Structure and biological activity of hagfish insulin

J. AIoZ. Biol.

(1979) 132, 85-100

Structure and Biological l

J. B. CUTFIELD?,

l

S. M. CUTFIELD?, AND

Activity

E. J. DODSON, C.D. REYNOLDS

Department

G.

G. DODSON,

2 S. F.

EMDIN

of Chemistry

University

Heslington,

of Hagf%h Insulin

York

of York

YOl

5DD,

England

l Laboratory of Molecular Biophyks University of Oxford Oxford, England 2 Department

of

University

Ume& a-90287, (Received

Pathology

of Umed Sweden

9 February

1979)

An isomorphously phased electron density map of hagfish (Myxine glutinosa) insulin has been calculated at a resolution of 3.1 A spacing. The molecule crystallises with one molecule per asymmetric unit but is organised as a symmetric dimer lying on a Z-fold crystal axis. The structure of the hagfish insulin monomer is much more similar to that of pig insulin molecule 2 than molecule 1 of the dimer that constitutes one third of the 2 Zn insulin hexamer. There are different conformations however at the N and C termini of the B-chain. At the C terminus the two final residues on hagflsh insulin partially obscure the Al glycine residue, which in pig insulin is exposed. This structural difference has been shown, however, not to be responsible for the reduced activity of the hagfish insulin.

1. Introduction There has been growing interest in the insulin from the Atlantic hagfish, MyxinP glutinosa, the most primitive vertebrate extant and the most primitive animal whose insulin has been characterised properly. Recent studies have established the hagfish insulin sequence (Peterson et al., 1975), see Table 1. and shown it to be consistent with a dimeric form but not with a zinc-binding hexameric organisation. Lowresolution X-ray studies were carried out on tetragonal crystals, space group P4,2,2 with a = b = 38.4 A and c = 85.3 A. The asymmetric unit of this crystal contains a monomer whose folding is very like that of the pig insulin molecule in the 2 Zn insulin crystal. The dimer is also organised in a very similar way to that in pig insulin but in this case lies on the S-fold crystal axis (Cutfield et al., 1974). Biologica’lly the hormone from the hagfish is substantially less potent than pig insulin, however, as found in the fat cell assay (Emdin et al., 1977). t Present

address:

Department

of Biochemistry,

University

of Otago,

Dunedin,

New

Zealand.

85 0022-2836/79/210085-16

$02.00/O

Q 1979 Academic

Press Inc.

(London)

Ltd.

STRUCTURE

AND

ACTIVITY

OF

HAGFISH

INSULIN

x7

The evident similarity in structure and preservation of sequence in the regions of the hormone considered to be involved in biological activity made us very interested in the detailed structure of hagfish insulin. Shortage of material however has restricted the present study to 3.1 A spacing. In spite of this rather limited resolution a number of observations can be made on the structure of hagfish insulin, its relationship Do the pig insulin crystal structures and on its reduced biological activity.

2. Materials (a)

and preparation

Crystallisation

and Methods of isomorphous

derivatives

Crystals were prepared by first dissolving 0.5 to 1 mg of the protein in 0.008 M-HCl and 16% (v/v) acetone. The solution was then warmed to 50°C and poured into a small centrifuge tube containing a second solution (0.04 M-sodium acetate, 16% acetone) also warmed to 50°C to prevent precipitation. The resulting mixture was known to have a pH of 5.7 which saved having to measure and adjust the pH of this very small volume. The protein concentration in the final solution was 2.5 mg/ml. Slow cooling to room temperature gave well-formed tetragonal bipyramids usually between 0.2 and 0.8 mm in their longest direction. Three heavy-atom derivatives were prepared, all by soaking the crystals in appropriate solutions. There was some variation in the behaviour of crystals from different preparations (obtained between 1973 and 1975) towards the heavy-atom solutions. The conditions for the 3 derivative preparations were as follows. (i) Lead

acetate

A solution of 0.01 M-lead acetate in 0.05 M-sodium acetate, The crystals of the later preparations (1975) were first plntaraldehyde for 4 min before soaking for 20 to 24 h in the (ii)

Gold

in O.Olo/ solution.

(v/v)

cyanide

A solution of 0.05 The crystals were pattern beyond this (iii)

pH 6.0. crosslinked lead acetate

77ranyl

M-gold soaked length

cyanide in 0.05 M-sodium citrate, for 48 1-1. There was no detectable of soaking time.

pH

5.0. change

in the

diffraction

acetate

A solution of 0*005 M-lxanyl acetate in 0.05 M-sodium acetate, pH The crystals were soaked for 24 h, further soaking damaging the some individual variation of up to 0.3 A in axial lengths after soaking. (b) Data

5.0. crystals.

There

was

collection

diffraction data for the 6 A series were re-collected for the native and derivative crystals. Native crystals gave reasonable data to 2.8 A, though at this spacing it was necessary to spend 2 to 3 min counting time per reflection. The derivative crystals diffracted less well and their data limits were at larger spacings. In the case of the lead series anomalous scattering measurements were also made at low resolution. The data were collected on a Hilger and Watts 4-circle diffractometer controlled by a PDP8 using scans of 20 to 40 steps of 0.02 to 0.03” in omega. Counting time varied hetwperr 1 and 3 s. The

X-ray

(c) Heavy

atom

parameters

and

their

refinement

The atomic co-ordinates and scattering factors for the lead and uranyl series had been determined earlier in the 6 A analysis (Cutfield et al., 1974). Difference Fourier electron density maps with coefficients 1Fp,l - (P,I and phases derived from the transformed pig insulin co-ordinates were used to redetermine the heavy-atom co-ordinates in the nem derivative series. The lead substitution proved to be at the same site, adjacent to Al7 glutamate, as ill the earlier analysis, so the different behaviour of the later crystals did not reflect a different

.I.

88

F.

(:UTE’IELU

BY’

.4L.

reaction. The uranyl substitution was more complex. First the 2 crystals used in data 001. lection had slightly different axial lengths (Table 2) and were therefort: treated separately. The lower resolution series ( az to 4.5 A) showed the uranyl ions had substituted at 2 sites, separated by 7.3 A. (In the earlier 6 a analysis only 1 diffuse site was detected.) No significant peaks were detected in the difference Fourier of t,he higher resohltion series (4.5 to 3.5 A) suggesting either that the calculated phases had divcqed too far from the t,rllr values or that the thermal parameters of the many1 ions were’ very Iligll. Lo\v-rc,solutiolr (4.5 AA) data from tllis crystal however showed 1 miijor and 3 minor peaks. Th(, tnajor peak corresponded to site 2 of tile 4.5 A! series. This site ant-l I of tllcl minor peaks I‘(‘fined well. When this major site was introduced into the highor rtxsolution scbricls it refined sensibly. A difference Fourier revealed the presence of another poak wljicll also refined sensibly. Both these sites had a much lower occupancy than the llrarryl sites i II hllcs 4.5 A series. The uranyl site common to both series (sitcx 2) provtbs t,c) IX, ndjacclnt t,o tllc* aspartate group of H 10.

TABLE

Cell

dimensions

oj the native

Native

Pb

2 and

deriuatiw

crystals

(ii)

(, ~~ b

38.4

38.4

38.45

3X.26

:k4.35

,

85.3

X5.2

tw(i

x 5 .*5

85. I

The data for tllc gold cyanide scrics extended or~ly to 4.5 :\ spacairtg: bryot~tl tllis resolution the isomorplrous changes in bllo particular cryst’als available were tlot significa,nt.. The difference Fourier based on the transformed pig insulin co-ordinates showed 1 peak above background which however did not refine. When the difference Fourier was calculated with phases derived from the isomorphous lead snd nranyl series 2 nt,her peaks in addition to the previously observed peak appeared. ‘i’h(lse peaks however, wlren introducrtl together, all refined sensibly. Refinement was carried out on both centric and acerlt,ric terms. Wit11 tile lead series tllc co-ordinates and as only phases available were calculated from the transformed pi0‘U insulin these were considered insufficiently accurate for refinement only cer&ric data wer,r used is tho Ileavy-atom st,rll(~t~~ir~c minimising the expression (K / FPHl - 1F, + fhi )2, where
(d)

Phasing

calculation

The phases for calculation of the electron density map were detormirled from tllcx ps,rameters listed in Table 3 using the procedrlres of Blow 8: Crick (1958). ‘l’lle avclrap fipw of merit was rattler low beyond 4 Lk spacing and reflectrd the weakness of tilts stnzctSrlr~~ amplitudes collected from small and in sorne cases det,eriorated crystals (Table 4). E’rorn 3.5 to 3.1 A spacing only the lead series contributes to the phasing. Since anomalous scattering observations were not made beyond 4.5 A4 resolution only the centric terms art’ likely to have a reasonably high figure of merit and the bulk of the information between 3.5 and 3.1 A spacing will be provided by these terms. Comparison of the 3.5 A and 3.1 AA spacing maps showed however that a, real improvemrrlt was obta,ined l)y this extension. Part of the isomorphously phased electron density map at 3.1 ‘4 resolution is ~IIOVI’II in Fig. 1.

STRUCTURE

AND

ACTIVITY

OF

TABLE Atomic

parameters

Y

z

n

OCC

0.427 0.005 0.086 0.080 0.010 0.400 0.200 0.385

0.184 0.047 0.090 0.110 0.460 0.218 0.377 - 0.030

0.194 0.020 0.084 0.083 0.050 0.148 0.257 0.340

34 30 15 15 20 20 30 30

0.25 0.24 0.18 0.07 0.06 0.17 0.13 0.11

and scale factors data.

Resolution

0.005 14 206


0.93

of merit)

61 27 9 103 40

F,, r.m.8. E,SXtl E &“Dlll

Ul

P, r.m.s. El,“, U2 i?, r.m.s. El,“, Au

for the derivative

40

details

0.02 7 136

0.04 5 144

0.063 4.6 169

0.08 3.5 126

0.10 3.1 85

0.8 38 21 7 31 19

0.71 27 16 5 26 20

0.6 22 17 5

0.48 18 15 6

0.36 14 12 4

15 11

12 6

31 21 2915

Z[F,,

obs -

r.m.s., root-mean-square. E is the error from the Blow

F,,

~alc)~/N]~:

& Crick

phasing

- 7.0 -20

1.08 1.03

5

1.46

1.0

1.06

from

refinement

0.048 (0.59) 0.13

0.05 0.064 0.049

0.055

LTV E fS”,,, =

K1

obtained

Phasing

31 18

66

data

B’

4

16 11

F, r.m.s. E isom

x9

3

TABLE

Pb

INSULIN

z

occ, occupancy factor. W, 1 Overall temperature K1 J against the native


HAGFISH

E,,,,

(I>20)

= Eisom/3

method.

3. Results (a) Electron In spite of the good. The central is clearly defined regions are also diffuse or absent

density

map

low average figure of merit the electron density map was surprisingly part of the dimer structure where the residues are very well ordered and there is appropriate density for all the side-chains. The surface generally readily interpreted though some of the side-chains have density, suggesting possible disorder. The electron density was most

90

J.

F.

CUTFIELl1

ET

Bl,.

826 Tv 625 Phe

Flo. density

1. Superimposed elect,ron density contours taken from the isomorphoualy phusctl elrctruu map at 3.1 A resolution in the region of residues (a) Al to A3, (b) A6 to Al 1, (c) B24 to B26.

difficult to interpret at the B-chain N and C termini. Some difficulty was also experienced at the residues A8, A9 and AlO, where some ext’ra electron density is present which may represent’ attached solvent structurct. The transformed pig insulin co-ordinates were superimposed on bhe electron density during the interpretation of the structure. They were found to correspond t’o the electron density rather closel,y and gave an immediate impression of the similarities and differences between the two structures. It was also interesting to see that the rotation and translation parameters deduced at 3 A spacing gave such an excellent match between the two structures. Hagfish insulin co-ordinates were obtained by adjusting. where necessary. the overlapping pig insulin residues whose chemical structures in the two insulins were

STRUCTURE

AND

ACTIVITY

OF

HAGFISH

INSULIN

91

the same. The regions where the structure and/or sequence differs in the hagfish sequence were built into the electron density. This procedure was carried out using transparent s&ions stacked along the c axis. Positions indicated on this map were then recorded and made to conform to the correct chemical geometry by application of the MODELFIT routine (Dodson et al.. 1976). (b) Atomic

podions

It is not possible to obtain atomic positions accurately in a 3.1 A resolution electron density map. The absence of detail often means that more than one peptide conformation can be made to match the electron density. With side-cha’ins the overall position can usually be defined quite well but again details of the conformation are sometimes uncertain. The regula,rised co-ordinates from the 3.1 A map were refined by the diagonal leastsquares method of Agarwal (1978) which uses fast Fourier transform techniques. Refinement based on 1724 reflections reduced R from 52.7% to 40.4% for the 412 atoms of the protein. Five cycles of co-ordinate refinement and two cycles of temperature factor refinement were made. Individual isotropic temperature factors (15.0 A2 initially) were assigned to all atoms and were refined in cycles separate from the co-ordinate refinement. The low-angle data (d > 8.2 A) were excluded from all the calculations. The structure was regularised with constraints on the positions after each cycle of refinement. These co-ordinates gave a good match to the electron density on the isomorphously phased map. We have recently collected a complete set of intensity data for hagfish insulin t’o a resolution of 1.9 A from one crystal. A refinement of the structure using these dat)a is in progress. (c) Description

of the molecular

structure

Where the superimposed pig insulin co-ordinates for both main-chain and sidechain are found to lie nicely on the electron density it seems probable that the two structures are closely similar. Differences in structure also clearly exist. These involve both main-chain and more frequently side-chains. Figure 2 shows the main-chain atoms for residues Al to A7 of hagfish insulin (co-ordinates refined with 3.1 a spacing data) and molecules 1 and 2 of 2 Zn insulin (co-ordinates refined with 1.5 A spacing data transformed to the hagfish insulin lattice using a programme written by Dr G. David Smith of the Medical Foundation. Buffalo). It is clear that pig and hagfish insulins have similar structures in spite of the difference in aggregation and large variation in sequence (19 changes in 51 residues), The similarities extend beyond the general folding and organisation of the molecule and many of t’he side-chains prove to be in the same positions in the two insulins. We set the most striking match in structure at the B-chain residues in the a-helix B9 to B19 (Fig. 3) and in the extended C-terminal residues B23 to B26. Interestingly these two regions are made up of largely invariant residues (Table 1) and contain the residues B12, B16, B24, B25 and B26 which are buried by dimer formation (see below) (Fig. 4). The A-chain lies between the N and C-terminal arms of the B-chain and is folded compactly over the B-chain a-helix. None of its residues are involved in dimer formation and even in pig insulins a high proportion (16 in 21) of its side-chains arc on the molecule’s surface at all levels of aggregation. Moreover there is no ext,ensive

STRUCTURE

BND

ACTIVITY

OF

HAGFISH

FIG. 3. A comparison of the atomic positions in hagfish insulin molecule 2 for the B-chain residues in the a-helix (B9 to B19).

9X

INSULIN

(solid

lines)

and

2 Zn insulin,

and well-defined secondary structure in the A-chain. Nonetheless the A-chain in hagfish insulin still exhibits generally similar folding of the polypeptide backbone to that in pig insulin. Some of the side-chains are displaced, however, from the pig insulin positions by up to 3 A. While it is possible that some of the differences we see at the periphery are a result of slight errors in the rotation applied to the pig insulin this ca.nnot account for the relative translation seen for example in Al4 tyrosine. There is a drastic change in amino acid sequence in hagfish insulin at the three adjacent surface residues A8, A9 and AlO. In pig insulin this sequence is threonine. serine, isoleucine; in hagfish insulin it is hist,idine, lysine, arginine, which is a sequence rather like that found in fish insulins. The main-chain is not very well-defined in the map but seems not to be very different. At the disulphide bond A6-All, however, there is evidence of structural movement (Fig. 5) of about 2 A. This relative shift extends down the A-chain and is particularly noticeable at Al3 leucine and Al4 tyrosine. To some extent these differences in structure could reflect the lower a,ggregat’ion state of t.he dimeric hag&h insulin. Tn hexameric 2 Zn insulin both Al3 and Al4 (as leucine and tyrosine) make close contacts between dimers. At $14 to Al5 the main-chain direction in hagfish insulin is again that’ seen in pig insulin. The similarity continues to the terminal A21 asparagine. There are two regions only where the hagfish and pig insulin backbone conformation are markedly different. In both of these regions the amino acid sequence is also much altered (Table 1). One of these is at the B-chain N terminus and the other at the Bchain C t’erminus. In 2 Zn pig insulin the B-chain N-terminal residues Bl to B4 arc closely involved in hexamer formation. The somewhat different path, in ha.gfish

FIG. 5. A comparison fish insulin (solid lines)

of the atomic positions for the .-I-chain residues (-41 to API) and 2 Zn insulin, molecule 1; and (c) 2 Zn insulin, molecule

AIN AIN

in (a) hagfkh 2 (solid lines)

-?@ A21C

insulin (solid and molecule

lines) 1.

and

2 Zn insulin,

,A2lC

molecule

-Q-49

2; (b) hag-

AIN

96

J.

F’. CCTFIELI)

E 7’ .A I,.

insulin, followed by these residues (Fig. 4) and their different amino acid therefore understandable. ,4t the B-chain C terminus the residues B28 to fish insulin turn towards Al glycine (Fig. 6). There arc no intermolecular made by corresponding residues within the pig insulin hexamer a,nd only ones in the crystal packing. Hence this alt,ered structure in hagfish insulin a feat’ure of the isolated monomer and reflects the changes in sequence at,

sequenw is B31 in hagcontacts rather wak is prol)al)l) B2R t)o B31.

This electron density map establishes that the dimer contains an exact Z-fold axis. unlike pig 2 Zn insulin, where the symmct’ry nibhin the dimer is only approximat(~. It follows from the close similarity between the hitgfish and pig insulin monomer structures that the contacts made within the corwsponding dimers are also vvr\ alike. It, is nice that, the refinement at 1.5 AA of pig r 2 %n insulin has shown that tlw residues along t’he line of local S-fold axis. apart from B25 side-cha,in, conform extremely closely to t,he Z-fold symm~~try--t tic%discwpancies are much more apparent away from t,he axis (Dodson et ccl.. nnpul&shrd work) (Fig, 4). In 2 Zn pig insulin the two molecult 3 in tlw ditncbta f,xhil)it distinct differenclv itt conformation at the N terminus of t,llcl A-(hain tint1 in thfx disposition of thci BT, histidine and B25 phenylalanine side-chains. In all t hew features UY find the hagfish insulin structure is closer to pig insulin molecult~ 2. As far ati the arrangement of B25

5’~. 6. Electron tlensity contours in t,hr rrgioll of th
STRUCTURE

AND

ACTIVITY

OF

HAGFISH

INSULIN

97

side-chain is concerned the situation is understandable. Whereas in molecule 1 the B25 phenylalanine side-chain actually lies across the non-crystallographic 2-fold axis forbidding exact symmetry; in molecule 2 the B25 phenylalanine benzene ring is turned away and falls in neatly against A19 tyrosine where it can conform to exact 2-fold symmetry, where we find it in hagfish insulin. Although each of these structural matches may seem individually unimportant the total impression is that the hagfish insulin dimer is constructed from two molecules very like molecule 2.

4. Structure

and Activity

The structural similarity between hagfish and pig insulin is close in spite of their different aggregation states and 19 changes in sequence. The diiferences between pig insulin molecule 2 and hag&h insulin are of the same order or less than those observed between the two crystallographically independent molecules (1 and 2) in 2 Zn pig insulin. There are, however, substantial differences in their biological activity. The binding capacity of hagfish insulin in the rat fat cell and liver cell is 25% and 5%: respectively, of that of pig insulin. Its ability to enhance the metabolism of the fat cell is only 5% of that of pig insulin (Emdin et al., 1977). The residues considered to be possibly involved in the expression of insulin’s activity are the dimer-forming residues and the adjacent A-chain N and C-terminal residues (Blundell et al., 1972; The Peking Insulin Group, 1974~~; Pullen et aE., 1976). These residues (which are illustrated in Figs 4 and 7) are all present with similar structures in hagfish insulin. The dimer-forming residues in both species are pa’rtitularly similar, consistent with the finding that the dimerisation constants of pig and hagfish insulin are the same (Frank, 1976; Wollmer, 1978; personal communications). This is evidence that ha,gfish insulin’s reduced biological activity is not produced by differences in the dimer-forming residues; therefore the expression of insulin’s biological activity must involve other parts of the molecule. Support for this conclusion comes from recent chemical studies on insulin derivatives: des B30-B26 insulin (despentapeptide) and des B30-B25 insulin (deshexapeptide). The Chinese report these two derivatives as having, respectively, nearly full and moderate biological activity (The Peking Insulin Group, 19743,1977). Other preparations of des B30-B26 insulin have a lower activity, of about 15%: which suggests even so that B26 tyrosine is not critically involved in the interactions at the membrane (Gattner, 1975). The extra residue at the B-chain C terminus on hagfish insulin is seen to be direct’ed towards the A-chain N terminus. Its presence here seemed a likely explanation for hagfish insulin’s lower activity since various bulky groups attached to the Al glycine also reduce insulin’s potency (Blundell et al., 1972; Pullen et al., 1976). If this idea were correct we would expect the removal of B31 methionine to elevate hagfish insulin’s biological activity in fat cells. It does not, however, nor does the further removal of B30 lysine, which model-building suggested might be interacting with A4 glutamic acid and the Al a-amino group (Emdin $ Gliemann, unpublished observation). Assuming the removal of B31 (and B30) d oes not perturb the nearby A-chain N-terminal structure (their removal in pig insulin does not lower activity) we must’ conclude that the additional B-chain residue is also not responsible for hagfish insulin’s lower potency. How then can we account for hagfish insulin’s reduced activity?

d

Bl6

STRUCTURE

AND

ACTIVITY

OF

HAGFISH

on

INSULIN

First there may be additional conformational changes in the insulin molecule when it interacts with the cell membrane which hagfish insulin is less able to undergo. Some evidence for pig and beef insulin’s structural flexibility comes from the 4 Zn insulin structure in which a very substantial rearrangement of the hormone is observed (Bentley et al., 1976). These involve the B-chain N terminus which we find can switch from an extended conformation to a helical conformation which is a continuation of the central stretch of u-helix. Associated with this rearrangement is a marked separation of the B-chain C-terminal residues and the A-chain N-terminal and C-terminal residues. The sequence changes in hagfish insulin are extensive at the Bchain N terminus and at the nearby A-chain residues A8, A9 and AlO. Although the residues in these regions vary considerably they may always possess a capacity foi some conformational change in highly potent insulins, necessary for full expression of biological activity, which is absent in hagfish insulin. Secondly it must be appreciated that the free energy fall corresponding to a reduction to 25--5% in hagfish insulin binding to the fat and liver cell, while measurable, is only of the order of 1 to 2 kcal mol- l, assuming the system is in equilibrium?. This means a small conformational change could be capable of producing this effect. The considerable structural differences that exist between pig insulin molecules in 2 Zn insulin (Blundell et a.l., 1972) and the even larger ones in 4 Zn insulin weaken this idea but it may be that small structural differences matter very much in some parts of the molecule. Finally we note that there is a region on the molecule of more variable residues where hagfish insulin is distinctly similar in its surface character to pig insulin. This region is made up of Al3 isoleucine (leucine in pig insulin), B6 leucine, B14 alanine a’nd B18 alanine (valine in pig insulin) and B17 isoleucine (leucine in pig insulin). At B21 the glutamic acid in pig insulin is replaced by the non-polar valine. The nonpolar character of this region is understandable in pig insulin since it is buried in hexamer formation. Its presence, and extension at B21, is interesting in hagfish insulin since it does not form hexamers, and suggests that perhaps more extensive interactions are made by the hagfish insulin molecule at the receptor or at some stage of its production.

5. Conclusion Our findings suggest that the relationship between insulin’s structure and its biological activity is subtle. Possibly a specific variation is needed in the hormone’s structure for correct binding or relatively small changes at particular regions may affect activity more than substantial changes elsewhere in insulin’s structure. Refinement of the hagfish insulin molecule will give us a considerably more accurate comparison on which to base future arguments. Much

of the crystallographic

work

was carried

out in the Laboratory

of Molecular

Biophysics at Oxford University. We are grateful to Professor Dorothy Hodgkin, Professor David Phillips and Professor Sture Falkmer for their interest and help in these studies. Our thanks are due to the Kristineberg Zoological Station, Fishebackskil, Sweden, where the material was collected. The work was supported by the Royal Society, the British t The dimerisation respectively.

and receptor

binding

constants

for insulin

are approx.

2 x lo”

and

10” >I-‘,

100

J.

Diabetes Association, the Swedish Medical Research (S. F. E.) received a travel

F.

CUTFIELD

EI’

dL.

Medical Research Council, the Science Council and the Swedish Diabetes grant from the Boncompagni-Ludowisi

Research Association. Foundation,

Council, the: One of us Sweden.

REFERENCES Agarawal, R. (1978). Acta Crystallogr. sect. A, 34, 791-809. Bentley, G., Dodson, E. J., Dodson. G. G.. Hodgkin, D. (‘. & Mercola. Nature (London), 261, 1666168. Blow, D. M. & Crick, F. H. C. (1959). Acta Crystullogr. 12, 7944802. Blundell, T. L., Dodson, G. G., Hodgkin, D. C. t Mercola, D. A. (1972). Chew.

26,

D.

A&an.

A.

( 1976).

Protein

279-402.

Cutfield, J. F., Cutfield, S. M., Dodson, E. J., Dodson, ($. (4. c(r; Sabesan, M. N. (1974). J. Mol. Biol. 87, 23-30. Dodson, E. J., Isaacs, N. W. & Rollett, .J. S. (1976). Scta Crystallogr. sect. A, 32, 31 I. Emdin, S. O., Gammeltoft, S. & Gliemann, J. (1977). J. Biol. Ckmn. 252, 602-608. Gattner, H. G. (1975). Hoppe-Xeyler’s 2. Physiol. Chem. 356, 1397 -1404. Peterson, J. D., Steiner, D. F.. Emdin, S. 0. & Falkmer, S. (1975). .I. Biol. Chem. 250. 518335191. Pullen, R. A., Lindsay, D. Q., Wood. 8. P., Tickle, 1. J ., Hlundell, T. L., Wollmer, A.. Krail, G., Brandenburg, D., Zahn, H., Gliemann. .J. & Gammeltoft, 8. (1976). Nature (London), 259, 369-373. The Peking Insulin Structure Research Group (1974a). Scientia Sin,. 17, 762.-778. The Peking Insulin Group (19745). Xcientia Sin. 17, 780-792. The Peking Insulin Group (1977). Acta Biochim. Biophys. Sin. 9. 1699173. Weitzel, G., Bauer, F. U. & Rehe, A. (1977). Hoppe-Seyler’s %. Physiol. Chewa. 358, 1673-1582.