Size and Shape of Bovine Interphotoreceptor Retinoid-binding

THE JOURNAL OF BIOLOGICAL CHEMISTRY I C ‘ 1987 by The American Society for Biochemistry and Molecular Biology, Inc

VOI. 262, No. 27, Issue of September 25, pp. 13198-13203, 1987 Printed in CJ S. A.

Size and Shape of Bovine Interphotoreceptor Retinoid-binding Protein by Electron Microscopy and Hydrodynamic Analysis*

(Received for publication, March 10, 1987)

Alice J. AdlerS, Walter F. Stafford 1114, and Henry S. Slaytern From the Eye Research Institute of Retina Foundation, the §Boston Biomedical Research Institute, and the 1Danu-Farber Cancer Institute and the Department of Physiology and Biophysics, Harvard Medical School, Boston, Massachusetts 02114

Individual molecules of interphotoreceptor retinoid- binding protein (IRBP), a protein likely to be impor- tant in the visual cycle, were visualized by means of electron microscopy. IRBP was coated with a very thin layer of tungsten and photographed by dark-field im- aging. IRBP is seen to be a flexible, elongated molecule about 24 nm in length by 3-4 nm in width (statistical modes). These dimensions agree very well with those calculated from the frictional ratio obtained from sedimentation data. Approximately half of these rod- shaped IRBP molecules are straight, and half are bent in the middle, usually with an angle of 60-90” between the two arms. A representation of IRBP as a bendable string of beads yields calculations of dimensions and of hydrodynamic parameters consistent with the electron microscopic and sedimentation data; the sedimentation coefficients derived from this representation are nearly insensitive to molecular bending.

When IRBP is bound to saturating amounts of its endogenous ligands, all-trans- or 1 1-cis-retinol, its sedimentation behavior is unchanged, and the same types of particles are visualized by electron microscopy as with the free protein; however, a greater proportion of the molecules are bent. Deglycosylation of IRBP (with peptide:N-glycosidase F) results in a somewhat smaller molecule that retains its rod-like shape, as shown by gel filtration and sedimentation data. The results indicate that IRBP is an elongated molecule and suggest that a structural change may occur upon ligand binding.

Transport of vitamin A between the vertebrate retina and its supportive pigment epithelium is essential for regeneration of visual pigment (1); this exchange proceeds through the interphotoreceptor matrix (2) lying between these two tissues. In this matrix, retinoids are carried by IRBP’ (3, 4). The amount of all-trans-retinol endogenously bound to IRBP in- creases about 5-fold (5, 6) when rhodopsin is bleached in the bovine eye. This evidence suggests that IRBP functions by shuttling vitamin A between the photoreceptors and the retinal pigment epithelium during the visual cycle.

*This research was supported by United States Public Health Service Grants GM-14237 (to H. S . S.) and EY-04368 (to A. J. A) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$To whom correspondence should be addressed Eye Research Inst. of Retina Found., 20 Staniford St., Boston, MA 02114.

I The abbreviations used are: IRBP, interphotoreceptor retinoid- binding protein; EM, electron microscopy; HPLC, high-performance liquid chromatography; R,, Stokes radius.

IRBP has recently been purified (7), and some aspects of its structural and ligand-binding properties have been char- acterized (5,8-11). With a molecular weight of about 132,000 ( 5 , 8), IRBP is the biggest of the retinoid-binding proteins. Its unusually large Stokes radius, 5.5 nm (5, 8), indicates that the molecule is asymmetric, not globular. Its high frictional ratio, calculated from sedimentation data (5, 8), is consistent with an elongated molecule, for which one possible model is an ellipsoid with length and width of approximately 22 and 3 nm, respectively. This idealized description of IRBP contains no information about the real shape of the protein; direct visualization is required for this. Furthermore, IRBP is a glycoprotein with about 10% asparagine-linked carbohydrate (8, 9); the chains are of both complex and hybrid types (10) and could conceivably affect the structure of the molecule.

Electron microscopy, given appropriately prepared samples, allows visualization of individual macromolecules and can provide high-resolution structural information even for relatively small proteins. The method applied here is metal rep- lication, in which the sample is sprayed onto a grid and then rotary-shadowed with a very thin layer of tungsten. Through this procedure, dimensions have previously been obtained even for a polypeptide of M, 21,000 (pituitary growth hor- mone); a conformational t,ransition has been observed in serum albumin (Mr 68,000); and the width of the myosin tail (only 2 nm) has been measured (all reviewed in Ref. 12). Several glycoproteins have been examined in this manner (13), but they are all larger than IRBP.

In this study, we subjected IRBP concurrently to EM by the dark-field tungsten-shadowing technique and to hydrodynamic studies. IRBP was seen to be an extended molecule, with most frequent dimensions of 24-nm length by 3-4-nm width, in agreement with the previous sedimentation model. The molecule is flexible and is often bent in the middle, especially when bound to its retinol ligands.

MATERIALS AND METHODS

ZRBP Preparations-IRBP was isolated from the interphotoreceptor matrix of bovine eyes basically as previously reported (7). Two minor modifications were made: impurities were removed by gel filtration on HPLC (see below), and protein solutions were concen- trated with Centricon microconcentrators (from Amicon). IRBP con- centrations were measured by absorbance at 280 nm (8). Purity was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (7).

One requirement of the EM methodology is that the protein sample must be prepared in a completely volatile solvent. Therefore, IRBP samples (0.5 ml) were transferred out of phosphate-buffered saline into 0.1 M ammonium acetate (J. T. Baker Chemical Co. crystal) (pH 7.0) by passage through a Sepharose CL-4B (Pharmacia P-L Bio- chemicals) gel filtration column (bed volume of 5 ml).

For some experiments, IRBP’s binding sites were saturated with ligand by incubation for 30 min at room temperature in the dark with a 10-fold molar excess of either all-trans-retinol (Sigma) or ll-cis-

13198

Electron Microscopy of IRBP 13199

retinol (prepared by sodium borohydride reduction (14) of 11-cis- retinaldehyde from Hoffmann-La Roche). Excess retinol was removed by passage of the sample through the same type of small Sepharose column as was used for salt exchange. The extent of ligand binding was determined by fluorescence (8).

Removal of IRBPs carbohydrate chains was accomplished with peptide:N-glycosidase F (from Genzyme, Boston, MA). This enzyme (15) cleaves glycoproteins between asparagine and the proximal N - acetylglucosamine of the sugar chain. The reaction, between 0.4 mg of native IRBP (no detergent present) and 1.2 units of enzyme, was carried out at 37 'C for 18 h in 0.15 ml of 0.2 M Tris buffer (pH 7.4) containing 0.01 M dithiothreitol. Deglycosylation was complete, as demonstrated by sugar analyses and by failure of the treated protein to bind to concanavalin A-Sepharose.

Electron Microscopy-IRBP samples were prepared at a concentration of 0.1 mg/ml in aqueous solutions containing 0.1 M volatile salt, ammonium acetate, and 50% (v/v) glycerol. The samples were diluted 4-fold with the same solvent and immediately sprayed onto mica.

Procedures used for electron microscopy have been presented in detail elsewhere (12, 16-18). Briefly, IRBP samples containing ammonium acetate and glycerol were applied as an aerosol to freshly cleaved mica. After 20 h of outgassing at Torr to evaporate the solvent, preparations were coated with a very thin layer of tungsten by electron beam evaporation. Mass thickness of the background film of tungsten was 9.3 X g/cm*. This metal layer was subsequently coated with 2.5 nm of carbon. Micrographs were recorded very close to focus on a JEM lOOCX electron microscope at 100 kV, generally at a magnification of X 40,000-53,000, Dark-field images were obtained from the same lightly shadowed specimens using matched annular condenser and objective apertures (17).

Analytical Size-exclusion Chromatography-Samples of IRBP and of molecular weight standard proteins were subjected to HPLC through a Beckman Spherogel TSK 4000 SW column using a Beck- man system 344 chromatograph monitored by absorbance at 280 nm. The Stokes radius for IRBP (under various conditions) was evaluated by the method of Ackers (19). (The same experimental procedure was used also to purify IRBP samples.)

Analytical Ultracentrifugation-Sedimentation velocity runs were performed at a speed of 52,000 rpm at 20 "C on a Beckman Model E analytical ultracentrifuge equipped with an ultraviolet scanner system at 280 nm. The loading concentration of IRBP samples in various solvents was 0.3 mg/ml.

High-speed equilibrium sedimentation runs for molecular weight determination were performed at 4 'C on the same instrument using Rayleigh optics and a speed of 16,000 rpm. The method has been presented (8, 20).

RESULTS

Because it was necessary to prepare IRBP samples in 0.1 M ammonium acetate for EM experiments, we examined some hydrodynamic properties of IRBP in this salt. The rationale was to see whether the change in solvent, from phosphate- buffered 0.15 M sodium chloride (8), caused any change in IRBP size or shape in solution and also to examine any effects of ligand binding. These results are presented, but the data are not shown except for a sedimentation equilibrium run (Fig. 1). In summary, no significant solvent differences were found. Therefore, any conclusions drawn from EM about the state of the protein in solution (13) should apply to IRBP in conventional saline as well. Furthermore, neither ammonium acetate nor the presence of 50% glycerol affected retinol binding, as monitored by fluorescence (8).

Stokes Radius from Size-exclusion HPLC-The partition coefficient, u, of IRBP on a TSK 4000 SW column was always found to be 0.48-0.50 and was not influenced by which salt was present or by whether retinol was bound. The calculated R, (taking into account scatter in the standard curve) was 5.4 f 0.2 nm, identical with that previously measured on a 3000 SW column (8). Removal of sugar chains from IRBP caused a small decrease in effective size; R, was reduced to 4.9 f 0.2 nm .

Sedimentation Coefficient-Sedimentation velocity analysis

1.0 1

X

n i 1 - 1 I

I I 0.0 0.5 1.0 1.5

CONCENTRATION (g/L)

FIG. 1. Sedimentation equilibrium experiment for IRBP in 0.1 M ammonium acetate. In this run, IRBP was saturated with 11-cis-retinol. The cell loading concentration of the protein was 0.25 mg/ml. Four types of molecular-weight averages are plotted Versus local cell concentration: W, M,; A, M.; +, Myl; 0, Ms. The inset is a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel of this IRBP sample showing the molecular weight range of 220,000 (top of gel) through 14,000 (bottom). Bovine IRBP runs at an apparent M, of 140,000.

of IRBP showed a single, symmetrical boundary. The value of s : , , ~ was 5.7 f 0.2 S, again (as with R,) independent of solvent or presence of ligand; previous measurements in sodium chloride yielded 5.8 S (8). Deglycosylated IRBP had a sedimentation coefficient of 5.4 S.

Molecular Weight-Equilibrium sedimentation analysis (by curve fitting) of IRBP in ammonium acetate buffer gave a molecular weight of 130,000 f 2,000 and showed only an insignificant amount of aggregation. The divergence of the molecular weight moments suggested the presence of a small amount of material with a molecular weight higher than that of IRBP monomer, probably not more than 10 f 5% in the original solution if present as the dimer. This run should be compared to that presented earlier (8), which led to M, 133,000 and showed considerably more aggregation at the same loading concentration. The reason for the difference in association is unclear. Perhaps the purification was better, the preparation was fresher, or the tendency to aggregate is less in acetate than in chloride (which may bind to the protein).

No difference was observed between apo-IRBP and the protein saturated with either of its retinol ligands; all samples appeared basically monodisperse. The gel depicted in Fig. 1 shows that minor modifications in the preparation of IRBP did not detract from its purity.

Observation of IRBP in the Electron Microscope-Fig. 2 shows a typical dark-field micrograph of individual IRBP molecules coated with tungsten. Most of the IRBP molecules appear as extended particles, either straight or bent in the middle, of about the same contour length. The remainder are more compact, globular particles. Beneath the field are shown arrays of the elongated structures, some straight, some bent. Thus, IRBP appears to be a rod-like but flexible molecule. The globular structures are probably representations of various projected views of IRBP viewed from different perspec- tives, including molecules that collapsed on the grid; this interpretation is supported by their size (see below), which indicates that they are not aggregates.

The particles in the photographs were measured. The ranges for several of IRBP's dimensions are given in the histograms of Figs. 3 and 4. Although the spread in all of the parameters is considerable, the lengths of the elongated molecules, both linear (Fig. 3a) and flexed (Fig. 3b) , are seen to

13200 Electron Microscopy of IRBP

2 5 nm -

FIG. 2. Dark-field electron micrographs of IRBP prepared by rotary shadowing with a very thin layer of tungsten. The field shows the variety of projected shapes that IRBP molecules display on the grid. The diamond outlinw indicate linear extended particles: the triangular outlines indicate bent ones. The circles encompass globular structures that are not clearly identifiable in shape; these were scored either as spheres (example toward l ~ f t of field) or as cleft particles (at bottom of field). The gallery below the field shows two rows of selected, individual particles illustrating (at higher magnification) the bent and linear forms of IRRP.

peak at about 24 nm. The mode for the rod's width (Fig. 4a) is 3-4 nm. The most frequent value for the angle between the two halves of the bent molecule (Fig. 46) is 60-75", although angles up to 90" are common.

The dimensions measured for IRBP are summarized in Table I. The most striking results are ( a ) that the modal values for length (24 nm) and for width (3-4 nm) are the same for hoth t-ypes of elongated molecules seen in EM (straight and bent) and (6) that these dimensions agree almost exactly with those calculated previously ( 5 , 8) from sedimentation data based on an assumed model of IRBP as a prolate ellipsoid of revolution with an axial ratio of 7:l or 8:l.

The broad distribution in length, seen in Fig. 3a, cannot be attributed to aggregation of IRBP in solution since very little was observed in the ultracentrifuge. One possible explanation for this variability in length is the natural flexibility of protein molecules; such extension over a large range of lengths has been observed in EM for nearly all proteins examined (13, 21), except for rigidly helical ones. Another possibility is that IRRP molecules may associate during drying on the grid, perhaps in a linear, overlapping manner (which would account for the secondary peaks at 36 nm and 48 nm).

The spread in the measurements is evident from the histograms and from the standard deviations (from the mean values) listed in Table I. The mean values are greatly skewed by outlying measurements and therefore are not tabulated. The dimensions given in Table I have not been corrected for the thickness of the tungsten coating; t.his could create considerable uncertainty, particularly in the width, for a molecule

as thin as IRBP. Although the average background thickness of the coating is only about one tungsten atom (0.5 nm), the coating is seen in EM to be discontinuous; the white spots in the background of Fig. 2, up to 2 nm in diameter, are aggregates of atoms. If the probable correction for tungsten thickness on the molecular replica, about 0.8-1.5 nm, were applied, it would reduce the measured IRBP width. However, given the relatively small size of IRRP and the rather complex tertiary structure reflected in the particle images, i t is considered appropriate simply to state that the width of the extended form of the molecule appears to vary irregularly along its length, in the range of 1-4 nm, presenting no particular pattern relating one particle to the next. That is, the particle's intrinsic roughness appears to represent a greater uncertainty in width than does the thickness of the metal coating.

It was stated above that the globular particles (classified as spherical and cleft) seen in EM appear to be various projec- tions of single, rod-like IRBP molecules and that they are probably neither aggregates nor species with vastly different tertiary structures. The evidence for this conclusion is that the modal maximum diameter for all the folded species (bent, cleft, and spherical) is the same, 12-13 nm, and that this measurement is exactly half the total length of the extended particles (straight and bent), 23-25 nm. These observations are consistent with the IRBP molecule's retaining its basic size and shape, but often folding, collapsing, and settling at various angles when dried on the grid.

A similar analysis could be applied to the variety of particles, seen in EM, that appear to be bent a t a range of angles

Electron Microscopy of IRBP 13201

8.98 16,W 32,W 48.09 Length (nn)

FIG. 3. Histograms showing distributions of IRBP lengths as measured from electron micrographs. a, length of extended IRBP particles in the straight configuration; b, contour length of extended, bent IRBP molecules.

a,@ 8.m 16.88 6% 128 Degrees

98 ’ ~ S E

Wldth (fill)

FIG. 4. Histograms of widths and angles. a, distribution of apparent widths of straight IRBP particles; b, distribution of the angles subtended by the two adjacent arms of bent IRBP particles. (Straight molecules were not included in this histogram; they would have given rise to an equivalent peak centered at 180”).

(Fig. 2 and 4 b ) . These forms would be indistinguishable by sedimentation analysis (see “Discussion”) and possibly may not exist in solution. However, it is very unusual for molecules to appear bent in the electron microscope unless they are actually flexible (13); this indicates that IRBP’s observed flexion most likely is not the result of its bending during drying. In any case, whereas the variety of shapes seen in EM may not exist in solution, the potential flexibility manifested by this variability suggests, but does not necessarily prove, that the IRBP particles are flexible. At the very least, even if IRBP is always straight in solution, it is likely to contain a swivel point allowing it to bend when settling on the grid.

EM of IRBP Bound to its Ligands-When IRBP was saturated with either all-trans- or 11-cis-retinal (which are its endogenous ligands) and examined by dark-field EM, the

TABLE I Dimensions of IRBP particles as seen by electron microscopy Projected Most shape of Measurement” frequent S.D. nb particle value

nrn nrn Straight Length 23.0 11.5 52

Width 3.5 7.0 54 Maximum diameter 18.5 8.7 52

Bent Contour length 24.6 4.4 56 Width 4.5 2.3 56 Maximum diameter 12.7 2.8 56

Cleft Length 19.0 6.1 43 Width 4.1/6.4‘ 1.9 43 Maximum diameter 12.9 3.4 43

Spherical Lengthd 12.6 3.3 38 No corrections have been made for thickness of tungsten coating. n, number of measurements. All observed particles in a given

field were classified and measured. Distribution was bimodal. For a sphere, length = width = maximum diameter.

TABLE I1 Effect of retinol binding on IRBP structure

Ligand Ratio, coefficient bent: n

Sedimentation Stokes radius

straight“ Meas- Calcu- Meas- Calcu- uredb lated’ wedd lated’

nrn None 1.1:l 189 5.7 -t 0.2 5.8 5.4 -t 0.2 5.3 All-trans-retinol 1.9:1 234 5.7 -t 0.2 5.9 5.4 f 0.2 5.3 11-cis-Retinol 3.1:l 663 5.7 -t 0.2 5.9 5.4 & 0.2 5.2

(I As determined by examination of n particles in EM. Measured by sedimentation velocity. The model of IRBP as a string of seven beads was used to calculate

so and R, for the straight and bent configurations. (See Fig. 5 and “Discussion.”) Weighted averages of these values were then obtained for each appropriate bentstraight ratio.

Measured by size-exclusion HPLC.

same set of particle shapes was seen as with the apoprotein. Furthermore, each particle dimension was unaltered (within experimental error). Extended particles (straight plus bent) comprised 55 f 2%, and globular particles (cleft plus spherical) comprised 45% of observed structures in all three cases.

However, the binding of ligands was associated with an increase in the number of bent, extended particles at the expense of straight, extended ones. 11-&-Retinol effected more of this apparent transition, expressed as a ratio of shapes in Table 11, than did the all-trans isomer. Since a large number of particles were measured, this finding is considered statis- tically significant.

DISCUSSION

An electron microscopic method capable of visualizing relatively small macromolecular features was applied to interphotoreceptor retinoid-binding protein. IRBP was seen to be an elongated but flexible molecule, about 24 nm long and 3- 4 nm wide, sometimes straight and sometimes bent in the middle. IRBP is one of the smallest proteins (12, 13) with complex tertiary structure to be successfully analyzed by EM. The dimensions found for IRBP agree with the conclusion reached from sedimentation experiments (5, 8) that IRBP is asymmetric and can possibly be approximated by an ellipsoid with axial ratio of 7 or 8. The EM observations, however, are not merely confirmatory, but yield more detailed information

13202 Electron Microscopy of IRBP

about shape and variability than is possible to deduce from hydrodynamic data. Most strikingly, in the present case, they demonstrate that IRBP is often flexed at its midpoint.

IRBP is capable of carrying two molecules of retinol under some conditions ( 5 , 9). It is possible that each binding site may be on a separate arm of the molecule. An additional finding suggesting the existence of such domains is that a cloned cDNA coding for human IRBP contains two long segments with high residue identity, implying an internal gene duplication (22). IRBP contains considerable secondary structure (8), estimated at -15% a-helix and -20% @-chain. Perhaps such conformations help maintain the relative rigid- ity of the molecule’s arms.

To determine whether the entities seen in EM actually correspond to single molecules of IRBP or were possibly aggregates of them, we compared the dimensions of the particles to a mathematical representation of the molecule. IRBP was approximated (Fig. 5 ) by a colinear arrangement of seven spheres to give a chain with an axial ratio of 7:l; this was based on previous estimates of shape derived from the frictional ratio (5,8), not from the EM data. From the measured mass (130,000 daltons) for the IRBP monomer and a partial specific volume of 0.73 ml/g, a diameter of 3.53 nm was calculated for each sphere; this gave the correct volume for the entire, dehydrated molecule. The dimensions of this representation (24.7-nm length x 3.5-nm width) are almost identical to those of the extended, straight particles seen in EM (Table I). The bent molecules were approximated by the same string of seven spheres, bent around the center sphere at an angle of 60”. (This angle was chosen since it corresponds to the most frequent value in the histogram of Fig. 4b.) These mathematical constructs, shown in Fig. 5 , are not intended as literal models for IRBP’s structure.

Sedimentation coefficients for both the straight and the bent “string-of-beads” representations for IRBP were calculated by the method of Garcia de la Torre and Bloomfield (23) using the computer program GENTRA of these authors for the translational frictional coefficient. The calculated values of the sedimentation coefficients were 5.5 and 6.1 S for the straight and bent models respectively, using a value for the hydration of 0.3 g of H20/g of protein. (These estimates are not particularly sensitive to the values chosen for hydra-

rn 3.53 nm

li/ 5.5s 6.1 S

FIG. 5. Mathematical representation used for calculation of sedimentation coefficients. The structural isomers of IRBP (straight and bent) were approximated with a string-of-beads model consisting of a set of seven spheres having the same total volume as a single IRBP molecule. The dimensions calculated agree very well with the dimensions measured for the straight particles seen in EM (Table I). The bent molecules were represented by the same string of spheres bent around the center sphere at an angle of 60”; this angle was chosen since it corresponds to the maximum on the histogram shown in Fig. 46. See text for explanation of how dimensions were obtained and how sedimentation coefficients were calculated.

tion.) Since the measured value of the sedimentation coefficient would be a weighted average of the sedimentation coefficients of the species present, these calculated values agree very well with the observed value of 5.7 S. Therefore, it seems likely that the particles seen in EM correspond to individual IRBP molecules.

If an axial ratio of 8:l instead of 7:l is chosen for IRBP, a string-of-beads calculation results in sedimentation coefficients of 5.5 and 5.9 S for the straight and bent models, respectively (even better agreement with experiment); however, the dimensions for this version of the model are 27.0 by 3.4 nm (somewhat worse agreement, but still very plausible). The sedimentation equation (24), which relates molecular weight, sedimentation coefficient, Stokes radius, and partial specific volume of a macromolecule to one another, can be used to calculate the Stokes radius for the IRBP models. (This equation is quoted and applied to IRBP data in Ref. 8.) The results of this calculation are 5.6 nm for the straight model and 5.1 nm for the bent one, in fine agreement with the measured value of 5.4 nm for IRBP (obtained independ- ently from size-exclusion chromatography).

A surprising outcome of the string-of-beads representation as applied to IRBP is the relative lack of sensitivity of the calculated so and R, values to bending of the molecule. This insensitivity to flexion explains how the mixture of particle shapes seen in EM (and unsuspected from the sedimentation results of Ref. 8) could have their dimensions fit so well by a single, conventional, ellipsoidal representation (5,8). Because of the predicted insensitivity of s to the shape of IRBP, we cannot make an unequivocal statement about the molecule’s flexibility in solution; however, the range of shapes seen in EM would indicate that IRBP has flexibility and possibly a swivel point near the middle. The calculations serve to show that the possible extreme shapes seen and modeled are consistent with the observed value of s.

Binding of ligands to IRBP appears to result in a partial straight-to-bent shape transition, as seen in EM and summarized in Table 11. These data illustrate that the bending of a greater fraction of IRBP molecules does not cause any experimentally detectable change in so or R,, as discussed above. This shape transition, which is more complete for 11- cis- than for all-trans-retinol, may be correlated with the number of binding sites filled. IRBP appears to bind 2 eq of 11-cis-retinol a t saturation, but only 1 eq of the all-trans isomer (25). However, ligand-induced flexion may be irrele- vant physiologically since IRBP’s endogenous load of retinol in the eye corresponds, a t most, to only 30% saturation of one site (4, 5 , 7). Although saturation with exogenous retinol causes a greater proportion of IRBP molecules to bend, it does not effect a noticeable change in secondary structure, as shown by circular dichroism data in the peptide region (8). Perhaps this constitutes additional evidence that the more structured regions of the molecule reside in its two arms (as suggested above) and not in the hinge region between the arms, which is subject to bending.

Removal of IRBP’s carbohydrate chains results in small decreases in Stokes radius and sedimentation coefficient. When these values are inserted into the sedimentation equation (24) (and 0.74 is used for the partial specific volume of the deglycosylated protein), the calculated molecular weight is 116,000 k 8,000. This M , is 12 5 1% smaller than that measured for control IRBP and can be accounted for simply by loss of the weight of the sugar components, which analysis shows to be 11% of IRBP (8). Therefore, removal of carbohydrate results in a somewhat smaller molecule with no apparent change in shape. The sugar chains (containingabout

Electron Mia

8-10 sialic acid residues/IRBP molecule) (8, 10) do not seem to be required for IRBP’s elongated structure. This situation can be contrasted to that of epiglycanin, a mucin-like glycoprotein, whose rod-like shape seen in the EM (26) collapses upon deglycosylation.2

In conclusion, this report establishes, through EM rneas- urements, that IRBP is an elongated molecule capable of bending and that this bending may constitute a structural transition that accompanies IRBP’s function in retinol binding.

Acknowledgments-We would like to thank Dr. P. F. Sorter of Hoffmann-LaRoche for a gift of 11-cis-retinaldehyde and Susan Spencer and Rosilyn Ford for expert technical assistance.

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*oscopy of IRBP 13203

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Documents

Size and Shape of Bovine Interphotoreceptor Retinoid-binding