fit the experimental data for the aggregated prepara- tions. Analysis of the end-to-end distance of negatively stained segment long spacing crystallites prepared.
THEJOURNAL
OF
BIOLOGICAL CHEMISTRY
Vol. 255, No. 19, Issue of October 10, pp, 9427-9433, 1980 Printed in U.S.A.
Type I Collagen in Solution STRUCTURE AND PROPERTIES OF FIBRIL FRAGMENTS* (Received for publication, August 3, 1979, and in revised form, January 14, 1980)
Frederick H. Silver$ and RobertL.Trelstad From the Department of Pathology, Shriners Burns Institute and Massachusetts General HospitalHarvard and Medical School, Boston, Massachusetts 02114
Several models of the substructure of collagen fibrils based We havemeasuredthe diffision coefficientand weight average molecular weight of type I collagen on theoretical and experimental considerations have been of rat tail proposed. The microfibril, the smallest structural unit, confibril fragments obtained by acid extraction of lathyritic chick skin, tains from 4 to 8 collagen molecules in cross-section (7-10). tendons and neutral extraction using laser light scattering techniques. The molecular Other evidence suggests that lateral order within a fibril is not weightandtranslationaldiffusioncoefficientwere long range (11) and may be associated with the asymmetry of found to be 8.05 f 0.40 X 10’ and 0.450 -C 0.04 X lo-’ the molecule. cm2/s,respectively, for preparations which contain agIn vivo collagen aggregation may beginbefore secretion gregates in 0.01 M HCl, and 2.82 0.20 X 10’ and 0.780 since intracellular vacuoles containing aggregates with lengths f 0.04 x lo-’ cm2/s for collagen single molecules. of 2 or more collagen molecules (12,13) have been observed. Using these data, as well as the theoretical difhsion Similar aggregates have been observed both intracellularly coefficient for a single collagen molecule, models for and extracellularly in a wide variety of tissues (14,15).In vitro various staggering modes and aggregate mixtures studies were of type I collagen fibrillogenesisindicate that initiation developed in an attempt to understand the structure of of the fibril components present in solution. It was foundcollagen fibril formation occurs by the conversion of single 1 to 4 D (D = 67 nm)staggers molecules into 4 D staggered dimers and trimers, suggesting that several models with fit the experimental data for the aggregated prepara- that the 4 D stagger may be thermodynamically preferred of negatively over other staggering modes (16). Further aggregation into D tions. Analysis of the end-to-end distance staggered aggregates is a secondary event (16). stainedsegmentlongspacingcrystallitesprepared The purpose of this investigation has been to characterize from solutionscontainingfibrilfragmentsandthe the physicochemical and ultrastructural properties of collagen bandingpatternofpositivelystainedsegmentlong spacing crystallites suggest that collagen solutionssolutions con- derived from solubilized type I collagen fibrils and 4 D andpossibly 4.4 D to assess the organization of such fragments in respect to their tainlinearaggregateswith staggers in agreement with light scattering Therdata. possible mode of packing in the native fibril. mal denaturation studies demonstrate that the agparMATERIALS AND METHODS’ of cross-linked linear aggregates, 33.5 ent melting point Rat Tail Tendon Collagen-Acid-soluble rat tail tendon collagen 2 0.5”C, is identical with that of single molecules atpH 2.0. We conclude that a linear filament with predomi- was prepared (see Fig. 1) by dissolving tendons from young rats in nant 4 D stagger is abasicunit of type I collagen 0.01 M HCI a t 4°C for 4 h followed by centrifugation for 30 min a t 30,000 X g. The supernatant was sequentially filtered through 0.8-, fibrillar structure. 0.65, and 0.45-micron Millipore filters. SDS’ electrophoresis and
*
amino acid analyses of this preparation showed that it contained predominantly type I collagen a chains and p and y components. The relative proportions of a,p, and y components were determined by The structure of collagen in solution has been studied chromatographing heat-denatured samples in 1.0 M CaCL on an extensively both at the molecular (1-6) and supramolecular agarose A-5m column (2.0 X 100 cm) and computing the peak areas levels (2-6). Early studies focused on the size and shape of of the effluent monitored at 230 n~ in a recording spectrophotometer. collagen single molecules (1,2),while some of the later studies Peak identification was achieved by estimating the molecular weight attempted to focus on supramolecular forms present in solu- using laser light scattering techniques. Lathyritic Chick Skin Collagen-Extracts of lathyritic chick skin tion (3-6). From these efforts, we know that extracts of type with 0.4 ionic strength potassium phosphate buffer, pH 7.6, were I collagen containing tissues are mixtures of single molecules purified by repeated NaCl precipitation at both neutral and acidic and high molecular weight aggregates. Since these fragments pH. The purified collagenwas desalted by dialysis at 4°C after are derived from native collagen fibrils,structural analyses of resolubilization in 0.01 M HCI. Evaluation of chemical purity was done these components may generate more information which can by amino acid and hexosamine analysis on HC1-hydrolyzed aliquots.
beused to develop a better understanding of localfibril structure.
’
* This work was supported by Grant PCM-7818903 from the National Science Foundation, Grants HL-18714 and GM 20007 from the National Institutes of Health, National Service Award No. 5 T32 HD 07092-01, and by the Shriners Burns Institute. 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, at the Shriners Burns Institute, 51 Blossom St., Boston, Mass. 02114.
+
Portions of this paper (including part of “Materials andMethods” and Tables 1 through 7) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, Md. 20014. Request Document No.79M-1568, cite author(s), and include a check or money order for $1.65 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press. The abbreviations used are: SDS, sodium dodecyl sulfate; SLS, segment long spacing crystallites.
9427
Type 1Collagen inSolution Preparation of Segment Long Spacing Crystallites (SLS)"sLS crystallites of rat tail tendon collagen were prepared by dialysis of collagen solutions in HC1, pH 2.0, a t 4°C uersus 0.01 M ATP in HC1, pH 2.0, overnight. A drop of the dialysate was placed on carbon/ Formvar-coated copper grids. Transmission Electron Microscopy-SLS of acid-soluble rat tail tendon collagen were stained negatively with phosphotungstic acid (PTA), pH 7.6, and positively with saturated aqueous uranyl acetate and viewed with a Philips 300 transmission electron microscope. The end-to-end length of monomeric collagen SLS was determined from negatively stained samples. The locations of the NH2 and COOH termini of negatively stained SLS were determined by measuring a molecular length from each free end. In most cases, the overlap of negatively stained polymeric SLS was obvious by the positions of the NH2 and COOH termini of 2 adjacent molecules, as is seen in Fig. 6.4. The molecular overlap was also determined by measuring the distance between Bands 3 and 4 and 51 and 52 (17) of positively stained polymeric SLS. Measurements on 10 different preparations of monomeric SLS indicated that Bands 3 and 4 and 51 and 52 are both about 7% from the ends of the molecule, while the distance between these bands is about 86%of the molecular length.
Tendon 1 g
I
HC1, pH 2.0 4"C, 4 h
Solubyized tendon
1
Centrifugation
pellet
discard
30 min @ 30,000 g
Supernatant Collagen solution Overall recovery of 0.609 g Recovery Filter through "91% 0.8-, 0.65-, and 0.45-p fdters
1
High molecular weight fraction (A?r
= 8.05 f 0.40 X lo5,D20.w= 0.45 f 0.04 x 10" cm2/s)
Overall recovery 0.554 g
RESULTS
I
Filter through Recovery The relative composition of the high molecular weight acid=M% 0.22-p filter soluble rat tail tendon as defined by chromatography on A5m was as follows: a chains, 33.6%;p components, 42.5%; and Single molecule fraction y components, 23.9%;a recovery of 88%was obtained. Amino (ar = 2.82 0.20 X lo5, DX).,,, = 0.80 f 0.04 X 10" cm2/s) acid analysis was typical of type I collagen; specifically,there were 320 residues of glycine, 2 residues of tyrosine, 93 residues Overall recovery of hydroxyproline, and 128 residues of proline. Noncollage0.277 g nous glycoproteins and proteoglycans were less than 1%based FIG. 1. Purification procedure for high molecular weight on galactosamine and glucosamine analysis. Amino acid analand single molecule fractions of acid-soluble (HCl, pH 2.0) rat ysis of the lathyritic chick skin preparation gave the following tail tendon collagen. composition: glycine,335 residues; tyrosine, 4 residues; proline, 118 residues; and hydroxyproline, 88 residues. SDS slab gels showed the presence of predominantly type I a chains; how40 ever, /3 components were present in the lathyritic preparation. In a previous study (16), we prepared neutral solutions of single collagen molecules from extracts of lathyritic type I collagen bycentrifugation and filtration through a 0.22-micron Millipore filter; here, we want to characterize the physical forms of collagens extracted by dilute acid. To accomplish this, it was essential to develop routine handling procedures to separate and characterize the different solution forms. In Bi@ 15 order to minimize the number of steps involved in the purifimr ao5,ooo cation procedure, it was necessary to study a tissue that was abundant, easily isolated, and as homogeneous as possible. 5 For these reasons, rat tail tendon collagen was chosen. l o > 0 Molecular Weights-As shown in Fig. 1, about 61%of the 0.4 0.5 0.6 0 0.1 0.2 0.3 dry tendon weight is soluble in 0.01 M HC1 at 4'C. Sequential coNcENruArloAf / m q / m l / filtration through OB-, 0.65- and 0.45-micron filters results in FIG. 2. Kc/Rs, versus concentration for high molecular a solution with a weight average molecular weight of 8.05 0.40 X (see Fig. 2), which represents 55%of the tendon weight (0)and single molecule fractions (0)of pH 2.0 HC1soluble rat tail tendon collagen. All measurements were made at by weight. This preparation will be referred to as a high a scattering angle of4' with respect to the transmitted beam a t a molecular weight fraction. Further filtration through a 0.22- temperature of 4°C with a Chromatix KMXS light scattering device. micron Millipore filter resulted in a single molecule fraction, The high molecular weight fraction was filtered through a 0.45-p which represented 28% filter, whereas the low molecular weight fraction was further filtered molecularweight 2.82 0.20 X of the initial tendon weight. The plots, for determination of through a 0.22-p filter. weight average molecular weight,are shown in Fig. 2 for high molecular weight and single moleculefractions. The negative correlation function was independent of sample time at sample slope of Fig. 2 suggests that attractive forces occur between times of 5 and 10 ms. The normalized correlation function at 2, 5, and 10 ms couldbe superimposed to form a single both single moleculesand aggregates in acid solution (20). Translation Difusion-Translation diffusion coefficients, correlation function which exponentially decayed to a baseof acid-extracted tendon preparations were determined line as shown in Fig. 3. The average diffusion coefficient, by photon correlation of scattered laser light. The correct was determined from the decay rate of the correlation funcbase-line of the homodyne correlation function was signifi- tion. The translational diffusion coefficientof the high moleccantly dependent on the sample time (see Equation la) at ular weight fraction at zero concentration was found to be 0.45 cm2/s (see Fig. 4). concentrations above 1mg/ml as if a continuous spectrum of f 0.04X The translational diffusion coefficientof the single molecule aggregates of different sizes were present in solution. A t concentrations below 0.05 mg/ml, the base-line of the normalized preparation was determined as a function of concentration on
*
r
-
*
*
Type I Collagen in Solution
9429
samples of known molecular weight (see Fig. 4). D20.,cextrapolated to zero concentration was 0.780 f 0.04 X 10" cm'/s. Thermal Stability-Thermal transitiontemperatures of high molecular weight and single molecule fractions were determined by measuring the temperature dependenceof the molecular weight. As shown in Fig. 5, the apparent melting 'OK P
$
099C
\
:
\"\x
i
&
0 ', #\.: \
0
E
A
~-
B
' 098Cl
0-
-Q"'""t&*"* 0 .
0
3CJ.
200
4 0 0
m
400
T I M /mer)
FIG. 3. Normalized experimental and theoretical correlation function of light scattered at an angle of 4' with respect to the transmitted beam at 4°C in HCI, pH 2.0. Experimental curves for lathyritic chick skin (A)and rat tail tendon (0)are composed of data obtained at sample times between 2 and 10 ms. The true base-line was obtainable only at samples timesof 5 and 10 ms. The theoretical normalized correlation function (0) was obtained using Equations 4 and 10.
0
0.2
0.1
0.3
05
04
CONCENTRATION f m g l m f FIG. 4. Translational difisioncoefficient back calculated to standard conditions, Dz0.,, versus concentration for high molecular weight (0)and single molecule fractions (0)of pH 2.0 acid-solubilized rat tail tendon collagen at 4°C. All measurements were made at 4' on solutions with Kayleigh factors in agreement with those reported in the legend to Fig. 3 using a Chromatix KMX-6 light scattering device modified for photon correlation.
r
FIG. 6. Transmission electron micrographs. A, negatively stained end overlapped (1) and end-to-end aggregated (2) SLS hased on the end-to-end distanceof a single collagen molecule, denoted by the hnr. B, positively stained end overlapped aggregate; overlap zone marked by arrows. C, positively stained end-to-end aggregated SLS (COOH terminus to COOH terminus); arrows, Bands 51 and 52 on adjacent molecules. All electron micrographs shown are a t a magnification of x 155.000.
'.O0
0
5
40
45
20
25
30
35
40
45
T€MP€RA TURE f "C/
FIG. 5. Molecular weight versus temperature for highmolecular weight (A) and single molecule fractions (0)of pH 2.0 solubilized rat tail tendon collagen. After denaturation, the high molecular weight and single molecule fractionshave molecular weights of 340,000 f 30,000 and 130,000 f 15.000, respectively.
temperature of both preparations was 33.5 f 0.5OC. In addition, this figure shows that the denatured molecular weights are 3.40 f. 0.30 X lo5 and 1.30 f 0.100 X 10" for the high molecular and low molecular weight fractions. Ultrastructural Properties-Transmission electron micrographs of SLS formed from the high molecular weight fraction are shown in Fig. 6, A, B, and C. This preparation contains linearly aggregated SLS with both 9% overlaps and end-to-
Solution Type in I Collagen
9430
end arrangements of adjacent molecules. Positively stained preparations indicate that most, if not all, of the overlapped SLS have common polarities of the overlapping molecules, whereas the end-to-end aggregates were predominately antipardel with the associating ends being the COOH termini. Acid-soluble versus Lathyritic Collagens-Weight average molecular weight and average translational diffusion coefficient of neutral extracted lathyritic chick skin collagen in0.01 M HCI are shown in Table 1. The lathyritic solutions were filtered through a 0.45-p fiter prior to examination but, as noted earlier, were subjected to a different purification process from that shown in Fig. 1. DISCUSSION
In an attempt todetermine the staggering mode(s) present at the macromolecular levels in the type 1 fibril, we have solubilized fibrilsinto constituent soluble forms and examined their physical properties in solution. The acid-extracted rat tendon collagen preparations were not salt-precipitated in an effort to preclude aggregation induced by the precipitations used in typical protocols. Because of the purity of the tendon as a tissue, the acid-solublematerial obtained from it revealed typical type I collagen properties by SDS-gel electrophoresis and amino acid analysis. Laser light scattering studies demonstrate that theweight average molecular weight, Br,of the high molecular weight fraction was 8.05 -t 0.40 X lo5 at 4°C. Using 2.82 X lo5 for the light scattering molecular weight of the single moleculepreparation, we calculate that theaverage aggregate contains 2.85 molecules. From this nonintegrdvalue of the molecular weight and the sample time dependence of the correlation function, it is apparent that this preparation contains at least two components. From experimental and theoretical values of M ? and Dm+,, models were generated of the several possible aggregate structures present in solution. The first models generated were for single component solutions of 0, 1, 2, 3, and 4 D staggered collagen molecules and are listed in Table 2. The value of the diffusion coefficient of single molecules at zero concentration was found to be 0.780 +- 0.04 X 10-7 cm2/s, which is close to the theoretical value of cm2/s for a 300 nm long, 1.5 nm wide rod 0.858 X calculated using Equation 4. Previously, Fletcher (5) found an anomalous dependence of the diffusion coefficienton concentration at acid pH with a maximum value of 0.86 f 0.2 X cm2/s near a concentration of 0.4 mg/ml. Ourdata also appear to maximize near that concentration. We have used the theoretical diffusion coefficient0.858 X 10-7 cm2/s for generating staggering models which falls between Fletcher’s (5) maximum experimental value and our experimental value at zero concentration. In order for the diffusion coefficientto be 0.450 X lo-? cm’/ s and the weight average number of molecules in the aggregate to be 2.85, the aggregate cannot be 0 D staggered since the diffusion coeffkient of all 0 D staggered models up to a hexamer is higher than theobserved value. On the other hand, using Table 1, it is apparent that a two-component mixture of monomers and tetramers, monomers and pentamers, and dimers and trimers with 1 to 4 D staggers can quantitatively explain these experimental observations. The best two-component models can be obtained by finding the staggering pattern which results in the experimentally observed values of weight average number of molecules and average diffusion coefficient, As model screening criteria, we used 0.45 -+ 0.06 X IO-’ cm2/s for the average diffusion coefficient and 2.85 f 0.35 for the weight average number of molecules. Tables 3 to 6 illustrate that mixtures of monomers and either tetramers or pentamers and mixtures of dimers andeither trimers, tetramers, or pentamers with 1, 2, 3, or 4 D staggering are
models. From these calculations, it is clear that other information is necessary to define the exact structure of collagen aggregates in acid solutions. In order to determine which of the staggering modes prevail in collagen solutions, we prepared SLS of solubilizedcollagenfrom the 0.45-p filtered preparation. Electron micrographs of these solutions revealed many monomeric,dimeric, trimeric, tetrameric, and even pentameric SLS. Careful analysis of negatively stained preparations demonstrated the presence of two types of SLS. The first type (Fig. 6A) consisted of crystallites with overlaps of about 9%of the length of a monomeric SLS; the second type were end-to-end or 4.4 D staggered SLS. In fact, 4.0 and 4.4 D staggered SLS interactions accounted for 92% of all interactions observed in negatively stained specimens. Positively stained samples wereanalyzed by determining the fraction of the molecular length between bands 3 and 4 and 51 and 52 to the NH2- and COOH-tenninal ends (see “Materials and Methods”). As shown in Fig. 6B, 4.0 D polarized staggered aggregates at least 2 molecules longas well as 4.4 D staggered antiparallel aggregates are observed (Fig. 6 0 . It is apparent from these SLS that the most common staggering patterns observed are 4.0 and 4.4 D alignment of neighboring collagen molecules.It is not clear whether 4.4 D interactions are induced during SLS formation. Other reports of head to tail or end overlapped SLS have been made (24, 25) supporting these observations. lt is important tonote that 4.0 and 4.4 D staggered aggregates would have about the same diffusion coefficientsand therefore either model or a mixture of these aggregates would fit the light scattering data. As noted earlier, most of the 4.4 D staggered preparations contained an antiparallel arrangement of the molecules. Although antiparallel components can be present in fibrillar forms, it is not known whether such components can be present in typical native fibrils without disrupting fibril structure. Of interest is the observation that antiparallel SLS with 0 D overlap have been described in odontoblast-derived tissues as well as near 4.0 to 4.4 D dimeric SLS in chick embryo tendon cells whose polarities have not been determined (13, 14). Refinement of the 4 D staggered model can be made by including additional components in the model and by fitting the theoretical correlation function to experimental measurements. Multicomponent 4 D models (Table 7) fit the diffusion and molecular weight data better but, in many cases, do not significantly change the fit of the correlation function. We have chosen the model whichhas weighting factors of 0.2,0.3, and 0.50 for monomers, dimers, and trimers to illustrate how well these models fit the correlation function. Fig. 3 compares the experimental and theoretical normalized correlation functions for a typical experiment. We conclude fromthese results that solubilized fibrilfragments are predominantly monomers, linear dimers, and trimers, the weight fraction of each of which is probably dependent on collagen concentration and other solution conditions. An estimate of the weight fraction of each species can be made from the weighting factors (see Equations 6 and 7), which we have calculated to be 0.387 monomers, 0.291 dimers, and 0.321 trimers. Multiangle studies on these solutions are needed to determine more accurately the weight fraction of each component. To determine whether the process of linear aggregation thermally stabilizes collagen in acid solution, denaturation studies were conducted on rat tail tendon solutions of single molecules (I& = 0.282 X lo6) and linear aggregates = 0.805 X lo6). The process of denaturation was followed by measuring the apparent molecular weight (Re/&, see “Materials and Methods”) as a function of temperature. Both solutions behaved similarly in that the molecularweight dropped significantly at a temperature of 33.5 f 0.5”C,which
(nr
Solution Type in I Collagen
GELATION
DISSOLUTION
\
n+
HE AT
'
9431
previous observations by Obrink (3) that themolecular weight by light scattering of citrate solutions (pH 3.7) of collagen from lathyritic and nonlathyritic rat skin are 7.40 X lo5 and 8.10 to 18.0 X lo5,respectively, and by Fletcher (5) of the twocomponent nature of acid solutions. In summary, our findings suggest that type I fibrils are at least partially composed of 4 D staggered filaments as diagrammed in Fig. 7 and that the 4 D stagger is probably more stable than other types of interactions in acid or neutral solutions at 4°C. The ability of collagen to reassemble into native fibrils probably derives from sequence information contained in the nonhelical ends and in the imino acid-poor regions about 0.4 D from each end. How or if this information facilitates fibril assembly in uzuo is uncertain; however, in uiuo morphological observations that tendon fibroblasts and corneal epithelial cells (12,13,15)contain intracellular aggregates 2 and 3 molecules in length may reflect this preference.
\----. "
t "
FIG. 7. Schematic representation of dissolution and gelation of type I collagen from a collagen fibril. The relationship between monomers and 4 D staggered dimers, trimers, and tetramers in the standard two-dimensional presentation of the collagen fibril is highlighted. The 0.4 D overlap or 4 D stagger of molecules reflects a particularly stable form of aggregation in solution for both lathyritic and nonlathyritic collagens. Dissolution of the fibrilrenders such aggregates soluble as indicated by the fragments in the lower portion. Gelation of such solutions apparently involves the same 4 D staggered intermediates (16).
is close to the melting point of collagen at pH 2.0 determined by viscometry (26, 27) and optical methods (26). After denaturation, the single molecule solution had a weight average molecular weight of 1.30 X lo5, suggesting that the weight fraction of a chains must be at least 0.6, assuming a molecular weight of 0.95 X lo5 and 1.80 X lo" for 01 chains and ,8 components, respectively. In comparison, the molecular weight after denaturation of the high molecular weight fraction containing linear aggregates was found to be 3.4 x lo5, which is greater than the molecular weight of a y component and therefore must reflect the fact that at least some of the linear aggregates are covalently cross-linked together. This fact is significant since, in the absence of cross-linking, it could be argued that linear aggregates are a quasi-stable solution state and do not accurately represent fibril fragments. In addition tothe experiments on acid-soluble rat tail tendon collagen, we have studied a purified neutral extract of lathyritic chick skin collagen in 0.01 M HCl. This preparation was salt-precipitated several times so that induced aggregation during purification was a distinct possibility. Table 1 and Fig. 3 compare the weight average molecular weight, average diffusion coefficient, and the experimental normalized correlation function to thevalues of these parametersobtained for the acid-soluble rat tail tendon solution at 4°C. These data indicate that the lathyritic skin collagen solution and the rat tail collagen in acid appeartobequite similar and that purification or the lathyritic state does not influence the state of aggregation. These results suggest that the type I native fibril from at least two different vertebrate tissues probably contains 4 D staggered filaments at least 3 or 4 molecules in length which appear tobe identical with filaments which form during the initial stages of collagen heat gelation (16). The specificity for the 4 D staggered interaction probably involves the imino acid-poor regions located about 0.4 D from each end of the moleculewhich from stereochemical considerations may be more flexible than theneighboring parts of the collagen helix. The importance of the nonhelical ends on fibril formation in vitro (28-30) probably reflects the necessity for the proper alignment of the telopeptides and imino acid-poor regions (30, 31). The presence of aggregates in acid solutions agrees with
REFERENCES 1. Boedtker, H., and Doty, P. (1956) J . Am. Chem. SOC.78, 42674280 2. Davison, P. F., and Drake, M. P. (1966) Biochemistry 5,313-321 3. Obrink, B. (1972) Eur. J. Biochem. 25,563-572 4. Yuan, L., and Veis, A. (1973) Biopolymers 12, 1437-1444 5. Fletcher, G . C. (1976) Biopolymers 15, 2201-2217 6. Thomas, J. C., and Fletcher, G. C. (1979) Biopolymers 18, 13331352 7. Smith, J. W. (1968) Nature (Lond.) 219, 157-158 8. Veis, A., Anesey, J., and Mussell, S. (1967) Nature (Lond.) 215, 931-934 9. Hosemann, R., Dressig, W., and Nemetschek, TH. (1974) J. Mol. Biol. 83,275-280 10. Piez, K. A. (1975) in Extracellular Matrix Influences on Gene Expression (Slavkin, H. C., and Greulich, R. C., eds) pp. 231236, Academic Press, New York 11. Hukins, D. W. L., and Woodhead-Galloway,J. (1977) Mol. Cryst. Liq. Cryst. 41, 33-39 12. Trelstad. R. L. (1971) J. Cell Biol. 48.689-694 13. Trelstad; R. L., and Hayashi, K. (1979) Deu. Biol. 71, 228-242 14. Weinstock, M. (1977) J. Ultrastruct. Res. 61, 218-229 15. Bruns, R. R., Hulmes, D. J. S., Therrieu, S. F., and Gross, J. (1979) Proc. Natl. Acad.Sci. U. S. A . 76, 313-317 16. Silver, F. H., Langley, K. H., and Trelstad, R. L. (1979) Biopolymers 18, 2523-2535 17. Bnms, R. R., and Gross, J. (1973) Biochemistry 12,808-815 18. Tanford, C. (1961) Physical Chemistry of Macromolecules, Chap. 5 and 6, John Wiley & Sons, New York 19. Schwartz, D., and Veis, A. (1978) Connect. Tissue Res.6,185-190 20. Doty, P., and Edsall, J. T. (1951) Adu. Protein Chem.6, 50-55 21. Ford, N. C., Jr. (1972) Chem. Scr. 2,193-206 22. Berne, B. J., and Pecora, R. (1976) Dynamic Light Scattering, Chap. 8, John Wiley & Sons, New York 23. Cohen, R. J., and Benedek, G. B. (1976) J. Mol. Biol. 108, 151178 24. Rauterberg, J., von Bassewitz, D. B. (1975) Hoppe-Seyler's 2. Physiol. Chem. 356,95-100 25. Veis, A., and Yuan, L. (1974) Biopolymers 14,895-900 26. Hayashi, T., and Nagai, Y. (1973) J. Biochem (Tokyo) 73, 9991006 27. Dick, Y. P., and Nordwig, A.(1966)Arch. Biochem. Biophys. 117, 466-468 28. Comper, W. D., and Veis, A. (1977) Biopolymers 16,2133-2142 29. Hayashi, T., and Nagai, Y. (1973) J. Biochem (Tokyo) 74, 253262 30. Helseth, D. L., Jr., Lechner, J. H., and Veis, A. (1979) Biopolymers 18,3005-3014 31. Silver, F. H., and Trelstad, R. L. (1979) J. Theor. Biol. 81, 515526
9432
By.
Type I Collagen in Solution
F . H . S ~ l v e land R.L. Tlrelrtad
EXPERIMENTAL AND THEORETICAL METHODS Laser Llght Scattering a,
MoleCUlaT Welght D e t e r m ~ n a t ~ o n
component
OD OD OD
on on
In
10 1D 10 2D 2D 2D 2D
0.858
1
0.718 0.758 0.717 0.717 0.717 0.642
2
0.559
0.316
4
40
0.233
..
2 3
4
4n
30 40 40
6
0.471 0.425 0.559 0.446 0.308 0.555 0.415 0.337 0.281 0.498 0.357 0.281
30 3n 3n
3 4 5
5 2 3 5 2 3
4 5 2
3 4 5
2a = 300. 2a = 3 0 0 . 2a = 3 0 0 . 300, La 300, 2a 300, 2a 368, 2a 436, 2a 504, 21 l a = 972, 2a = 436, 2a = 572, Za = 708. 2a 844, 2a 504, 2a = 708,
--~
--
--
Zb = l.5nm
2b 2h 2h Zb 2b Zh 2h
2h
3.0nn 3.0nm
= 4.Onm = 4 Onm = 4.Onm = 3.0nm = 3.Onrn
--
2b 2h = 2b = 2b = 2b = ?h 2h =
4.0nm 4.0nm :.Om
3.0nm 4.0nm 4.Onm 1.5nm l.5nm
Za = 9 1 2 , 2b = 1 5nm
2a = 1116, 2h = I S n m La = 572. 2h 1.Sna 2a = 844. 2h = 1.5nm Za = 1116, 2h = 1.1nm 2a 1388, 2h = 1.5nm ~
-
Calculated u r l n g e q u a r l o n 4 bee Methods n IS the number Of molecules 1" fhe a g g r e g a t e
""" D
~
=
b7 nm
1s
used for all calculations
Table 3
rrlners
x cd/sec
1.667 1.439 1.250
1.111 2.14 1.667 1.364 1.154
0.8 0.6 0.4 0.2
2.5
0.8 0.0 0.4 0.2
1.818 1.750 1.240 0.8 0.6
0.b19
0.079 0.734 0.798
11.548 0 .6 2 6 0.703
0.7 8 0 0.~12
1.923 1.470 1.279 2.14
u.svn
0.6
2.31 2.50
0.009 0.192
0.8
2.73
0.576
0.4 0.2 0.2
0.4
0.685 0.771 0.625
0.2
0.8
3 33
0.501
0.4
O.b 0.4 0.2
2.86 2.50 2 22
0.539 0.574 0.608
0.6
0.8 where
2.78
0.685
0.728 0.772 0.815
.2 0.4
0.8
0.6 0.8
0.2
,I
O.b 0.4
3.85 3.13 2 b3 2.27
a
408
0.112 0 555
0.599
Type I Collagen in Solution
9433
Table 4
MO"Olller
nlmer
Trlrner
Tetramer
Table 6
Tetramer
Pentamer 0.8
0.2 0.4 0.6 0.8 0.2 0.4 0.6 0.8
0.8 0.6 0.4 0.2 0.8 0.6 0 4 0.2
0.2 0.4 0.6 0.8
0.8 0.6 0.4 0.2
0.2 0 4
0.8 0.6 0.4 0.2
O h
0 8 0.2 0.4 0.6 0.8
0.8 O b 0.4 0.2
0.2 0.4 0.6 0.8
0.8 0.6 0.4 0.2
0.2 0.4 0.6 0.8
0.8 0.6 0.4 0.2
1.667 1.429 1.250 1.111 2.14 1.667 1.364 1.154
0.619 0.679 0.723 0.798 0,528 0.611 0.693 0.716
2.50 1.818 1.429 1.176
0.456
0.2 0.4 0.6 0.8 0.2 0.4 0.6 0.8 0.2 0.4
0.2
0.6 0.4
0.8 0.6 0.4 0.2 3.85
0.557 0 . 657 0.758
2.78 1.923 1.470 1.279 2.73 2.50 2.31 2.14 3.33 2.86 2.50 2.22
0.397 0.437 0.478 0.518
3.85 3.13 2.63 2.27
0.358 0.408 0.459 0.509
%O,w
0.8 0.6 0.4 0.2
3.13 2.63 2 27 1,667 1.429 1.250 1.111 1.155 1.364 1 .667 2.144 2.500 1.818 1.429 1.176 2.78 1.923
0.8 0.6 0.4 0.2
0.4 0.6 0.8 0.2 0.4 0.6 0.8
crn2/se, 0.385 0.413 0.442 0 470 0.324 0.368 0.411 0.455 0.285 0.339 0.392 0.445 0.570 0.642 0.714 0.780 0.758 0.658 0 557 0.457 0.396 0.512 0.h?7 0.743 0.358 0.483 0.608 0.733
2.73 2.50 2.31 2.14 3.33 2.86 2 . 50 2.22
0.8 0.6 0.4 0.2
1.470
1 279
Table 7
4D Staggered F l b r l l F r a g m e n f Models Tahle I
Weighrlng Factors Olner Trlmer
M o d e l s 3~ D S t a g g e r Weighting F a c t o r s
retramer
D~~
x
IO'?
.sl
rTlmer
ti20
Y
0.5
0.428
* c ( ~ A ~ )
a,
C.j/SeC
0.5
I
2.5
1.~20
0.1
0 4
0.4Z7
2.9
0.946
0.2
0.3
0.5
0.475
2.8
1.1180
0.1
0.4
0.5
0 4b5
2.4
1 064
0.2
0.3
0.5
0.502
2 3
1.210
0.2
0.3
0.25
0 25
U.489
2.55
1.100
0.3
0.2
0.25
0.25
O . 5 l 1b. 3 5 0
2.45
0.1
0.4
0.25
0.25
0.446
2,bS
0 5
C d l S e C
.
0.2
11 8
0.4 0 0
0.4
0 8 0.2 !I 4
