Author(s) Nishikawa, Ken; Oobatake, Motohisa; Citation University (1970), 48(2-3):

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(1970), 48(2-3): 102-110 ssue Date 1970-08-22 URL http://hdl.hle.

1 TitleTertiary Structures Proteins : A Author(s) Nishikawa, Ken; Oobatake, Motohisa; Citation Bulletin nstitute for Chemi University (1970), 48(2-3): ssue Date URL Right Type Departmental Bulletin Paper Textversion publisher Kyoto University

2 Bull. nst. Chem. Res., Kyoto Univ., Vol. 48, Nos. 2-3, 1970 Tertiary Structures Proteins : Analysis Conformations Ken NSHKAWA, Motohisa OOBATAKE Tatsuo Oo* Received May 16, 1970 Outlines tertiary s myoglobin lysozyme determined x-ray analysis expressed simply diagrams a distance from center gravity to each residual Ca atom plotted against residue number. se diagrams exhibit some characteristic features se proteins, e. g. hydrophobic residues a-helical regions located at rar inner part molecules. tertiary a protein can also be computed dihedral about N-CC bonds C'-C' bonds, ce 0. calculated conformations using several sets for co cj, including those given Kendrew Phillips, show little similarity to native ones, especially in ir overall shapes. Refinement dihedral has been tried to reproduce native conformations, we have been able to obtain reasonable sets for co c.'); calculated conformations in good agreement with native one, agreement which is shown in diagrams. NTRODUCTON During last two decades, remarkable developements have been made on investigations for s proteins ; determination amino acid sequences begun with nsulin has now been extended to a larger molecule more than 200 residues," stable secondary s a-helix R- which could account for x-ray diffraction patterns fibrous proteins, have been found actually in tertiary protein molecules complete determination steric arrangement atoms in some proteins, was successfully done on myoglobin, haemoglobin, lysozyme, RN ase, so on.2' One most important features revealed so far on a protein, may be specific arrangement constituent atoms, arrangement which gives rise to appearance corresponding specific function protein, e. g., enzymic activity. re experimental results which strongly suggest that a protein has one only one specified for protein, although a large number or conformations possible. For purpose to elucidate formation stable s proteins polypeptides, various experiments such as renaturation experiments have been done.3,4' Appntly, it seems to be quite strange that a native protein molecule a definite primary determined genetic codes, chooses one specific conformation. completely denatured molecule, presumably corresponded to linear chain molecule just synsized on ribosomes, has been shown to be folded up itself into native compact conformation under a physiological environment. refore, it is quite likely that native con- * p)11, )(Ai : Laboratory Physical Chemistry Enzyme, nstitute for Chemical Research, Kyoto University, Uji, Kyoto. (102)

3 Tertiary formation a without tion must stable Structures protein is rmodynamically aid or materials. be at a minimum free a protein plete chemical synsis specific for protein.'$' n order enables us Proteins : Analysis stable,'' When this energy.s' molecule a protein been thus forming is true, This has was Conformations view believed in success a native specific conforma- rmodynamically to be true to show since enzymatic com- activity to verify above postulate oretically, a high speed computer to calculate coordinates atoms in a protein, interatomic ener- gies, refore conformation stability protein. For polypeptides, simpler model compounds proteins, method would be shown to be useful, since stability a-helix its helical sense for various homopolymers Lamino acid have taking into account hydrogen biopolymers made clearly been bond generating bonds. energy. now on protein elucidated explained energy, Such successfully molecules.11' yet from a native being energy or energy minimum,') energy, electrostatic analyses on developed10' However, energetic analyses approach total tortional conformational conformation n this article, some myoglobin an in terms non-bonded some energy, s trials have been stability protein molecules is not point view, due probably to difficulty given rotational known to derivation freedoms co tertiary s native conformations d about lysozyme will be described. Molecular tertiary s lysozyme13' were determined or complete x-ray atomic analysis. in determination coordinates would coordinates over or all atomic not so reliable atomic pairs conformation in to calculate molecule, molecule coordinates Because be although as a whole, an total myoglobin121 experimental accuracy order internal 1A energy arrangements is correct. or less,n' summing atoms, stereographic Fig. 1. native myoglobin, drawn connecting successively Cd carbon to neighboring one. N-terminal end is designated as N. One can see stereographic picture with aid a stereoscope. (103)

... Tertiary Structures Proteins : Analysis Conformations formation a protein is rmodynamically stable,'' thus forming a specific without aid or materials.

4 ... Tertiary Structures Proteins : Analysis Conformations formation a protein is rmodynamically stable,'' thus forming a specific without aid or materials. When this is true, native conformation must be at a minimum free energy.s> This view rmodynamically stable a protein molecule has been believed to be true since complete chemical synsis a protein was in success to show enzymatic activity specific for protein.'01 n order to verify above postulate oretically, a high speed computer enables us to calculate coordinates atoms in a protein, interatomic energies, refore conformation stability protein. For polypeptides, simpler model compounds proteins, method would be shown to be useful, since stability a-helix its helical sense for various homopolymers L- amino acid have been explained in terms total energy or energy minimum," taking into account non-bonded energy, tortional energy, electrostatic energy, hydrogen bond energy. Such conformational analyses on s biopolymers now successfully being developed10' some trials have been made on protein molecules.l1' 3owever, stability protein molecules is not clearly elucidated yet from energetic point view, due probably to difficulty generating a native conformation given rotational freedoms co SG about bonds. n this article, some analyses known tertiary s lysozyme myoglobin an approach to derivation native conformations will be described. Molecular tertiary s or complete atomic coordinates myoglobin12' lysozyme131 were determined x-ray analysis. Because experimental accuracy in determination atomic coordinates would be an order 1 A or less,'n coordinates not so reliable to calculate total internal energy summing over all atomic pairs in molecule, although arrangements atoms, or conformation molecule as a whole, is correct. stereographic o X. e~, Fig. 1. native myoglobin, drawn connecting successively C" carbon to neighboring one. N-terminal end is designated as N. One can see stereographic picture with aid a stereoscope. (103)

5 K. NSHKAWA, M. 0OBATAKE T. OM Fig. 2. native lysozyme, drawn as Fig. 1. views both proteins shown in Figs. 1 2, where peptide main chain is represented successive lines connected Ca atom to neighboring Ca atom. molecules nicely folded in compact shape. As can be seen in figures, myoglobin contains high portion (about 75 %) a-helix141 in it, whereas lysozyme has nearly half as much as helical content (30 %)13) in addition to antiparallel /3-, non-helical parts peptides make conformation globular delicate orientation each peptide. n main chains, peptide unit, -CONH-, has approximately planar. However, about Ca carbons not always tetrahedral."' globular shape molecules may be expressed comparing a distance each Ca carbon from center gravity. n Figs. 3 4, those distances plotted against residue number for myoglobin lysozyme, respectively. Roughly speaking, every point is not so apart from mean radius \v/ G -- H Residue number Fig. 3. tance diagram from number, thin shown line. with representing center root-mean Open circles letters gravity squ indicate to native each myoglobin, Ca is atom plotted in which against distance averaged over all residues hydrophobic residues a-helical A, B, C etc., following (104) Kendrew's notation. dis- residue is shown regions

6 y K. NSKAWA, M. OOBATAKE T. 001 Fr, i ` Fig. 2. native lysozyme, drawn as Fig. 1. views both proteins shown in Figs. 1 2, where peptide main chain is represented successive lines connected Cd atom to neighboring Cd atom. molecules nicely folded in compact shape. As can be seen in figures, myoglobin contains high portion (about 75 %) a-helix141 in it, whereas lysozyme has nearly half as much as helical content (30 %)13) in addition to antiparallel a-, non-helical parts peptides make conformation globular delicate orientation each peptide. n main chains, peptide unit, -CONH-, has approximately planar. However, about Cd carbons not always tetrahedral.1 ) globular shape molecules may be expressed comparing a distance each Cd carbon from center gravity. n Figs. 3 4, those distances plotted against residue number for myoglobin lysozyme, respectively. Roughly speaking, every point is not so apart from mean radius A: F-1 G a l H Residue number Fig. 3. diagram representing native myoglobin, in which distance from center gravity to each Cd atom is plotted against residue number, root-mean squ distance averaged over all residues is shown thin line. Open circles indicate hydrophobic residues a-helical regions shown with letters A, B, C etc., following Kendrew's notation. (104)

7 Tertiary Structures Proteins: Analysis Conformations h.. h. -13-a - 'h - h~ h Residue number Fig. 4. diagram for native lysozyme. symbols, la 3, st for a-helix 3-, respectively. thin line represents root-mean squ distance open circles indicate hydrophobic residues. gyration, A for myoglobin A for lysozyme ; in or words, some residues may be buried inside molecule ors exposed to surface. refore, residues having shorter distances than mean radius regarded as located at inner part molecule. t is interest to see wher hydrophobic groups may exist inside or not, because hydrophobic residues generally constitute inner core protein.") Circles in figures represent hydrophobic residues in molecules, most which have shorter distances than mean radius. To examine more quantitatively, mean radius gyration is calculated for hydrophobic groups separately. result is that mean radius for hydrophobic residues A for 63 groups myoglobin, A for 39 groups lysozyme, respectively. corresponding values for hydrophilic 90 groups for both proteins A A. This result listed in Table 1 shows appntly afore-mentioned nature. nterestingly, helical parts molecules found in rar inner region except one short helix D myoglobin. ncidentally, this helix region is deleted in a-chain haemoglobin, which has a similar to myoglobin."' Table 1. Rootmean Squ Radius Gyration. MyoglobinLysozyme <r,> 15.13A (153)13.73A (129) >r5>7,fi A (63)11.81 A (39) <r5>i, A (90)14.32 A (60) re is some difference in detailed patterns shown in figures for myoglobin lysozyme ; i. e., deviations from mean value much larger for lysozyme especially leucine 56 located very close to center gravity seems to be deeply buried. On or h, re is no such a minimum for myoglobin but it has a periodic. difference may be due to that in helical content, to that in its biological function, former has a capacity (105)

8 K. NSKAWA, M. OOBATAKE T. 00 to bind oxygen latter acts as an enzyme. t is, in addition, interest to mention that locations catalitic sites, glu 35 asp 52 interier lysozyme molecule.13' difference is also clearly seen deviation <rg>, h from <ry> or 0.7 A for myoglobin 1.9 A for lysozyme. Calculation protein conformation Once a sequence a protein is known, chemical can be determined completely. refore, a certain conformation protein may be calculated as a function freedoms in molecule. When rotational freedom about a bond, bending bonds, stretching a bond taken into account, complete description conformational analysis may be made.11' However, re too many variables to deal with problem, so that it may be better to impose some reasonable restrictions. First all, all bond lengths fixed according to crystal data for small molecules as listed in Table 2. Second, all bond chosen to fit average ones crystal data for small molecules which have similar chemical s as listed in Table 2 also. geometry a peptide is taken from one obtained crystal as usually employed in calculation, i. e., transplanar frozen rotation about C'-N bond.l") refore, only freedoms left rotation about N-Cd, Cd-C' bond, co, respectively. Under such restrictions, every conformation can be computed as a function all co,,, which reproduce conformation. When set co 0 could be found, conformational analysis a protein molecule would be hopefull, because we will be able to examine wher conformation corresponds to energy minimum or not calculating energy as a function co 0. f we fail to find any appropriate set co 0, some loosening restrictions imposed must be taken into consideration in next step. Table. 2. Equilibrium Values for Bond Lengths Bond Angles. Bond lengths (A)Bond Cd C' 1.53r (CdC'N) 114 C'=0 1.24r(NC'O) 125 Cd N 1.47z (OC'Cd) 121 C' N 1.32r (C'NCd) 123 N H 1.00z (CdNH) 114 C H 1.00r (HNC') 123 r (NCdC') To begin with, several sets co % available for calculation ; that is, values for myoglobin given Kendrew,"' those for lysozyme Phillips,"' both being approximate values with uncertainty about 10.10' se values thought to be given measuring each angle model building."' Or sets dihedral could be computed from atomic coordinates C',_1, N,, C,d, C1', N,+1. Atomic coordinates corresponding to conformation determined above could be obtained according to computation poly- (106)

9 Tertiary Structures Proteins : Analysis Conformations /11\1\ willaa Fig. 5. diagram for conformation myoglobin calculated from dihedral Kendrew is shown thick line, as well as its native, thin line, for comparison y Fig. 6. diagram for conformation lysozyme calculated from values Phillips is shown thick line, comparing native one ( thin line) \\lk Fig. 7. diagram for conformation lysozyme calculated from computed dihedral (See text), comparing native one ( thin line). (107)

10 K. NSHKAWA, M. 0OBATAKE T. 001 a L Fig. 8. Stereographic Fig. 9. Stereographic chains. gravity for one. extended from dihedral view lysozyme calculated from dihedral from similar figures one though not resemble principal angle, computation, origin main chain , discrepancy apart conformations from is use 8- due in crystal (108) to short variey calculation angle confor- seems radius folded in angle mean maximum be gyra. Figs. 8 dihedral in to better shown show. segments seen 5. Phillips as from mean Fig. values Phillips be proteins computed conformations to in different center shown respectively given seems is quite 7, far be applying for to conformations ones, proteins, carbon Kendrew 6 a-helices both Figs. computed global native lysozyme in Ca seems dihedral ends e. g., Despite for each values molecule for shown both Even use because obtained views do distance use stereographic 9. form, calculated shapes computed tion. computed mation myoglobin calculated myoglobin Appntly, Phillips. peptide native view Kendrew. reproduced figures. NCdC' value along for deviation this

$K. NS-KAWA, M. OOBATAKE T. OO drt_, ^ 'c ~.y FA~_ f mm++ z \h E~ Fig. 8. Stereographic view myoglobin calculated from dihedral Kendrew. Fig. 9.$

11 K. NS-KAWA, M. OOBATAKE T. OO drt_, ^ 'c ~.y FA~_ f mm++ z \h E~ Fig. 8. Stereographic view myoglobin calculated from dihedral Kendrew. Fig. 9. Stereographic view lysozyme calculated from dihedral Phillips. peptide chains. calculated distance each Ca carbon from center gravity for myoglobin use values Kendrew is shown in Fig. 5. Appntly, shapes molecule seems to be quite different from native one. similar figures for lysozyme applying values Phillips computed shown in Figs. 6 7, respectively. conformation computed use dihedral given Phillips seems to be extended form, because both ends far apart from mean radius gyration. one obtained computed seems to be folded better. stereographic views conformations for proteins shown in Figs Even though global conformations computed dihedral do not resemble native ones, conformations short segments reproduced computation, e. g., a-helices as seen in figures. principal origin discrepancy is due to variey angle NC5C' along main chain. Despite use in calculation mean value for this angle, 112.5, for both proteins, crystal angle show maximum deviation (108)

12 ^ Tertiary Structures Proteins : Analysis Conformations 20 from this value. When all NCdC' derived from crystal data computed co used in calculation, for lysozyme, approximate conformation can be reproduced. However, we could not obtain correct conformation for myoglobin even putting crystal into computation. This discrepancy is due to twists peptide planes. Although we can improve conformations putting more variables in a calculation, calculation seems to be less meaningful, since re sever strains in crystal which may be absent in possible native conformations. Staring from se values, we have tried to obtain a reasonable set co 0 for native conformation. Details procedures results will be described elsewhere. computations done so far show that a reasonable set dihedral with fixed NCdC' angle does exist for myoglobin for lysozyme. generation native conformation is shown in Figs , where distances Ce carbon from center gravity coincide with one for native conformation although slight differences present. Since we have not imposed any stress for bending bonds, stretching bonds, io _ \-Alv Fig. 10. diagram for conformation myoglobin calculated from refined dihedral (See text). thin line is same one in Fig. 3, for comparison ' zo- \,/'/,1 10 /1 a. F\r\ Fig. 11. diagram for conformation lysozyme calculated from refined dihedral (See text). thin line is same one in Fig. 4, for comparison, (109)

13 K. NSHKAWA, M. OoBATAKE T. OOt energy may be calculated only mutual interatomic energies, such as non-bonded energy, hydrogen bond energy etc.. Preliminary energy calculation showed, however, a few steric hindrances occur for conformation computed use refined dihedral, so that furr studies to remove hindrances necessary. study along this line is now in progress. ACKNOWLEDGMENTS authors grateful to express ir sincere thanks to Dr. J. C. Kendrew to Dr. D. C. Phillips for sending us atomic coordinates proteins, which enable us to perform present study. This work supported partially research grant from nstitute Fundamental Physics, Kyoto University. REFERENCES (1) "Atlas Protein Sequence Structure" Vol. 4 (1969). (2) M. F. Perutz, Europ. J. Biochem., 8, 455 (1969). (3) C. B. Anfinsen E. Haber, J. Biol. Chem., 236, 1361 (1961). (4) T. semura, T. Takagi, Y. Maeda K. Yutani, J. Biochem., 53, 155 (1963). (5) C. B. Anfinsen, Brookhaven Symposia in Biology, 15, 184 (1962). (6) K. D. Gibson H. A. Scheraga, Physiol. Chem. Phys., 1, 109 (1969). (7) B. Gutte R. B. Merridield, J. Am. Chem. Soc., 91, 501 (1969). (8) R. G. Denkewalter, R. Hirshmann et al, J. Am. Chem. Soc., 91, 502, 503, 505, 506, 507 (1969). (9) T. Ooi, R. A. Scott, G. Verkoii H. A. Scheraga, J. Chem. Phys., 46, 4410 (1967). (10) G. N. Ramachran V. Sasisekharan, Adv. in Port. Chem., 23, 283 (1968). (11) M. Levitt S. Lifson, J. Mol. Biol., 46, 269 (1969). (12) C. L. Nobbs, H. C. Watson J. C. Kendrew, Nature, 209, 339 (1966). (13) C. C. F. Blake, G. A. Mair, A. C. T. North, D. C. Phillips V. R. Sarma, Proc. Roy. Soc., Lond., B (1967). C. C. F. Blake, L. N. Johnson, G. A. Mair, A. C. T. North, D. C. Phillips V. R. Sarma, Proc. Roy. Soc., Lond., B 167, 378 (1967). (14) J. C. Kendrew, Brookhaven Symposia in Biology, 15, 216 (1962). (15) M. F. Perutz, H. Muirhead, J. M. Cox L. C. G. Goaman, Nature, 219, 131 (1968). (16) L. Pauling R. B. Corey, Proc. Natl. Acad. Sci. U, S., 37, 241 (1951). (17) Personal communication. (110)

Protein Structure. W. M. Grogan, Ph.D. OBJECTIVES

Protein Structure. W. M. Grogan, Ph.D. OBJECTIVES Protein Structure W. M. Grogan, Ph.D. OBJECTIVES 1. Describe the structure and characteristic properties of typical proteins. 2. List and describe the four levels of structure found in proteins. 3. Relate