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

We havemeasuredthe diffision coefficientand Several models of the substructure of collagen fibrils based
weight average molecular weight of type I collagen on theoretical and experimental considerations have been
fibril fragments obtained by acid extraction of rat tail proposed. The microfibril, the smallest structural unit, con-
tendons and neutral extraction of lathyritic chick skin, tains from 4 to 8 collagen molecules in cross-section (7-10).
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 ag- In 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
the fibril components present in solution. It was foundcollagen fibril formation occurs by the conversion of single
of
that several models with 1 to 4 D (D = 67 nm)staggers molecules into 4 D staggered dimers and trimers, suggesting
fit the experimental data for the aggregated prepara- that the 4 D stagger may be thermodynamically preferred
tions. Analysis of the end-to-end distance of negatively over other staggering modes (16). Further aggregation into D
stainedsegmentlongspacingcrystallitesprepared staggered aggregates is a secondary event (16).
from solutionscontainingfibrilfragmentsandthe The purpose of this investigation has been to characterize
bandingpatternofpositivelystainedsegmentlong the physicochemical and ultrastructural properties of collagen
spacing crystallites suggest that collagen solutionssolutions con- derived from solubilized type I collagen fibrils and
tainlinearaggregateswith 4 D andpossibly 4.4 D to assess the organization of such fragments in respect to their
staggers in agreement with light scattering Ther- data. possible mode of packing in the native fibril.
mal denaturation studies demonstrate that the agpar-
ent melting point of cross-linked linear aggregates, 33.5 MATERIALS AND METHODS’
2 0.5”C, is identical with that of single molecules atpH Rat Tail Tendon Collagen-Acid-soluble rat tail tendon collagen
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.
tion (3-6). From these efforts, we know that extracts of type Lathyritic Chick Skin Collagen-Extracts of lathyritic chick skin
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. ’ Portions of this paper (including part of “Materials andMethods”
and Tables 1 through 7) are presented in miniprint at the end of this
* This work was supported by Grant PCM-7818903 from the Na- paper. Miniprint is easily read with the aid of a standard magnifying
tional Science Foundation, Grants HL-18714 and GM 20007 from the glass. Full size photocopies are available from the Journal of Biolog-
National Institutes of Health, National Service Award No. 5 T32 HD ical Chemistry, 9650 Rockville Pike, Bethesda, Md. 20014. Request
07092-01, and by the Shriners Burns Institute. The costs of publication Document No.79M-1568, cite author(s), and include a check or
of this article were defrayed in part by the payment of page charges. money order for $1.65 per set of photocopies. Full size photocopies
This article must therefore be hereby marked “advertisement” in are also included in the microfilm edition of the Journal that is
accordance with 18 U.S.C. Section 1734 solely to indicate this fact. available from Waverly Press.
+ To whom correspondence should be addressed, at the Shriners The abbreviations used are: SDS, sodium dodecyl sulfate; SLS,
Burns Institute, 51 Blossom St., Boston, Mass. 02114. segment long spacing crystallites.

9427
 Type 1Collagen inSolution
Preparation of Segment Long Spacing Crystallites (SLS)"sLS Tendon 1 g
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
I HC1, pH 2.0
4"C, 4 h
Solubyized tendon

Centrifugation -
pellet
discard
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
1 30 min @ 30,000 g
Supernatant
termini of negatively stained SLS were determined by measuring a
molecular length from each free end. In most cases, the overlap of Collagen solution
negatively stained polymeric SLS was obvious by the positions of the Overall recovery of 0.609 g
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
Recovery
"91%
1 Filter through
0.8-, 0.65-, and 0.45-p fdters
High molecular weight fraction

both about 7% from the ends of the molecule, while the distance (A?r = 8.05 f 0.40 X lo5,D20.w= 0.45 f 0.04 x 10" cm2/s)
between these bands is about 86%of the molecular length. Overall recovery
RESULTS 0.554 g
The relative composition of the high molecular weight acid-
soluble rat tail tendon as defined by chromatography on A-
5m was as follows: a chains, 33.6%;p components, 42.5%; and
y components, 23.9%;a recovery of 88%was obtained. Amino
acid analysis was typical of type I collagen; specifically,there (ar
Recovery
=M%
I
Filter through
0.22-p filter
Single molecule fraction
*
= 2.82 0.20 X lo5, DX).,,, = 0.80 f 0.04 X 10" cm2/s)

were 320 residues of glycine, 2 residues of tyrosine, 93 residues Overall recovery

of hydroxyproline, and 128 residues of proline. Noncollage-
0.277 g
nous glycoproteins and proteoglycans were less than 1%based
on galactosamine and glucosamine analysis. Amino acid anal- FIG. 1. Purification procedure for high molecular weight
and single molecule
ysis of the lathyritic chick skin preparation gave the following tail tendon collagen. fractions of acid-soluble (HCl, pH 2.0) rat
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; how-
ever, /3 components were present in the lathyritic preparation.
40 r
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 purifi-
cation procedure, it was necessary to study a tissue that was
mr ao5,ooo -
abundant, easily isolated, and as homogeneous as possible. 5
For these reasons, rat tail tendon collagen was chosen.
Molecular Weights-As shown in Fig. 1, about 61%of the lo>
0
0 0.1 0.2 0.3 0.4 0.5 0.6
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
a solution with a weight average molecular weight of 8.05 *FIG. 2. Kc/Rs, versus concentration for high molecular
0.40 X (see Fig. 2), which represents 55%of the tendon weight (0)and single molecule fractions (0)of pH 2.0 HC1-
soluble 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
*
molecularweight 2.82 0.20 X which represented 28% filter, whereas the low molecular weight fraction was further filtered
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
both single moleculesand aggregates in acid solution (20). 2, 5, and 10 ms couldbe superimposed to form a single
Translation Difusion-Translation diffusion coefficients, correlation function which exponentially decayed to a base-
of 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 func-
base-line of the homodyne correlation function was signifi- tion. The translational diffusion coefficientof the high molec-
cantly dependent on the sample time (see Equation la) at ular weight fraction at zero concentration was found to be 0.45
concentrations above 1mg/ml as if a continuous spectrum of f 0.04X cm2/s (see Fig. 4).
aggregates of different sizes were present in solution. A t con- The translational diffusion coefficientof the single molecule
centrations below 0.05 mg/ml, the base-line of the normalized preparation was determined as a function of concentration on
 Type I Collagen in Solution 9429
samples of known molecular weight (see Fig. 4). D20.,cextrap-
olated 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 4 0 0 200 3CJ. 400 m

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 0.1 0.2 0.3 04 05

CONCENTRATION f m g l m f
FIG. 4. Translational difisioncoefficient back calculated to
standard conditions, Dz0.,, versus concentration for high mo-
lecular weight (0)and single molecule fractions (0)of pH 2.0
acid-solubilized rat tail tendon collagen at 4°C. All measure-
ments were made at 4' on solutions with Kayleigh factors in agree-
ment with those reported in the legend to Fig. 3 using a Chromatix
KMX-6 light scattering device modified for photon correlation.

'.O0 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 magni-
fication of x 155.000.

temperature of both preparations was 33.5 f 0.5OC. In addi-

0 5 40 45 20 25 30 35 40 45 tion, this figure shows that the denatured molecular weights
T€MP€RA TURE f "C/ 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.
FIG. 5. Molecular weight versus temperature for highmolec- Ultrastructural Properties-Transmission electron micro-
ular weight (A) and single molecule fractions (0)of pH 2.0
solubilized rat tail tendon collagen. After denaturation, the high graphs of SLS formed from the high molecular weight fraction
molecular weight and single molecule fractionshave molecular are shown in Fig. 6, A, B, and C. This preparation contains
weights of 340,000 f 30,000 and 130,000 f 15.000, respectively. linearly aggregated SLS with both 9% overlaps and end-to-
9430 in I Collagen
Solution Type
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 anti-
pardel with the associating ends being the COOH termini.
Acid-soluble versus Lathyritic Collagens-Weight average
molecular weight and average translational diffusion coeffi-
cient 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 dem-
onstrate 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 solu-
tions 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
0.858 X cm2/s for a 300 nm long, 1.5 nm wide rod
calculated using Equation 4. Previously, Fletcher (5) found an
anomalous dependence of the diffusion coefficienton concen-
tration 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 the-
oretical diffusion coefficient0.858 X 10-7 cm2/s for generating
staggering models which falls between Fletcher’s (5) maxi-
mum 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 di-
mers and trimers with 1 to 4 D staggers can quantitatively
explain these experimental observations. The best two-com-
ponent 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 infor-
mation 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 pre-
pared SLS of solubilizedcollagenfrom the 0.45-p filtered
preparation. Electron micrographs of these solutions revealed
many monomeric,dimeric, trimeric, tetrameric, and even pen-
tameric SLS. Careful analysis of negatively stained prepara-
tions demonstrated the presence of two types of SLS.
The first type (Fig. 6A) consisted of crystallites with over-
laps 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. Posi-
tively 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 con-
tained 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 measure-
ments. 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 func-
tions 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 (nr =
0.805 X lo6). The process of denaturation was followed by
measuring the apparent molecular weight (Re/&, see “Ma-
terials 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
 in I Collagen
Solution Type
'
DISSOLUTION
n+
GELATION
\ HE AT
\--
---.-
-
"
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 high-
lighted. 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 dena-
turation, 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 frac-
tion 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 corre-
lation 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 colla-
gen 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
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 two-
component nature of acid solutions.
In summary, our findings suggest that type I fibrils are at
least partially composed of 4 D staggered filaments as dia-
grammed 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 cor-
neal epithelial cells (12,13,15)contain intracellular aggregates
2 and 3 molecules in length may reflect this preference.
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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, 515-
526
9432 Type I Collagen in Solution

By. 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
0.858
0.718
1
2
2a = 300.
2a = 3 0 0 . 2b --
Zb = l.5nm
3.0nn

--
OD 0.758 3 2a = 3 0 0 . 2h 3.0nm
OD 0.717 4 La 300, 2h = 4.Onm
on
on
0.717
0.717
5
6
2a
2a - 300,
300,
Zb
2b
= 4 Onm
= 4.Onm
In
10
1D
0.642
0.559
0.471
2
3
4
2a
2a
21
--
~ 368,
436,
504,
Zh
2h
2h
= 3.0nm

--
= 3.Onrn
4.0nm
10 0.425 5 l a = 972, 2b 4.0nm
2D 0.559 2 2a = 436, 2h = :.Om
2D 0.446 3 2a = 572, 2b = 3.0nm

-- -
2D 0.316 4 Za = 708. 2b = 4.0nm
2D 0.308 5 2a 844, 2b = 4.Onm
30 0.555 2 2a 504, ?h 1.5nm
3n 0.415 3 2a = 708, 2h = l.5nm
3n 0.337 4 Za = 9 1 2 , 2b = 1 5nm
30 0.281 5 2a = 1116, 2h = I S n m
40 0.498 2 La = 572. 2h 1.Sna~

40 0.357 3 2a = 844. 2h = 1.5nm

40
4n
0.281
0.233
4
5 2a -
Za = 1116, 2h = 1.1nm
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 0.685
1.439 0.728
1.250 0.772
1.111 0.815
0.8 2.14 0.b19
0.6 1.667 0.079
0.4 1.364 0.734
0.2 1.154 0.798
0.8 2.5 11.548
0.0 1.818 0 .6 2 6
0.4 1.750 0.703
0.2 1.240 0.7 8 0
0.8 2.78 0.~12
0.6 1.923 u.svn
0.4 1.470 0.685
0.2 1.279 0.771
0.2 2.14 0.625
0.4 2.31 0.009
0.6 2.50 0.192
0.8 2.73 0.576

0.2 0.8 3 33 0.501

0.4 O.b 2.86 0.539
0.6 0.4 2.50 0.574
0.8 0.2 2 22 0.608

where .2
,I 0.8 3.85 a 408
0.4 O.b 3.13 0.112
0.6 0.4 2 b3 0 555
0.8 0.2 2.27 0.599
 Type I Collagen in Solution 9433
Table 4
Table 6

MO"Olller nlmer Trlrner Tetramer Pentamer Tetramer %O,w

crn2/se,
0.8 0.2 2.73 0.385
0.2 0.8 1.667 0.619 0.4 0.6 2.50 0.413
0.4 0.6 1.429 0.679 0.6 0.4 2.31 0.442
0.6 0.4 1.250 0.723 0.2 0.8 2.14 0 470
0.8 0.2 1.111 0.798 0.2 0.8 3.33 0.324
0.2 0.8 2.14 0,528 0.4 0.6 2.86 0.368
0.4 0.6 1.667 0.611 0.6 0.4 2 . 50 0.411
0.6 0 4 1.364 0.693 0.8 0.2 2.22 0.455
0.8 0.2 1.154 0.716 0.2 3.85 0.8 0.285
0.4 0.6 3.13 0.339
0.2 0.8 2.50 0.456 0.4 2.63 0.392
0.4 0.6 1.818 0.557 0.2 2 27 0.445
0.6 0.4 1.429 0 . 657 1,667 0.570
0.8 0.2 1.176 0.758 1.429 0.642
1.250 0.714
0.2 0.8 2.78 1.111 0.780
0 4 0.6 1.923 1.155 0.758
O h 0.4 1.470 1.364 0.658
0 8 0.2 1.279 1 .667 0 557
0.2 0.8 2.73 2.144 0.457
0.4 O b 2.50 0.8 2.500 0.396
0.6 0.4 2.31 0.4 0.6 1.818 0.512
0.8 0.2 2.14 0.6 0.4 1.429 0.h?7
0.8 0.2 1.176 0.743
0.2 0.8 3.33 0.397 0.2 0.8 2.78 0.358
0.4 0.6 2.86 0.437 0.4 0.6 1.923 0.483
0.6 0.4 2.50 0.478 0.6 0.4 1.470 0.608
0.8 0.2 2.22 0.518 0.8 0.2 1 279 0.733
0.2 0.8 3.85 0.358
0.4 0.6 3.13 0.408
0.6 0.4 2.63 0.459
0.8 0.2 2.27 0.509

Table 7
4D Staggered F l b r l l F r a g m e n f Models
Tahle I Weighrlng Factors
M o d e l s 3~ D S t a g g e r
Olner Trlmer retramer D~~ x IO'? .sl * c ( ~ A ~ )
Weighting F a c t o r s C.j/SeC a, I
0.5 0.5 0.428 2.5 1.~20
rTlmer ti20 Y 0.1 0 4 0 5 0.4Z7 2.9 0.946
C d l S e C
0.2 0.3 0.5
0.2 .
11 8 1.667
0.475 2.8 1.1180
0.4 O.b 1.429 0.1 0.4 0.5
0 0 0 4b5 2.4 1 064
0.4 1.250
0 8 0.2 1.111 0.2 0.3
0.2 0.5 0.502 2 3 1.210
0.8 2.14
!I 4 . 0.0 1.667 0.2 0.3 0.25 0 25
<I 6 . 0.4 1.364
U.489 2.55 1.100
u.n 0.2 1.154 0.3 0.2 0.25 0.25 O . 5 l 1b. 3 5 0 2.45
0.: 0.8 2.50
0.4 0.6 1.818 0.1 0.4 0.25
(I .h 0.25 0.446 2,bS 1.090
0.4 1.429
U.8 0.2 1.176
(I. 2 0.8 2.78
0 4 0.6 1.923
!J h 0.4 1.470
0.2 0.8 1.279
0 2 0.8 2.73
0.4 0.6 2.50
D,b 0.4 2.31
0 .8 0.2 2.14
0.2 3.33
0.4 2.86
0.6 2.50
0 8 2.22
0 2 3.85
0.4 3.13
0.0 2.63
0.n 2.27