J. Mol. Biol. (1965) 14, 586-590

Hydrogen-bonding within the Tropocollagen Triple Helix

In native collagen, at least some of the polypeptide chains have a specific configurationand are aggregated together in a specific way. Two pairs of alternative structures,which are quite similar, are at present suggested. Rich & Crick (1961), on the basis ofwide-angle X-ray diffraction photographs and physical and chemical evidence,proposed that the individual polypeptide chains are twisted into a left-handed helix.Three of these chains are then twisted together in a gradual right-handed helix. Theirtwo structures, collagen I and collagen II, differ from each other in the way the chainsin the triple helix are phased with respect to each other. Collagen II is the structurewhich will accommodate amino acid side-chains more easily. Structures I and IIboth permit every third residue along a polypeptide chain to be hydrogen-bondedthrough the CO and NH groups to each of the other chains of the triple helix. Two outof every three residues could be hydrogen-bonded to the other chains of the triplehelix only if the centres of some atoms approached to what were regarded as unsatis­factorily short distances. This was true of structures I and II.

The structures proposed by Ramachandran, Sasisekharan & Thathachari (1962) aresimilar to the Rich & Crick structures in that the individual protein chains are twistedin left-handed helices and three ofthese are then twisted together into a gradual right­handed helix. However, Ramachandran et al. maintain that it is possible for twoout of every three residues (unless one of them is proline or hydroxyproline) to behydrogen-bonded through their NH and CO groups to the other main chains of thetriple helix. They believe that this is possible without introducing contact distancesbetween atoms less than those in other well-characterized systems, and that anyinstability introduced into the structure by the short contact distances is more thanbalanced by the great increase in stability of the structure as a result of the secondset of systematic hydrogen bonds. They favour the structure similar to collagen II.

Harrington (1964) has shown that if the total pyrrolidine content of a collagen isknown, and if reasonable assumptions are made about the enthalpy and entropychanges per mole of peptide residue on denaturing collagen in aqueous solution, thenit is possible to calculate denaturation temperatures of collagen assuming either oneor two sets of systematic hydrogen bonds. The agreement between the observedand the calculated denaturation temperatures of several collagens is striking if twosets of hydrogen bonds are assumed. If one set is assumed, the calculated values are140°C or more below the observed denaturation temperatures.

Bensusan & Nielsen (1964) have mentioned the difficulties of interpreting quantita­tively the changes in the infrared absorption spectrum when thin films of collagenor collagen in solution are deuterated. Results from these experimen ts have been heldto support the structures with a single set (Bradbury, Burge, Randall & Wilkinson,1958), or two sets (Bensusan & Nielsen, 19(4) of systematic hydrogen bonds withinthe triple helix.

In our experiments we have measured the increase in dry weight of freeze-driedtropocollagen, prepared as described in the caption to Table 1, when it is exposed todeuterium oxide vapour, using a quartz spring microbalance. We have made an amino

586

AlanineArginineAspartic acidGlutamic acidGlycineHistidineHydroxyprolineIsoleucineLeucineLysineMethioninePhenylalanineProlineSerineThreonineTyrosineValineAmide groups

LETTERS TO THE EDITOH

TABLE 1

Amino acid analysis of tropocollagen

H atoms attached to N, 0 or S atoms

m-moles/gequivalentsj l Of g

Imide Side-chain

1-137 0·11370·511 0·0511 0·20440·436 0·0436 0·0763t0·796 0·07963·659 0·36590·049 0·0049 0·00490·920 0·09200·119 0·01190·240 0·02400·280 0·0280 0·05600·058 0·0058 0·00580·125 0·01251·3900·362 0·0362 0·03620·159 0·0159 0·01590·012 0·0012 0·00120·229 0·02290·469 0·0938

Totals: 0·8172 0·5865

587

t 0·0763 is the sum of the values for aspartic and glutamic acids minus half the value for amidegroups.

Mean of duplicate amino acid analyses (Technicon Auto Analyzer) of tropocollagen. The serineand threonine values have been corrected (20 and 13%, respectively) for decomposition duringhydrolysis. Amide nitrogen was estimated by the method of Corfield & Robson (1955). Tropo­collagen was prepared at O°C. Calfskin, minced in 0·5 M-sodium acetate, was extracted severaltimes with 0·5 M-sodium acetate until the supernatant solution was clear. The residue was washedwith distilled water and then extracted with citrate buffer (0'1 M-citric acid-0'05 M-sodium citrate,pH 4,3) for 24 hr. The extracted material was sieved and centrifuged for 1 hr at 40,000 g to removeextraneous matter. The supernatant solution was then dialysed against 0·02 M-Na2HPO. for7 days, with frequent changes of dialysing solution. The precipitated tropocollagen was centri­fuged at 3000 rev./min for 10 min and then re-dissolved in citrate buffer. The solution in citratebuffer was dialysed against several changes of 0·05% acetic acid, and then the solution of tropo­collagen in 0'05% acetic acid was freeze-dried. A very accurately known dry weight of tropo­collogen was available from the quartz spring microbalance for the amino acid analysis: recovery ofamino acids from the column was 94·4%.

acid analysis of the tropocollagen sample and discovered that the weight increase isless than would be expected if every H atom attached to N, 0 or S atoms in tropo­collagen was replaced by a deuterium atom. Our results are in favour of two sets ofsystematic hydrogen bonds within the collagen triple helix, rather than a single set.

The quartz spring microbalance is an apparatus described by Burley, Nicholls &J. B. Speakman (1955), modified by surrounding the spring (sensitivity 1000 em/g)with a water jacket at a controlled temperature. The spring chamber can beevacuated by means of a mercury diffusion pump protected by liquid nitrogen trapsand backed by a rotary pump.

The tropocollagen samples were intensively dried on the spring at 20°0 in a vacuumbetter than 10- 4 mm mercury for 23 hours. To remove the last traces of water, thetemperature of the jacket was raised to 37°0 and drying was continued for one hour.

588 B. J. JORDAN AND P. T. SPEAKMAN

In a control experiment, drying at 37°C was continued for four hours but no furtherdecrease in weight occurred after one hour. The temperature was then lowered to20°C and deuterium oxide vapour carefully admitted to the quartz spring tube. Afterone hour in D20 vapour, the drying procedure was repeated and the percentage weightincrease of the tropocollagen sample found to be 0·71. In one experiment the samplewas exposed to D20 vapour for a further hour and dried again, when the weightincrease was 0·72%. After a further four hours and 34 hours exposure to D 20 vapour,the weight increases were 0·71%and 0·72%, respectively. The reproducibility of theseresults indicates that the drying procedure does not cause any significant denatura­tion. The weight increase after similar times in D 20 vapour in a duplicate experimentwas also 0·72%.

From the amino acid analysis of the tropocollagen (Table 1), it is possible to calcu­late the number of equivalents of H attached to N, °or S atoms in amino acid side­chains per 100 g tropocollagen (last column), and the number of equivalents ofimide H in the main protein chains per 100 g tropocollagen (penultimate column).These two numbers added together (1·40) give the percentage increases in weightif every H atom attached to an N, °or S atom in tropocollagen is replaced by D.In our experiments, we can therefore clearly distinguish two classes of potentiallyexchangeable (attached to N, °or S) H atoms: those which exchange rapidly, in lessthan one hour, and those which do not exchange even if exposed to D 20 vapour for40 hours; and we believe it is reasonable to make the assumption that the H atomswhich do not exchange with D 20 vapour in 40 hours are those taking part in syste­matic imide-carbonyl hydrogen bonds between tropocollagen main chains, and thatall the potentially exchangeable H atoms in the side-chains exchange under theseconditions. The interpretation of results from H exchange experiments has beendiscussed by Benson, Hallaway & Lumry (1964).

The amino acid analysis shows that the weight increase due 'to the potentiallyexchangeable side-chain H atoms, if all are replaced by D, is 0'59%. The observedweight increase due to the replacement of some imide H atoms by D in the maintropocollagen chains must be 0·72 - 0·59 = 0·13%. If all the main chain imide Hatoms were replaced by D atoms, the amino acid analysis shows that the weightincrease due to this source would be 0·82%. Thus the experiment shows that a fraction,

0·13 = 0.16, ofthe main imide H atoms are free to exchange. We have not investigated0·82the possibility that an acetic acid molecule could be attached to each arginine, histi­dine and lysine residue during freeze drying from 0·05% acetic acid. This would in­crease by one the number of exchangeable H atoms these side-chains contain. Theincrease in weight of tropocollagen after exposure to D 20 vapour and drying wouldmean, in this case, that the fraction of main-chain imide H atoms free to exchangewith D would be 0·06 instead of 0·16.

Analyses of small peptides from collagen indicate (see Josse & Harrington, 1964)that the sequence -Gly-X-Y-Gly-X-Y- is almost universal in collagen. Also,the hydroxyproline residues appear to be invariably in position Y; but since only 88residues per 1000 (calculated from Table 1) are hydroxyproline, some of the Ypositions are occupied by other residues. Proline residues, 132 per 1000, occur in boththe X and Y positions.

In the Rich & Crick (1961) collagen II structure, the single set of systematic hydrogenbonds involves only the carbonyl and imide groups of glycine residues. The imide H

LETTERS TO THE EDITOR 589

atoms of residues in the X and Y positions (unless they are proline or hydroxyproline)are free to exchange with D20. Thus the imide hydrogen atoms of 333 residues per1000 are not free to exchange; 88 hydroxyproline + 132 proline = 220 residues per1000 do not contain imide H atoms; and therefore 447 residues per 1000 have imide Hfree to exchange with D20. Thus the fraction of main-chain imide H free to exchange

with D20 in the collagen II structure is 447 = 0,57, whereas the fraction

333 + 447calculated from the experiments is only 0·16.

In the Ramachandran structure analogous to collagen II, the second set of syste­matic hydrogen bonds involves the imide and carbonyl groups of the residues in theX position. Wherever there is a proline residue in the X position, one hydrogen bondwill be missing from this second set of hydrogen bonds. Ifwe assume proline is randomlydistributed between the X positions, and the Y positions not occupied by hydroxy­proline, then the number of proline residues in position Y per 1000 =

132 X (333 - 88)---'---- = 56.

(667 - 88)

The number of residues in position Y which are not pyrrolidine residues and thereforehave imide H atoms free to exchange with D20 is 333 - (88 + 56) = 189. Therefore,the fraction ofimide H atoms which is free to exchange in this Ramachandran structure

189IS . = 0·24. This is much closer to the result calculated from the experi-

333 + 447mental weight increase.

Our interpretation of the experiments has been based on the assumption that all theH atoms attached to N, °or S atoms in amino acid side-chains will exchange rapidlywith D20 vapour under our conditions. If, however, some of these side-chain H atomsattached to N, °or S atoms in fact take part in stable hydrogen bonds within orbetween triple helices, and do not exchange appreciably with D20 vapour in 1 or 40hours, then the fraction of main-chain imide H atoms which exchanges, calculatedfrom the experimental weight increase, would be brought even closer to the fractionexpected on the Ramachandran model. Gustavson (1957) has suggested that the OHgroups of hydroxyproline might be involved in such hydrogen bonds, but in collagen IIand the Ramachandran analogue the hydroxyproline OH group cannot form hydrogenbonds with other main chains ofthe same triple helix. Rich & Crick (1961) suggest thatthe arginine side-chain may form hydrogen bonds with another main chain of thesame triple helix. It is possible that lateral aggregation of tropocollagen molecules inthe dry preparation would allow the formation of hydrogen bonds between differenttriple helices. On the whole, we believe that such hydrogen bonds would be likely toexchange in one or 40 hours in D20 vapour. Bensusan & Nielsen (1964) point out thatif an imide H atom occupies a predominantly hydrophobic region within the protein,then penetration of D20 molecules to the H atom may be slow. The number of imideH atoms which may be protected in this way within the triple helix or withinaggregates of tropocollagen molecules is not known. The relative rates ofexchange of hydrogen-bonded and protected imide H atoms in D20 vapour is alsounknown.

We suggest that the small discrepancy between the fraction of main-chain imideH atoms which exchange, calculated from the experimental result and predicted bythe Ramachandran model, could most easily be explained if more than the statistically

590 B. J. JORDAN AND P. T. SPEAKMAN

expected amount of proline were in the Y position, leaving more amino acids withimide H atoms for systematic hydrogen bonds in the X position.

A further conclusion from our experiments is that a very high proportion of thetropocollagen structure is in fact in hydrogen-bonded triple helices. The amount notin a definite structure must be very small.

We thank Dr George Stainsby for a gift of tropocollagen; we had many helpful discus.sions with him while these experiments were being carried out. This work was supportedby the International Wool Secretariat.

Department of Textile IndustriesThe UniversityLeeds, 2, England

Received 13 August 1965

B.J.JORDANP. T.· SPEAKMAN

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Soc. 25, 173.Burley, R. W., Nicholls, C. H. & Speakman, J. B. (1955). J. Textile Lnst, 46, T427.Corfield, M. C. & Robson, A. (1955). Biochem. J. 59, 62.Gustavson, K. H. (1957). In Connective Tissue, ed. by R. E. Tunbridge, p. 185. Oxford:

Blackwell.Harrington, W. F. (1964). J. Mol. Biol. 9, 613.Josse, J. & Harrington, W. F. (1964). J. Mol. Biol. 9,269.Ramachandran, G. N., Sasisekharan, V. & Thathachari, Y. T. (1962). In Collagen, ed. by

N. Ramanathan, p. 81. New York and London: Interscience Publishers.Rich, A. & Crick, F. H. C. (1961). J. Mol. Biol. 3, 483.