumes of the residues in alpha-helical conformation. The resulting structure has two well defined alpha-helical regions, 168-191 and 198-227, with root-mean-square deviation 44 pm and 69 pm, respectively, between the backbone atoms in 14 final energy refined conformations. The alpha-helices correspond to transmembrane segments F and G of bacteriorhodopsin. The segment F contains proline 186, which introduces a kink of about 25 degrees with a disruption of the hydrogen bond with the NH group of the following residue. The segments are connected by a flexible loop region 192-197. Torsion angles chi 1 are unequivocally defined for 62% of side chains in the alpha-helices but half of them differ from electron cryo-microscopy (ECM) model of bacteriorhodopsin, apparently because of the low resolution of ECM. Nevertheless, the F and G segments can be packed as in the ECM model and with side-chain conformations consistent with all NMR data in solution.

Amino Acid Sequence↗

PubMed1993

[Structure-functional organization of the angiotensin II molecule. III. Polyfunctional nature].

Conformational properties of the molecules of angiotensin II and some of its analogs are analysed with regard to the quantitative characteristics of their biological activities. Connections between low-energy conformations of angiotensin II and its biological functions are established. The hormone's spatial structures plausible in terms of interactions with the specific receptors located in smooth muscles and adrenal cortex tissues have been identified.

Amino Acid Sequence↗

PubMed1992

Determination of the nuclear magnetic resonance solution structure of the DNA-binding domain (residues 1 to 69) of the 434 repressor and comparison with the X-ray crystal structure.

The DNA-binding domain of the phage 434 repressor consisting of N-terminal residues 1 to 69 (434 repressor(1-69)), was expressed in Escherichia coli with natural isotope abundance, uniform 15N-labeling and biosynthetically directed fractional 13C-labeling in extent of about 10%. With these protein preparations the three-dimensional structure was determined in solution. The techniques used were nuclear magnetic resonance (n.m.r.) spectroscopy for the collection of conformational constraints, calculation of the protein structure from the n.m.r. data with the program DIANA and structure refinements by restrained energy minimization with a modified version of the program AMBER. A group of 20 conformers characterizes a well-defined structure for residues 1 to 63, with an average of 0-6 A for the root-mean-square deviations (RMSD) calculated for the backbone atoms of the individual conformers relative to the mean co-ordinates. The spatial structure of C-terminal residues 64 to 69 is not defined by the n.m.r. data. The molecular architecture of the 434 repressor(1-69) in solution includes five alpha-helices extending from residues 2 to 13, 17 to 24, 28 to 35, 45 to 52 and 56 to 60, which enclose a well-defined hydrophobic core. The n.m.r. structure is closely similar to the reported crystal structure of the 434 repressor(1-69), with an RMSD value of 1.1 A for the backbone atoms of residues 1 to 63. Small differences between the two structures in regions of the first helix and the loop between helices 3 and 4 were analyzed relative to possible correlations with protein-protein contacts in the crystal lattice and the different milieus of pH and ionic strength in the crystals and n.m.r. samples. Further systematic comparisons of local conformational features indicated that there are correlations between amino acid types, local precision of the structure determination by both techniques and local differences between the structures in the crystals and in solution. Overall, hydrophobic residues are most precisely characterized and agree most closely in the two environments.

Amides↗

PubMedDate not supplied

[ Fungal extracellular ribonucleases (Review)].

Results of studies of certain fungal extracellular ribonucleases mainly isolated from representatives of the genera Aspergillus and Penicillium are summarized. The isolation of these enzymes in highly purified states in the 1970-1980s strongly stimulated further studies of their structure, functions, and mechanisms of action. This also promoted the use of ribonucleases as catalysts in oligoribonucleotide syntheses and as objects of comparative and evolutionary biochemistry and other research works. Results of studies of the primary, secondary, and spatial structures of guanyl-specific fungal ribonucleases are reviewed. These studies revealed a high homology within the subfamilies of fungal, bacterial, and actinomycete RNases. Characteristics of the nonspecific Pb2 RNase are considered.

Aspergillus↗

PubMedDate not supplied

[Local structure of cytochrome c from horse heart in solution. Conformational analysis using data of two-dimensional nuclear Overhauser effect spectroscopy].

Using the earlier suggested method the calculation of the backbone conformations of horse heart cytochrome c in oxidized (ferricytochrome c) and reduced (ferrocytochrome c) states has been performed by the two-dimensional nuclear Overhauser effect spectroscopy data. For both protein forms the secondary structure elements have been revealed and the conformations of the irregular polypeptide chain segments have been analysed. The similarity of the secondary structures of ferri- and ferrocytochrome c in solution was established from the comparison of their conformations. Small differences between the conformations of two molecule forms are shown to be localized within the polypeptide chain fragments situated in the spatial structure near the heme crevice. The comparison of the dihedral phi and psi angles in the calculated conformations of horse cytochrome C with the corresponding characteristics of X-ray structures of tuna ferri- and ferrocytochrome c made for the oxidized and reduced protein forms using the quantitative criteria testifies the similarity of their conformations in solution and crystal. In is shown that the conformational changes of the separate amino acid residues which take place as the result of the "solution-to-crystal" transition occur on the surface fragments of protein globule and do not lead to essential alterations of the secondary molecule structure.

Amino Acids↗

PubMed2005

NMR structures and orientation of the fourth transmembrane domain of the rat divalent metal transporter (DMT1) with G185D mutation in SDS micelles.

DMT1, also known as Nramp2, is an iron transporter, and belongs to the family of Nramp proteins. Disease-causing mutations both in Nramp1 and Nramp2 occurring at the conserved two adjacent glycine residues located within the fourth transmembrane domain (TM4) suggest that TM4 may serve an important biological function. In the present study, we have determined the high-resolution structures of a synthetic peptide, corresponding to the sequence of the fourth transmembrane domain of rat DMT1 with G185D mutation, in membrane-mimetic environments (e.g., SDS micelles) using NMR spectroscopy and distance-geometry/simulated annealing calculations. The spatial structures showed alpha-helices without a kink in the middle portion of the peptide, with a highly flexible and poorly defined N-terminus. Both the N-terminus and the helical core of the peptide were embedded into the SDS micelles. Interestingly, the folding and membrane location of the C-terminus was pH dependent, being well-folded and inserted into SDS micelles only at a low pH value (4.0). The peptide exhibited amphipathic characteristics, with hydrophilic residues (Asp7, Thr11, Asp14, and Thr15) lying in one side of the helix, which provide a basis for the formation of water-filled channel architectures through self-associations. The significant broadening of the resonances of the hydrophilic residues Asp7, Thr11, and Asp14, which are buried inside SDS micelles, upon addition of Mn2+ further verified the possibility of the formation of a channel through which metal ions pass. The substitution of Gly7 by an aspartate residue neither significantly altered the structure and membrane location of the peptide nor abolished its properties of channel forming and metal permeation compared with the wild-type peptide.

Amino Acid Sequence↗

PubMed2026

Pattern Formation in a Spatial Public Goods Dilemma due to Diffusive or Directed Motion.

The costly provision of public goods serves as a model problem for the evolution of cooperative behavior, presenting a social dilemma between the collective benefits of shared resources and the individual incentive to free-ride in resource production. The spatial structure of populations can also impact cooperation over public goods, as diffusion of public goods and intentional motion of individuals towards regions with greater resources can interact with population and public goods dynamics to produce heterogeneous patterns in the spatial distribution of strategies and resources. In this paper, we build off a model introduced by Young and Belmonte for the reaction dynamics of interacting individuals and an explicit public good, deriving a system of PDEs that describes the spatial profiles of strategies and the public good in the presence of both diffusive motion of individuals and resources and chemotaxis-like directed motion of individuals in response to gradients in the concentration of public goods. Through linear stability analysis, we show that spatial patterns in strategic and public goods profiles can emerge due to either Turing instability with high defector diffusivity or a directed-motion instability through strong sensitivity of cooperators towards increasing resource concentration. We further explore the emergent spatial patterns with a mix of weakly nonlinear stability analysis and numerical simulation, showing that, for a wide range of reaction parameters, diffusion-driven instability appears to increase cooperation and public goods across the spatial domain, while directed motion of cooperators towards public goods tends to decrease cooperation and environmental quality across the environment.

Models, Biological↗

PubMedDate not supplied

Spatial scales and evolution of stormwater plumes in Santa Monica Bay.

Rainfall during winter storms produces extensive turbid, freshwater plumes in the coastal waters of the Southern California Bight. When the plumes result from urban runoff they contain toxic pollutants along with pathogenic bacteria and viruses, often resulting in closure of public beaches. We examined the spatial structure and evolution of stormwater plumes in Santa Monica Bay in 1996. The plumes resulted from freshwater discharge from the Ballona Creek and Malibu Creek watersheds which supply approximately 60% of the freshwater runoff to Santa Monica Bay. The spatial scales of the plumes were determined using shipboard measurements of water properties obtained from towyo transects and surface underway sampling. Salinity maps showed that the plumes typically extended 4-7 km offshore, consistent with scaling by the internal Rossby radius of deformation. Plumes extended along shore 10 km or more. Generally the plumes occupied the upper 10 m of the water column. The persistence time of a plume offshore of Ballona Creek was about three days based on a sequence of surveys in March 1996 following rainfall of about 21 mm. Limited comparison of plumes from Ballona Creek, which drains a developed watershed, and Malibu Creek, which drains a rural watershed, suggested that Malibu Creek required greater rainfall to produce an offshore plume. A stormwater plume offshore of Malibu Creek was observed on both sides of the creek mouth, possibly due to freshwater discharge from smaller surrounding watersheds or advection of freshwater discharges from the east and south. Plumes offshore of Ballona Creek mainly resulted from the creek itself and usually extended northward from the creek mouth, consistent with the wind forcing and the Coriolis acceleration.

California↗

PubMed2006

Spatial and temporal patterns of herd somatic cell score in france.

Spatial and temporal patterns of annual milk somatic cell score (ASCS) were explored in French dairy herds between 1996 and 2000 to detect regional singularities for risk of mastitis. A new cluster detection method was used, which was adapted to continuous variables and which allowed ASCS variation factors to be taken into account. The statistical unit was the herd-year. A linear regression model for each year allowed adjustment for breed, mean parity, number of calvings for each season, herd size, and farm altitude. Cluster detection was performed on raw data and on residuals of the model through a method based on the Hellinger distance between spatial distributions. The Hellinger distance between farm distributions was computed at different levels of ASCS (or residuals). Temporal ASCS patterns were explored using a computation of correlations and comparisons between spatial structures of the different years. The general ASCS trend over the study period was a decrease. The global Hellinger distance, which was higher than what could have been randomly expected for each of the 5 yr, indicated a significant spatial cluster formation. Cluster mapping over the 5 yr identified several areas, which sometimes differed between detection using raw data and that using ASCS residuals. Temporal correlations between ASCS residuals for each year were positive and decreasing, and 1996 and 2000 appeared spatially different from the other years. The more affected areas were regions that were not specialized in dairy production. During the study period, 2 progressive movements were detected, corresponding to a disappearance of clusters in the northwest and an increase of clusters in the southwest. Cluster detection could aid in the identification of new risk factors that are relevant at different spatial scales, and could help local organizations to supervise the risk of mastitis, and improve udder health management.

Animals↗

PubMed2005

[Population structure and community characteristics of Pseudotaxus chienii in Fengyangshan National Natural Reserve].

Pseudotaxus chienii, an endemic plant in China, is one of the second grade state protection wild plants, and distributes in Fengyangshan Natural Reserve as one of its concentrative dwelling places. A survey in the region was carried out on 10 communities, which were dominated by P. chienii. The analysis on its size structure, spatial distribution pattern and community characteristics showed that the populations of P. chienii could grow in the communities dominated by Rhododendron simiarum, Fokienia hodginsii-R. simiarum, and evergreen broad-leaved forests. In R. simiarum communities, the size structure, survival curve, and overwhelming community distribution pattern of P. chienii showed a sustaining development, while in communities dominated by F. hodginsii-R. simiarum, though the size structure was declining, the survival curve was Deevy- III type. Plenty of plantlets were still existed, and the populations kept steady. Two types were considered to be the most suitable ones for P. chienii populations. The important value of P. chienii reached 5% - 10%. In evergreen broad-leaved forests, the populations of P. chienii showed to be a declining type, and the survival curve was Deevy- II type. The distribution pattern was random, indicating that P. chienii populations could not fit for survive in this kind of communities. Correlation analysis showed that P. chienii populations had a positive correlation with R. simiarum and F. hodginsii, but a negative correlation with Cyclobalanopsis stewardiana and Schima superba. P. chienii could associate to the habits with the community canopy density of about 0.6 - 0.8. From the results mentioned above, the populations of P. chienii could be able to sustain and develop, and the existing habitat in Fengyangshan should be protected effectively. The protection of P. chienii requires more basic work to establish efficient measures to protect its habitat.

China↗

PubMed1999

Retinylidene ligand structure in bovine rhodopsin, metarhodopsin-I, and 10-methylrhodopsin from internuclear distance measurements using 13C-labeling and 1-D rotational resonance MAS NMR.

Rhodopsin is the G-protein coupled photoreceptor that initiates the rod phototransduction cascade in the vertebrate retina. Using specific isotope enrichment and magic angle spinning (MAS) NMR, we examine the spatial structure of the C10-C11=C12-C13-C20 motif in the native retinylidene chromophore, its 10-methyl analogue, and the predischarge photoproduct metarhodopsin-I. For the rhodopsin study 11-Z-[10,20-(13)C(2)]- and 11-Z-[11,20-(13)C(2)]-retinal were synthesized and incorporated into bovine opsin while maintaining a natural lipid environment. The ligand is covalently bound to Lys(296) in the photoreceptor. The C10-C20 and C11-C20 distances were measured using a novel 1-D CP/MAS NMR rotational resonance experimental procedure that was specifically developed for the purpose of these measurements [Verdegem, P. J. E., Helmle, M., Lugtenburg, J., and de Groot, H. J. M. (1997) J. Am. Chem. Soc. 119, 169]. We obtain r(10,20) = 0.304 +/- 0.015 nm and r(11,20) = 0.293 +/- 0.015 nm, which confirms that the retinylidene is 11-Z and shows that the C10-C13 unit is conformationally twisted. The corresponding torsional angle is about 44 degrees as indicated by Car-Parrinello modeling studies. To increase the nonplanarity in the chromophore, 11-Z-[10,20-(13)C(2)]-10-methylretinal and 11-Z-[(10-CH(3)), 13-(13)C(2)]-10-methylretinal were prepared and incorporated in opsin. For the resulting analogue pigment r(10,20) = 0.347 +/- 0.015 nm and r((10)(-)(CH)()3())(,)(13) = 0.314 +/- 0.015 nm were obtained, consistent with a more distorted chromophore. The analogue data are in agreement with the induced fit principle for the interaction of opsin with modified retinal chromophores. Finally, we determined the intraligand distances r(10,20) and r(11,20) also for the photoproduct metarhodopsin-I, which has a relaxed all-E structure. The results (r(10,20) >/= 0.435 nm and r(11,20) = 0.283 +/- 0.015 nm) fully agree with such a relaxed all-E structure, which further validates the 1-D rotational resonance technique for measuring intraligand distances and probing ligand structure. As far as we are aware, these results represent the first highly precise distance determinations in a ligand at the active site of a membrane protein. Overall, the MAS NMR data indicate a tight binding pocket, well defined to bind specifically only one enantiomer out of four possibilities and providing a steric complement to the chromophore in an ultrafast ( approximately 200 fs) isomerization process.

Animals↗