lvor Smith,' Margaret J. Smith, and Lynne Robertsg Courtauld Institute of Biochemistry Middlesex Hospital ,Medical School London, WIP 5PR England
I I
Models for Tertiary W U ~ U ~ ~ S Myoglobin and lysozyme
In 1953 Sanger (1) deduced the primary structure, the sequence of aminoacids, in the protein chain of insulin which was then the simplest, pure protein available. This herculean task was subsequently reduced to a routine in many laboratories and some hundreds of protein primary structures are now known and collated (3). The most recent success has been the determination of the structure of an immune globulin (3) containing 1320 aminoacids. More recently the primary structures of a number of RNA's have been deduced (3). As the primary structure gives no indication of the three dimensional or tertiary structure of the molecule there has been little incentive to build models of these compounds. However, in 1960, Kendrew, et al. (4), deduced the first tertiary structure of a protein, namely myoglobin, which has 153 aminoacids linked in sequence in a single chain. Subsequently, about half a dozen other proteins have been successfully examined and it will not be long before the tertiary structure of a transfer RNA is deduced. Here the incentive to build models is high, as not only does this tend to confirm the data if the model can be constructed, but also it becomes possible to examine the chemical and biological properties of the structure, e.g., enzyme-substrate interactions. Most models have been built with the KendrewWatson skeletal type of atomic unit, although the Courtauld and CPK space-filled models have also been used. These types of models require some weeks of full-time building so that having constructed a particular model it is retained as such; they are also extremely costly and consequently not generally available for student use. For teaching purposes, and also for much research, there is an increasing need for simple, inexpensive models, and Lande (5) has recently described a set of modules which consists of prefabricated groups of atoms such as the benzene ring, methylene, carbonyl, and peptide units which are on a much smaller scale and much less costly than the above types. Recently an entirely new type of model particularly suitable for the construction of large proteins and nucleic acids has been designed (6, 7), and we describe here the use of these Biobits models in the construction of myoglobin and lysozyme which will serve also to indicate the techniques of construction of any macromolecule from the X-ray crystallographic data. In general one must strive for the maximum accuracy
on principle, and so it is worth discussing the meaning of the term in this context. First, the X-ray data tends to suggest a certain accuracy implicit in a static molecule in crystalline form but this is not necessarily so in solution, or even in the crystal itself when one considers the two (or more) free ends of a molecule. Indeed, many of the aminoacid side chains must be in random motion in relation to the main chain particularly if they are hydrogen-bonded to water. Secondly, as the most accurate atomic models are based on some approximated bond lengths and angles it is found that in a model of a helix of less than ten residues an inaccuracy is introduced with the net effect that overall accuracy may well be less than 1 A. I n practice it is not possible to build any model which will correspond exactly with all the data and the more correct one part of the molecule is, the less correct will another part be. I n the models discussed here, the accuracy of particular parts is low but, nevertheless, the overall accuracy is high and the comprehensibility is outstanding. When presenting this type of data to the student what is required is not the detail but the general principles of structure and function. The models shown have all been built by students and each requires about two or three days of work. Each model can be viewed completely, as the superstructure is practically invisible, and can readily be rotated around any axis as it weighs only a few hundred grams. Once the 'bit' code is known, the primary, secondary, and tertiary structures are immediately obvious. The heliies can be seen, when present, to be joined by non-helical segments; the cleft into which, say, heme or substrate fits is not only readily observed but can be readily manipulated by insertion or removal of the substrate. Models, although in themselves static, should not be viewed statically. The learning process is greatly strengthed if the individual has built his own model as there is a constant reinforcement of theoretical knowledge. Those illustrated here are designed intentionally for individual student use and are in every sense a true visual aid. For such a purpose they must be extremely inexpensive, and this has been found to be possible in practice as a result of considering the truly basic requirements of molecular models for macromolecules rather than trying to upgrade atomic models designed originally to illustrate small molecules.
' To whom reprint requests should he addressed. 'Bedford College, London (Miss Roberts was a. vacation student at the Courtadd Institute).
The frame is of a collapsible packaway design. The molecular backbone is supported on spring-loaded wire; the springs fitting easily over '/la-in. mesh and the
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Frame Construction
wires being practically invisible. Small ferrules slide along the wires and the backbone is held by a further piece of wire which loops into the ferrule (see Fig. 1).
Figure 1. Details of frome construction. The frame consists of two merhwork "nib lying horimntally mt the top and bottom, ioined b y four corner vertical mds whish screw rigidly to hold the framework or a unit. Vertical wpportr are spring-loaded, top and bottom, to hold the wirer taut. The flgure rhowr a loop of wire running through o ferrule ar a means of supporting the backbone chain ond a piece of choin secured in position b y means of a comprrrrod or pinched ferrule. The tripeptide shown hmr three carboxyl callarr to indicate the direction of the choin ond the centrol wire [left) and nylon joiner [right) can also bereen.
the whole group points in relation to the chain. The number of suspension wires required is between twelve and twenty for molecules such as myoglobin and lysozyme and obviously depends on the exact shape of the molecule. In general, for a molecule with a high helical content, the mires are best placed just outside the ends of each helix, but, for long helixes, a support in the middle helps with the final alignment. For uon-helical molecules the choice is determined by convenience of building. When commencing building, four to six verticals are placed in position and a fifty-R-group length, 100 cm, of backbone is built into the system. Subsequently the R groups are inserted into one of the four holes available around the circumference of each residue. Building is continued in this manner until the model is complete. Helixes can be constructed simply and quickly in the following way. One end of the helix tubing is held by the thumb on a piece of glass tubing about 2 cm in diameter and the blue backbone tubing is wound around the glass in a clockwise manner working away from the thumb. When the correct number of residues has been wound, remove the glass tube, adjust the backbone so that between 3.5 to 4 residues make one complete turn, stretch the helix lengthwise so that the two ends now correspond to the correct coordinate dimensions w d assemble on the frame. Final adjustments can be made for each a-carbon and the R groups added to complete that part of the model. Myoglobin Model
The three thicknesses of wire make a fairly tight fit in the ferrule diameter so that, at this stage, no slipping occurs. When the whole is judged to be correctly placed along the vertical coordinate, the ferrule is pinched with a special tool fixing it permanently in position. The scale is 3 A/2 cm and three such scales are used to fix the XYZ coordmates. The vertical wire is first positioned in XY axes a t top and bottom of the frame and the Z coordinate is then defined by the ferrule position. The frame mesh is currently a/8 in. (0.96 em); but if the squares are considered as 1 cm this aids construction without appreciable loss in accuracy. Where heliixes run approximately vertical, the suspension wires may need to pass through one or more turns of the same or another helix in order to preserve an accurate structure. This is easily accomplished by unhooking a spring and threading through the helix as required. Model Construction
The basic design and coding of this type of model has been described elsewhere (6). Briefly, the backbone is composed of a length of blue plastic tub'mg carrying an internal wire to preserve the shape once it has been formed. At 2-cm intervals four holes are punched m u n d the circumference for the insertion of R groups which are color and size coded. Adjacent to each hole is a red collar indicating the carboxyl of each peptide link (or of each aminoacid in the chain), thus enabling the direction of the chain to be seen from any point along it. Two sets of the X-ray coordinates are required to position any aminoacid namely one for the or-carbon and a second from some point on the R group which serves to indicate the relative direction in which
XYZ coordinates of each or-carbon and of one position within its attached R group have been t a b ~ l a t e d . ~ The peptide backbone contains 153 residues in a single chain, about 30 of which are in non-helical conformation and the remainder exist in eight separate helixes named A-H. Stretches of non-helix occur a t each end of the molecule and serve also to separate helical segments where these make sharp changes in direction. The molecule contains no sulfur (cystine) bridges. For the heme group, a thin, transparent, red sheet of Lucite, 6 cm square is generally found to be sufficiently adequate. It is suspended between the proximal and distal histidines (residues 64 and 93) and pushed well in towards the nonpolar R groups. However, a shaped piece of Lucite can be constructed on the same scale as the main model in order to illustrate the side chains on the four pyrrole units and their hydrogen bond contacts with amino acid R groups. The model (Fig 2) illustrates many of the points of protein chemistry. First, all the helixes are immediately visible as are the nou-helical portions which occur a t the "bends" in the chain where the molecule folds back on itself. Second, the heme cleft is readily observed together with the proximal and distal histidines, and the general nonpolar nature of the heme-protein contacts. Third, almost all of the polar side chains are seen to be on the outside of the molecule thus confering on it its extreme solubility while almost all the nonpolar side chains are buried within the structure. This property can be highlighted in the following way. All the white nonpolar chains fluoresce white and if the polar a This table will be supplied with reprints or separately from the author.
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projecting red wires as can be seen in Figure 3. The cleft is again obvious and viewing in ultraviolet light after applying the red fluorescent paint as described above is equally instrnctive. Hydrogen Bond Interactions
Hydrogen bonding may occur between a number of diierent types of grouping. Within any given helix the carbonyl oxygen of the first residue bonds with the NH group of the amino acid four residues along. Indeed this deFigure 2. A rtereoriew of o model of myoglabin. Each figure con be viewed ~ e p o m t e l ybut, with the fines the olassical a-helix but, in With core, the whole choin con be followed through rtereoviewer, a three dimensional rtructure is evident. practice, all the residues of what from the N-terminal end (upper right) to the C-terminol end (lower right) and the eight heliies con b e ob. is considered to be a helical ~ e w e d ioined , by non-helical stretches The planar heme is seen in the centralcleft. ort ti on of the molecule may not chains are painted with a dot of red fluorescent paint be so bonded or may be bonded to other parts of the molecule. Such hydrogen bonds are not norand the model viewed in ultraviolet light an external mally shown in these models. Bonding may also shell of red dots is seen to enclose the white bits comoccur between the CO or NH of a peptide link and pletely; this makes an exceedingly effective and nnfora suitable R group and these are not generally shown gettable demonstration of this general property of proeither. A third type of bonding occurs in which two R teins. groups are connected when these groups have an OH, NH2, COOH, or CONHzresidue, or an imidazole, guaniLysozyme Model dine, or indole group which all contain N or NH or NH2 groups. It is often useful to insert a bond between The tertiary structure of hens egg lysozyme was d e these latter groups as they serve to hold the molecule duced by Phillips (8, 9). The molecule contains 129 together and because they cannot normally be rememamino acids in a single chain which is crosslinked by bered whereas the helix bonds can readily be assumed to four pairs of sulfur bridges. Phillips conceives the be present. A further point to note is that two hydromolecule in a number of parts which can be usefully gen bonds may derive from a single oxygen atom betaken as a guide to the model building (Fig. 3). The cause it possesses two lone pairs of electrons or from first forty residues (from the N-terminal end) form a groups such as COOH, CONHZ,guanidine, etc. The compact structure containing most of the helical part table lists a number of these bonds known to exist in of the molecule, and this can be built first. The next lysozyme and myoglobin. fourteen residues form an anti-parallel pleated sheet We have built a number of models of other tertiary which points away from the first section forming a sort of and primary structures based on the X-ray and priwing and making the first part of the cleft; for the mary structure data available. From the 3D strucmodel, however, it is convenient to build a unit of tures, the current generalizations of the shape of protwenty residues into the structure. Residues 61 to 87 form a separate third segment and residues 88 to 129 complete the structure. If desired, these four segments can be constructed using four colors of backbone tubing instead of the single blue backbone normally used. At this stage the four pairs of cysteines can be crosslinked as they all lie sufficiently close to the surface to enable this to be done without derahging the model. Seven main hydrogen bonds are known to bind the substrate and these may be indicated by green wires projecting from the R group bits. Two bonds, one from as~articacid 52 and the other from glutamic acid 35,are known ~ i g u r e3. A ~ t ~ e w i eofwthe lymryme model. The whole three dimensional structure con be readily obto take part in the enzymic at- sewed and the chain followed through from the N-terminal end (lower left) to the C-terminal end (lower right). The central cleft i s readily observed and the six moin hydrogen bonds whim bind the rubrtrate hove been tack On the substrate and these indicated with projecting wire,, in the upper port of the cleft. In the center of the cleft, two wirer project may be indicated by means of frorn t h e h o ominoocidrof the $ite, ASP 52 GI" 35. 304
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Hydrogen Bonds- Joining R Groups
Lysosyme
Myoglobin
1-7, 15-89, 27-111, 60-69 87-89 lor-40 13-129 COOH. 46-50, 59-52 4 6 5 2 , 51-53-66-69
4-79, 18-77 and 18-44 20-118-27, 79-3836-109 60-45-Hm, 59-62, 82-141 102-105, 64-116, 108-139 140-151
-Bonds are made by inserting coated wires into the boles of psirs of groups.
teins (and nucleic acids) are readily apparent. Indeed from a model of the primary structure of the one, completely known, immune globulin it is possible to build reasonably possible tertiary structures. Further, using such models in primary structure form it is possible to demonstrate aU the chemical and enzymatic techniques used to determine such structures. The chains may be readily cut and rejoined to indicate splitting. Alternatively, a series of peptides from, say, a range of enzymic digestions can be built and aligned as a permanent demonstration to indicate how such information is used in the elucidation of primary structure. Again, models of peptides obtained by enzymic digests of chains before and after splitting sulfur bridges show how the connection between pairs of cysteines is determined for a molecule which may have a number of such bridges. The primary structure models are, of course, extremely flexible while being completely stable and 'Biobits protein and nucleic acid models are available from Ealing Scientific Corporation, 2225 Massachusetts Avenue, Cambridge, Mass. 02140, and Capital Bioteehnic Developments, 35 St. Dunstans Avenue, London W.3, England. The latter also supply stereoviewers.
easily manipulatable so that they may be used to compare homologues by lining up two or more chains and simply moving them in parallel chains like snakes. Similar models for the construction of nucleic acids have been described (6, 10) and form an equally instructive companion set to the protein Biobits4 described here. A further advantage of this type of model is that the effect of a change of one amino acid residue for another may be studied in relation to the total structure. Thus a conservative change from, say, leucine to valine or arginiie to lysine may often readily he seen to have little effect on the three-dimensional structure and thus to explain why such changes produce little or no biological effect; whereas, a change from arginine to leucine m9.y have a minor, moderate, or major effect depending on whether the substitution has occurred at a position of relatively little importance or of major importance such as an active site. Literature Cited
0. P.. BiochernJ.. 53.353 (1953). ( 2 ) DIYHOFI,M. O., AND ECI, R. v.. "Atlas of Protein Sequence and Structure." 1967-68. National Biornadioal Research Foundation, ~aryl%nd 1968. . u. 307. (3) EDELMAN. G. M., CONNIN~HAM, B. A,. G A ~W., E., G o m m ~P. , D., Ru~rsx~amn U., , *wn N ~ x o ~ M. n , J., Pmc. Nat. Acad. Sci.. in press, (1969). 8 (4) K=ND,EW,J. C.. DroKaason. R. E.. S m m o a ~ n oB. , E., H ~ n rR. G.. DAVIEB, D. R.. P H I ~ I P D. B . C. AND SHOBE, V. C.. N C I ~ I I 185,422 IC. (1) SAN(IER.F., AND TBOMPBON. E.
:. F., KOENIO,D. F.. MAIR,G.A,, NORTH, 06,751 (1965). v. 1966). , J., J . B i d . Eduo., 3, 193 (1969).
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