Crystal structure of rhodopsin: a template for cone visual pigments and other G protein-coupled receptors.

The crystal structure of rhodopsin has provided the first three-dimensional molecular model for a G-protein-coupled receptor (GPCR). Alignment of the molecular model from the crystallographic structure with the helical axes seen in cryo-electron microscopic (cryo-EM) studies provides an opportunity to investigate the properties of the molecule as a function of orientation and location within the membrane. In addition, the structure provides a starting point for modeling and rational experimental approaches of the cone pigments, the GPCRs in cone cells responsible for color vision. Homology models of the cone pigments provide a means of understanding the roles of amino acid sequence differences that shift the absorption maximum of the retinal chromophore in the environments of different opsins.

Infant light adaptation shows Weber's law at photopic illuminances.

In the present study we investigate the dependence of photopic contrast thresholds on retinal illuminance in infants and adults. Contrast thresholds were measured at five retinal illuminances between about 6 and about 20,000 Td in subjects in both age groups. The forced-choice preferential looking technique was used in 3-month-old infants, and standard forced-choice techniques were used in adults. The stimulus was a 0.25 cy/deg squarewave grating phase alternated at 6 Hz. Infants' contrast thresholds were more than two log units higher than those of adults at all retinal illuminances. Contrast thresholds had a similar dependence on retinal illuminance in both infants and adults. For both age groups, contrast thresholds initially decreased with increasing retinal illuminance. However, at both ages, above a critical illuminance of about 200 Td, contrast thresholds remained constant, following Weber's law. Thus a vertical shift was sufficient to bring the two data sets into correspondence. In the context of a two-site model of light adaptation, our results imply that infants' elevated contrast thresholds cannot be explained solely on the basis of photoreceptoral immaturities. Later physiological immaturities must also limit infants' photopic contrast thresholds.

The transition from scotopic to photopic vision in 3-month-old infants and adults: an evaluation of the rod dominance hypothesis.

The scotopic to photopic transition was tested in adults and 12-week-old infants using a large field motion nulling technique at a series of luminances between -3.57 and 2.70 log cd m(-2). The stimuli were composed of 0.25 cyc deg(-1), 5.6 Hz blue/black and yellow/black sinusoidal grating components, superimposed and moving in opposite directions. The contrasts of the two components were traded off to determine motion nulls at each luminance level. An eye movement based response measure was used for infant subjects, whereas self-report was used in adults. In both age groups, the motion null values approached a scotopic asymptote consistent with V'(lambda) at the lowest luminance levels, and a photopic asymptote consistent with V(10)(lambda) at the highest luminance levels. The scotopic to photopic transition was gradual and occurred over about 3 log units between about -2 and 1 log cd m(-2) in both groups. The null values for infants and adults were highly similar at each luminance level, and the shapes of the transition curves were virtually identical at the two ages. These data suggest that at each different luminance level, the balance between rod-initiated and cone-initiated signals in the extrafoveal luminance channel is similar or identical in 12-week-old infants and adults.

Crystal structure of rhodopsin: A G protein-coupled receptor.

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) respond to a variety of different external stimuli and activate G proteins. GPCRs share many structural features, including a bundle of seven transmembrane alpha helices connected by six loops of varying lengths. We determined the structure of rhodopsin from diffraction data extending to 2.8 angstroms resolution. The highly organized structure in the extracellular region, including a conserved disulfide bridge, forms a basis for the arrangement of the seven-helix transmembrane motif. The ground-state chromophore, 11-cis-retinal, holds the transmembrane region of the protein in the inactive conformation. Interactions of the chromophore with a cluster of key residues determine the wavelength of the maximum absorption. Changes in these interactions among rhodopsins facilitate color discrimination. Identification of a set of residues that mediate interactions between the transmembrane helices and the cytoplasmic surface, where G-protein activation occurs, also suggests a possible structural change upon photoactivation.

Spatial frequency tuned covariance channels for red-green and luminance-modulated gratings: psychophysical data from human adults.

Both chromatic and luminance-modulated stimuli are served by multiple spatial-frequency-tuned channels. This experiment investigated the independence versus interdependence of spatial frequency channels that serve the detection of red-green chromatic versus yellow-black luminance-modulated stimuli at low spatial frequencies. Contrast thresholds for both chromatic and luminance-modulated gratings were measured within 12 individual subjects using a repeated-measures design. Spatial frequencies ranged from 0.27 to 2.16 c/deg. A covariance structure analysis of individual differences was applied to the data. We computed statistical sources of individual variability, used them to define covariance channels, and determined the number and frequency tuning of these channels. For luminance-modulated gratings, two covariance channels were found, including one above and one below 1 c/deg [cf. Peterzell, & Teller (1996). Individual differences in contrast sensitivity functions: the coarsest spatial pattern analyzer. Vision Research, 36, 3077-3085]. For chromatic gratings, correlations between thresholds for most spatial frequencies were uniformly high, yielding a single covariance channel covering all but the highest spatial frequency tested. A combined analysis of both data sets recovered the same three covariance channels, and showed that detection thresholds for low-frequency red-green chromatic and luminance-modulated stimuli are served by separate, statistically independent processes.

Spatial frequency tuned covariance channels for red-green and luminance-modulated gratings: psychophysical data from human infants.

This study concerns the spatial-frequency-tuned channels underlying infants' contrast sensitivity functions (CSFs) for red-green chromatic stimuli, and their relationship to the channels underlying infants' CSFs for luminance-modulated stimuli. Behavioral (forced-choice preferential-looking) techniques and stationary stimuli were used. In experiment 1. contrast thresholds were measured in 4- and 6-month-olds, using isoluminant red-green gratings with spatial frequencies ranging from 0.27 to 1.53 c deg. In experiment 2. contrast thresholds were measured in 4-month-olds. using both red-green and luminance-modulated gratings in the same low spatial frequency range. Covariance analyses of individual differences were performed. Experiment 1 revealed one dominant covariance channel for the detection of red-green gratings, with a second channel contributing to detection of the highest spatial frequencies used. Experiment 2 revealed two to three channels serving color and luminance: but surprisingly these channels were not statistically separable for luminance versus chromatic stimuli. Thus, covariance channels for color and luminance that are independent for adults [Peterzell & Teller (2000). Spatial frequency tuned covariance channels for red-green and luminance-modulated gratings: psychophysical data from human adults. Vision Research, 40, 417-430] are apparently interdependent in infants. These data suggest that for infants, detection thresholds for chromatic and luminance-modulated stimuli may be limited by common mechanisms.

Infant color vision: sharp chromatic edges are not required for chromatic discrimination in 4-month-olds.

In our previous demonstrations of chromatic discrimination in infants, we have used test and surround fields of different chromaticities that abutted each other at sharp chromatic edges. In order to see whether sharp chromatic edges are necessary for infants to make chromatic discriminations, 16-week-old infants were tested with three stimulus configurations in which sharp chromatic edges were eliminated. The three edge manipulations involved black borders, a dark surround, or blurred edges around the chromatic test field. In each case red, green, and violet test fields were used. Although performance decreased when sharp chromatic edges were eliminated, observers' percent correct scores remained clearly above chance for eight of the nine discriminations (three colors x three edge manipulations). We argue that all three edge manipulations reduce the likelihood of mediation of chromatic discrimination by M (magnocellular) cells. These data thus provide evidence that young infants have functional P (parvocellular) pathways, and use them for making chromatic discriminations.

Analysis of red/green color discrimination in subjects with a single X-linked photopigment gene.

Many subjects despite having only a single X-linked pigment gene (single-L/M-gene subjects) are able to make chromatic discriminations by Rayleigh matching, especially when large fields are used. We used a combination of psychophysics (Rayleigh match), electroretinograms (ERG), and molecular genetic techniques to rule out several possible explanations of this phenomenon. Use of rods for chromatic discrimination was unlikely since strong adapting fields were employed and the large-field match results were not consistent with rod participation. A putative mid- to long-wavelength photopigment that escapes detection by current molecular genetic analysis was ruled out by finding only a single L/M photopigment in flicker ERGs from 16 single-L/M-gene subjects. Large-field match results were not consistent with participation of S cones. Amino acid sequence polymorphisms in the S-pigment gene that might have shifted the S cone spectrum towards longer wavelengths were not found on sequencing. The mechanism of chromatic discrimination in the presence of a single photopigment therefore remains unknown. Further possible explanations such as variations in cone pigment density and retinal inhomogeneities are discussed.

Infants code the direction of chromatic quadrature motion.

The present experiment uses a quadrature motion paradigm to investigate the motion correspondence cues used by young infants for coding the direction of motion of red/green isoluminant gratings. Three-month-old infants and adults were tested with 0.25 c/d luminance-modulated or red/green isoluminant gratings, either moving continuously or shifted in spatial quadrature. Both direction-of-motion and detection thresholds were measured, and motion:detection (M:D) threshold ratios were examined. Infants, like adults, could code the direction of motion of red/green quadrature-shifted gratings. In adults, M:D ratios were similar for continuous and quadrature motion. In infants, M:D ratios were higher for quadrature than for continuous motion, but elevations of similar magnitude were seen for both luminance-modulated and red/green gratings. The results suggest that frequency-doubled signals, such as those often seen in the magnocellular (M-cell) pathway, are not necessary for coding the direction of motion of isoluminant gratings in infant subjects. Two other theoretical options--mediation by the scatter of isoluminance points in the M-cell population, and parvocellular (P-cell) mediation--are discussed.

Identification of the calcium binding site and a novel ytterbium site in blood coagulation factor XIII by x-ray crystallography.

The presence or absence of calcium determines the activation, activity, oligomerization, and stability of blood coagulation factor XIII. To explore these observed effects, we have determined the x-ray crystal structure of recombinant factor XIII A2 in the presence of calcium, strontium, and ytterbium. The main calcium binding site within each monomer involves the main chain oxygen atom of Ala-457, and also the side chains from residues Asn-436, Asp-438, Glu-485, and Glu-490. Calcium and strontium bind in the same location, while ytterbium binds several angstroms removed. A novel ytterbium binding site is also found at the dimer two-fold axis, near residues Asp-270 and Glu-272, and this site may be related to the reported inhibition by lanthanide metals (Achyuthan, K. E., Mary, A., and Greenberg, C. S. (1989) Biochem. J. 257, 331-338). The overall structure of ion-bound factor XIII is very similar to the previously determined crystal structures of factor XIII zymogen, likely due to the constraints of this monoclinic crystal form. We have merged the three independent sets of water molecules in the structures to determine which water molecules are conserved and possibly structurally significant.

Structural determinants of the bifunctional corn Hageman factor inhibitor: x-ray crystal structure at 1.95 A resolution.

Corn Hageman factor inhibitor (CHFI) is a bifunctional 127 residue, 13.6 kDa protein isolated from corn seeds. It inhibits mammalian trypsin and Factor XIIa (Hageman Factor) of the contact pathway of coagulation as well as alpha-amylases from several insect species. Among the plasma proteinases, CHFI specifically inhibits Factor XIIa without affecting the activity of other coagulation proteinases. We have isolated CHFI from corn and determined the crystallographic structure at 1.95 A resolution. Additionally, we have solved the structure of the recombinant protein produced in Escherichia coli at 2.2 A resolution. The two proteins are essentially identical. The proteinase binding loop is in the canonical conformation for proteinase inhibitors. In an effort to understand alpha-amylase inhibition by members of the family of 25 cereal trypsin/alpha-amylase inhibitors, we have made three-dimensional models of several proteins in the family based on the CHFI coordinates and the coordinates determined for wheat alpha-amylase inhibitor 0.19 [Oda, Y., Matsunaga, T., Fukuyama, K., Miyazaki, T., and Morimoto, T. (1997) Biochemistry 36, 13503-13511]. From an analysis of the models and a structure-based sequence analysis, we propose a testable hypothesis for the regions of these proteins which bind alpha-amylase. In the course of the investigations, we have found that the cereal trypsin/alpha-amylase inhibitor family is evolutionarily related to the family of nonspecific lipid-transfer proteins of plants. This is a new addition to the group which now consists of the trypsin/alpha-amylase inhibitors, 2S seed storage albumins, and the lipid-transfer family. Apparently, the four-helix conformation has been a successful vehicle in plant evolution for providing protection from predators, food for the embryo, and lipid transfer.

Coagulation factor XIIIa undergoes a conformational change evoked by glutamine substrate. Studies on kinetics of inhibition and binding of XIIIA by a cross-reacting antifibrinogen antibody.

Coagulation factor XIIIa, plasma transglutaminase (endo-gamma-glutamine:epsilon-lysine transferase EC 2.3.2.13) catalyzes isopeptide bond formation between glutamine and lysine residues and rapidly cross-links fibrin clots. A monoclonal antibody (5A2) directed to a fibrinogen Aalpha-chain segment 529-539 was previously observed from analysis of end-stage plasma clots to block fibrin alpha-chain cross-linking. This prompted the study of its effect on nonfibrinogen substrates, with the prospect that 5A2 was inhibiting XIIIa directly. It inhibited XIIIa-catalyzed incorporation of the amine donor substrate dansylcadaverine into the glutamine acceptor dimethylcasein in an uncompetitive manner with respect to dimethylcasein utilization and competitively with respect to dansylcadaverine. Uncompetitive inhibition was also observed with the synthetic glutamine substrate, LGPGQSKVIG. Theoretically, uncompetitive inhibition arises from preferential interaction of the inhibitor with the enzyme-substrate complex but is also found to inhibit gamma-chain cross-linking. The conjunction of the uncompetitive and competitive modes of inhibition indicates in theory that this bireactant system involves an ordered reaction in which docking of the glutamine substrate precedes the amine exchange. The presence of substrate enhanced binding of 5A2 to XIIIa, an interaction deemed to occur through a C-terminal segment of the XIIIa A-chain (643-658, GSDMTVTVQFTNPLKE), 55% of which comprises sequences occurring in the fibrinogen epitope Aalpha-(529-540) (GSESGIFTNTKE). Removal of the C-terminal domain from XIIIa abolishes the inhibitory effect of 5A2 on activity. Crystallographic studies on recombinant XIIIa place the segment 643-658 in the region of the groove through which glutamine substrates access the active site and have predicted that for catalysis, a conformational change may accompany glutamine-substrate binding. The uncompetitive inhibition and the substrate-dependent binding of 5A2 provide evidence for the conformational change.

Spatial and temporal aspects of infant color vision.

The present paper constitutes a review of the literature on young infants' chromatic discrimination capabilities. A series of early studies showed that infants as young as two months postnatal can make at least some chromatic discriminations between stationary, homogeneous fields of different wavelength compositions. Current studies of spatial and temporal contrast sensitivity functions (CSFs) for red/green isoluminant stimuli suggest that spatial chromatic CSFs show developmental changes in sensitivity and spatial scale, but not curve shape; while temporal chromatic CSFs (tCSFs) show developmental changes in sensitivity and curve shape, but not temporal scale. Infants can also code the direction of motion of moving isoluminant red/green gratings, for both continuous and quadrature motion. The possible mechanisms that underlie infants' chromatic discriminations are discussed.

Development of the spatio-chromatic visual evoked potential (VEP): a longitudinal study.

Most prior visual evoked potential (VEP) research on the development of color vision has employed pattern-reversing stimuli that are not optimal for producing chromatic responses. We measured infant VEPs using low spatial frequency, onset-offset stimuli, modulated along the three axes of a cone-based color space (Derrington et al. [J. Physiol 1984;357, 241-265.]). Three color-normal infants were tested in a longitudinal design over the first postnatal year. One red/green color-deficient infant was also tested at 197 days. We found that VEP responses to S-axis (tritan) stimuli have their initial onset later than responses to red/green (L-M) or achromatic stimuli, and that developmental changes in VEP waveforms are more complex and longer lasting for chromatic than for achromatic stimuli. Possible mechanisms underlying these changes are discussed.

Severity of color vision defects: electroretinographic (ERG), molecular and behavioral studies.

Earlier research on phenotype/genotype relationships in color vision has shown imperfect predictability of color matching from the photopigment spectral sensitivities inferred from molecular genetic analysis. We previously observed that not all of the genes of the X-chromosome linked photopigment gene locus are expressed in the retina. Since sequence analysis of DNA does not necessarily reveal which of the genes are expressed into photopigments, we used ERG spectral sensitivities and adaptation measurements to assess expressed photopigment complement. Many deuteranomalous subjects had L, M, and L-M hybrid genes. The ERG results showed that M pigment is not present in measurable quantities in deutan subjects. Using these results to determine gene expression improved the correlations between inferred pigment separation and color matching. Furthermore, we found a subject who had normal L and M genes and normal proximal promoter sequences, yet he had a single photopigment (M) by ERG and tested as a protanope. These results demonstrate the utility of ERG measurements in studies of molecular genetics of color vision deficiencies, and further support the conclusion that not all genes are expressed in color deficient subjects. In particular, deuteranomaly requires a presently unknown mechanism of selective expression which excludes normal M genes and allows expression of L-M hybrid genes in one cone type, and the normal L in another.

Development of pattern visual evoked potentials: longitudinal measurements in human infants.

PURPOSE: This experiment used longitudinal testing to trace the emergence of the major components of pattern visual evoked potentials (VEPs) in infants, using two paradigms: large-checkerboard pattern reversal and low spatial frequency pattern onset. METHODS: Testing with both pattern-reversal and pattern-onset stimuli was performed on the same infants. Testing was conducted at weekly intervals during the first three postnatal months, and at intervals of 2 weeks to 1 month thereafter. RESULTS: The pattern-reversal and early pattern-onset responses recorded within individual subjects showed remarkably systematic developmental sequences. The broad, positive component seen at 200 to 250 ms in infants could be traced readily through the developmental sequence, to become the more sharply tuned positive component seen at about 100 ms in adults. Responses to low spatial frequency pattern onsets in infants were larger and more reliable than those in adults. The late components of the pattern-onset response, generally attributed to pattern offset, emerged later and with more complex changes. In all cases, response amplitude was much more variable than response latency, both within and between subjects. CONCLUSIONS: Frequent VEP recording in a longitudinal design can reveal systematic and detailed transitions of wave-form during development.

Infant color vision: temporal contrast sensitivity functions for chromatic (red/green) stimuli in 3-month-olds.

In order to investigate the development of temporal contrast sensitivity functions (tCSFs) for chromatic (red/green) stimuli, we obtained chromatic contrast thresholds from 3-month-old infants and adults using behavioral techniques. Stimuli were moving or counterphase-reversing sinusoidal gratings of 0.25 c/deg. Five temporal frequencies were used: 0.7, 2.1, 5.6, 11 and 17 Hz (corresponding speeds = 2.8, 8.4, 22, 44 and 67 deg/sec). In order to compare chromatic results with those obtained under luminance-defined conditions, luminance tCSFs were also obtained from adults, and previously obtained infant luminance tCSFs were used (from Dobkins & Teller, 1996a). In accordance with previous studies, adults exhibited bandpass luminance tCSFs with peaks near 5 Hz and lowpass chromatic tCSFs that declined rapidly at temporal frequencies greater than 2 Hz, and the two curves crossed one another near 4 Hz. By contrast, infants exhibited bandpass rather than lowpass chromatic tCSFs with peaks near 5 Hz. These chromatic curves were quite similar in peak frequency and general shape to previously obtained infant tCSFs for luminance stimuli. Moreover, both chromatic and luminance tCSFs in infants were found to be quite similar in peak and shape to luminance tCSFs observed in adults. These findings point to the possibility that, for 3-month-old infants, both chromatic and luminance stimuli are detected by the same underlying mechanism under these conditions. We propose that such a mechanism is probably a physiological pathway dominated by magnocellular input. Earlier studies of infant color vision are discussed in this context.