Conformational flexibility and its functional significance in some protein molecules

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TIBS - December 1979 6 Amtzen, C. J. (1978) in Current Topics in Bioenergetics (Sanadi, D. R. .and Vernon, L. P., eds), Vol. 8, pp. 11 I-160, Academic Press, New York 7 Henriques, F. and Park, R. B. (1975) Plant Physiol. 55,763-767 8 Armond, P. A., Arntzen, C. J., Briantais, J. M. and Vernotte, C. (1976)Arch. Biochem. Biophys. 17554-63 9 Ape], K. (1977) Biochim. Biophys. Acta 462, 390-402 F. (1979) J. Biol. 10 Chua, N.-H. and Blomberg. Chem. 254,215-223 11 Siiss, K.-H., Schmidt, 0. and Machold, 0. (1976) Biochim. Biophys. Acta 448.103-l 13

12 Steinback, K. E., Burke, J. J., Mullet, J. E. and Am&en, C. J. (1978) in International Symposium on Chloroplast Development (Akoyunoglou, G., Elsevier/North-Holland 389-400, ed.), pp. Biomedical Press, Amsterdam 13 Bennett, J. (1977) Nature (London) 269, 344-346 14 Bennett, J. (1979a) Eur. J. Biochem. 99,133-l 37 15 Bennett, J. (1979b) FEES Len. 103,342-344 16 Gillham, N. W., Boynton, J. E. and Chua, N.-H. (1978) in Current Topics in Bioenergetics (Sanadi, D. R. and Vernon, L. P., eds), Vol. 8, pp. 21 I-260, Academic Press, New York 17 Apel, K. and Kloppstech, K. (1978) Eur. J. Biochem. 85,581-588

Conformational flexibility and its functional significance in some protein molecules Robert Huber Crystallography has contributed much to our knowledge ofprotein flexibility and here are described some examples of large-scale segmental flexibilities discovered by X-ray diffaction. These examples indicate that flexibility may be required for enzymatic catalysis and other functions ofproteins, and also for their regulation.

X-ray diffraction provides a static, timeaveraged picture of the molecule in a crystal lattice. The recent development of refinement methods [ 1,251 provides precise atomic coordinates and allows one to determine atomic temperature factors, which are a measure of flexibility, as is discussed in the following section. Spectroscopic methods, such as NMR, ESR, Mossbauer and fluorescence spectroscopy, can give direct and detailed information about dynamic behaviour. Usually such information can be integrated only with the aid of the X-ray structure. Molecular dynamics calculations offer promise of correlating different types of experimental information and of providing a theoretical framework for understanding the dynamic behaviour [3,4]. The small protein pancreatic trypsin .inhibitor (PTI) (58 amino acid residues) has been the most thoroughly studied protein, both by X-ray diffraction, by spectroscopic techniques, and by molecular dynamics calculations [3-71. Probably the phenomena observed in PTI also exist in larger proteins, but often it is difficult to test this. In some larger proteins X-ray diffraction is the major source of information about dynamic properties. R. Huber is ar the Max-Planck-lnstitut fir Biochemie, D-8033 Martinsried, F.R.G.

The temperature factor in protein crystallography

‘Because the energy of lattice vibrations (phonons) is very small compared to the energy of X-ray photons (about six orders of magnitude smaller), lattice vibrations cannot be observed directly by X-ray spectroscopy but they do affect scattering of X-rays. The intensity of X-rays scattered by a crystal with thermal motion, compared to a crystal without thermal motion, is reduced by an exponential factor (the Debye-Waller factor) which depends on the (instantaneous) mean square displacement of an atom [8a,b]. As X-ray scattering is an instantaneous process compared with lattice vibrations, the effect on the scattered X-rays of a large number of closely related conformers (microstates), which are randomly packed into a crystal lattice, are indistinguishable from the effects of a single vibrating conformer. Cooling is the obvious means to distinguish between them, as only the thermal vibrations are frozen out. This is general practice in the crystallography of small molecules. The finding that protein structures can be refined [1,2,5] provides a means of determining temperature factors of individual atoms. The errors are still large compared with data obtained from small molecule structures, but the values are physically reasonable. This is shown by the

18 Schmidt,

G. W., Cashmore, A. R., Broadhurst, M. K., Bartlett, S. and Chua, N.-H. (1979) in Cold Spring Harbor Symposia on Quantitative Biology (Membrane Biogenesis) (in press)

19 Chua, N.-H. and Schmidt, Biol. 81,461-483

G. W. (1979) J. Cell

20 Griffiths, W. T. and Mapleston, R. E. (1978) in Chloroplast Symposium on International Development (Akoyunoglou, G., ed.), pp. 99-104, Elsevier/North-Holland Biomedical Press, Amsterdam 21 Hoober, J. K. and Stegeman, Biol. 70,326-337

W. J. (1976) .r. Cell

22 Apel, K. (1979) Eur. J. B&hem.

97,183-188

observations that: (1) external polypeptide loops have temperature factors which are higher than average; (2) amino acid side chains of external residues show temperature factors which increase along the side chain; and (3) protein molecules which have been crystallized and analysed in different lattices show the same trend in temperature parameters along the chain [1,2,5,9a,lOb,lOc, llb, 12a]. The problem of decomposing the observed temperature factor into its contributions from static and dynamic disorder remains, however. Cooling is difficult, as protein crystals contain a large proportion of solvent which may not freeze. Even in the most favourable case, where the protein crystal is stable in 70% methanol, a rather narrow temperature range between room temperature and about -70°C may be attained [9b,9c]. A systematic study of the temperature parameters within this range is under way [lOa,b]. Mossbauer spectroscopy of myoglobin crystals indicates that a substantial part of the observed temperature factor is due to static disorder [l la]. The static disorder is composed of two factors: a component due to imperfect lattice formation (lattice defects) and a component due to differences in the structure of the individual molecules (microstates). Lattice defects are a property of the crystal and are not relevant to the molecules in solution. Whether microstates exist in protein molecules is presently under discussion [3,4,10b]. ‘Few and widely separated’ conformers have been clearly defined in a few cases of two-fold side chain disorder 11lb]. The following examples are characterized by apparent disorder of a substantial part of the molecule. In these cases drastic effects are observed: there is no significant electron density for the disordered segments whereas the ordered domains are well defined. In terms of the previous discussion, ‘invisibility’ may be caused by any of the following limiting situations: thermal @Else&r/North-Holland Biomedical Press 1979

TIBS -December

272

1979

which is well characterized from studies of the trypsin-PTI complex [2] (Fig. 2). A number of residues of the activation domain are directly involved in substrate binding. Asp-189 which interacts with lysines or arginines of the substrates is responsible for the specificity of trypsin for basic residues. One would expect that binding of substrates to trypsinogen should be impaired because trypsinogen lacks the complementary binding surface. The flexibility of this domain suggests that transformation to a trypsin-like state should be possible with sufficiently strong ligands. This is found to be the case in the TRYPWL-SEGMENTS WHICH ARE FLEXBLE IN TRYP!3NOGEN. trypsinogen-PTI complex [9a]. Here trypFig. 1. Stereo drawing of rhe C” carbon positions of trypsin. Residues linked by single lines are flexible in trypsinogen is forced to adopt a trypsin-like sinogen. Residues linked by double lines are well defined. Catalytic residues and hinge residues are indicated. conformation at the cost of some of the association energy. In the trypsinogen-PTI mobility with a root mean square dis- lized and has been analysed in the same , complex the binding pocket for the Ile-16 placement of more than about 0.8 A, or crystal form as trypsinogen, shows an N-terminus is formed, but remains empty. When exogenous Ile-Val dipeptide is microstates with a similar spread, or three ordered activation domain [ 12b]. Flexibility starts rather abruptly at single added, it is strongly bound by the complex or more different, widely separated conformers. These conclusions are drawn from residues, usually glycines. Glycine, which [21]. The structure of the ternary complex considerations of significance in refined has no side chain, is a preferred candidate is virtually indistinguishable from that of electron density maps. In less well- to mediate flexibility. A distinctive feature the trypsin-PTI complex. The Ile-Val pepdetermined cjstal structures the limits of of the activation domain is the complete tilde acts as an effector: it can induce the significance might be lower. As most of the lack of aromatic residues. This holds also transformation to a trypsin-like state in the molecules considered are well defined, lat- for the switch and hinge region in antibody presence of much weaker ligands than PTI tice defects are excluded as the major molecules and the flexible region in [22]. These structural and thermodynamic source of disorder. It is unclear which type tobacco mosaic virus protein [ 16,171. It data allow the construction of a scheme of disorder (static or dynamic) prevails. appears that aromatic residues confer rigid- shown in Fig. 3. When the activation Possibly, spectroscopic studies will provide ity to protein molecules. There are more domain forms without the supporting insight into these problems in the future, than 20 hydrogen bonds cross-linking the effect of endogenous N-terminal Ile-16, segments of the activation domain in tryp- binding energy must be supplied. Shaping despite the size of the molecules. the activation domain may be regarded as a carboxylate:Ile-16 In the following, the experimental evi- sin. The Asp-194 dence and the functional implications of ammonium ion pair appears to act as a folding process: the activation domain flexibility are discussed in some detail for clamp. The absence of this ion pair in tryp- folds around the Ile-Val N-terminus. The trypsinogen-trypsin, citrate synthase and sinogen may destabilize the activation activation domain is covalently linked in domain. The activation domain is cross- seven places to the rigid body of the antibody molecules. linked by the disulfide bridge 191-220 molecule, and so its conformational freeTrypsinogen compared to trypsin and their which is reducible in trypsinogen [20] but dom must be very limited. Folding of the complexes with trypsin inhibitor (PTI) not in trypsin. Segments of the activation activation domain is therefore a final shapdomain from the substrate binding site, ing of the chain and not the folding of a This system has been studied in great detail, and the various crystal structures have been defined at the highest resolution allowed by the crystalline order [12a]. The proenzyme trypsinogen is converted to trypsin by cleaving the chain between residues 15 and 16, liberating the new N-terminus, Ile-16. Trypsin and trypsiiogen are virtually identical in structure in about 85% of the polypeptide chain, but there are large differences in the remaining 15%. The parts which differ consist of four segments which are tightly interdigitated in trypsin: the N-terminus to Gly-19, Gly-142 to Pro-152, Gly A-184 to Gly-193 and Gly-216 to Asn-223 (Fig. 1). In trypsinogen, these segments are disordered. The disordered region is termed the activation domain. Crystal lattice forces are rigorously excluded as the source of this disorder since trypsin, which can be crystal-

Asp

189

Ser 190

Glv 193

QY

Cys.138

Glyl-37 GlyI36

Fig. 2. Scheme of PTI-trypsin interaction seen in the complex. Residues of PTI are indicated by I.

TIBS - December 1979

273 TRYPSINOGEN

chain flexibility appears essential for His57. Enzymes might even be designed so that particular vibrational modes enabling transfer are energetically favoured. Proton transfer over short distances appears mechanistically simple compared to the transfer of large intermediates over large distances in multi-enzyme complexes 1261.We hope to study some features of an enzyme with large-scale transfer in citrate synthase.

TRYPSIN

Citrate synthase

1 Fig. 3. Simplified equilibrium scheme for the trypsinogen PTI Be-Val system (left) and the trypsin PTI system (right), &A) flexible segments. (w Be-Val dipeptide. The various species observed crystallographically are A, B, C, D and E. Equilibrium constants are experimentally determined (large numbers) or have been inferred (small numbers) on the assumption that the equilibria are identical for species with the same structural features [2I].

random coil polypeptide chain. It is a fast process [231. The differences between trypsin and trypsinogen discussed so far concern the substrate binding site. Functional data show that the inactivity of trypsinogen is indeed due to its inability to bind substrate [24]. There are few differences between the arrangement of the catalytic residues in trypsin and in trypsinogen [ 121. A discussion of the subtle differences in the free molecules, if they are significant, seems irrelevant. The relevant species is an enzyme-substrate complex. The trypsinPTI and trypsin-ST1 (soya bean trypsin inhibitor) [2,2.5] complexes have many characteristics of such a species. In the trypsin-PTI complex the schematic arrangement of catalytic residues and the scissile peptide (Lys-15 -Ala-16)* is characterized by a half bond between seryl-195 0 y of the enzyme and the car-

bon of the scissile peptide Lys-15 of the inhibitor. The carbonyl group of Lys-I.5 is tetrahedrally distorted. As regards flexibility, the behaviour of His-57 is particularly interesting. His-57 is hydrogen-bonded to Ser-195, but at some stage of the catalytic sequence it must protonate the leaving NH group of Ala-16. This requires a conformational change after which the imidazole is hydrogen bonded to the NH of Ala-16. We know from NMR experiments with free PTI that even tightly packed, internal, aromatic residues can flip at a considerable frequency of a few thousand times per second [7]. The proton transfer by His-57 in serine proteases is one of many examples of similar activities in other enzymes. Some side * Abbreviations: See IuPA-ImB commission on Biokhemical Nomenclature, Biochemistry 9, 3471 (1970). Residues of the inhibitor are indicated by (I) or italics.

CITRATE SYNMASE

(a)

-

MONOMER ’

oW POSITIONS, - SOUND CITRATE

”

Citrate synthase, the condensing enzyme, catalyses the formation of citrate from oxaloacetate and acetyl-CoA. The enzyme from pig heart is a dimer with a mol. wt of 100,000. The folding of this globular molecule is characterized by the absence of P-sheets and the predominance of helices, some.of which are largely buried in the interior of the molecule [13]. The electron density per monomer accounts for only about 360 residues of the 430 required by molecular weight. The invisible part is disordered, is probably N-terminal and is involved in binding CoA (Fig. 4a,b). This last conclusion is drawn from the absence of difference density when crystals with and without CoA are compared. This analysis provides no information on the conformation of the disordered part, but CoA binding should be associated with a rigid fold. The functional significance of disorder is not yet established, but there are indications of two active sites in citrate synthase [ 131: CoA, sitting on a flexible protein arm, might transfer the intermediate (citroyl-CoA) from one site to the other. Acyl carrier proteins are commonly found’as separate subunits in large multi-enzyme complexes where they serve this purpose [26]. The citrate synthase problem requires more structural studies to define what function the disorder serves. There is a small N-terminal’domain flexibly attached to the

I . (b)

26-30

Fig. 4a. Stereo drawing of chainfolding ofa monomer subunit ofcitratesynthase [13]. The disordered N-terminal segment must continue to the right from residue 1 on. Fig. 46. Several sections through the electron density map around residue 1. There is ample space in the crystal lattice for the disordered domain.

TIBS -December

274 antgen

UY

COO'

Fig. 5. Structure of antibody (IgG) molecule and enzyme cleavage products. (V~)variable half of light chain, (Ctjconstant half of light chain, (Vn)variable part of heavy chain, (Cnl ,Cn2, Cu3) the three constant homology regions ofthe heavy chain. Fab-antigen-binding fragment, consisting oflight chain and halfofthe heavy chain (VL, V,, CL, CHI), FCC-terminal halfofthe heavy chain with the interheavy chain disulftde bond intact. Hinge peptide: the segment connecting CuI and Cn2 and containing the inter-heavy chain disulfide linkage; switch peptides: the segments connecting V and Cparts comprising residues at I IO (light chain) and I1 9 (heavy chain).

main body of the molecule in such diverse proteins as tomato bushy stunt virus protein [18] or lac repressor [27a,b]. The common function in all three cases is nucleotide binding! The antibody molecule (Fig. 5) The structure of the intact IgG molecule Kol provided the first example of large-

McPC803Fab

Fig. 6. Fab arm of intact IgG Kol (top), Fab fiagment Kol (middle), Fab fragment McPC 603 (bottom), seen along an axis through the switch peptides.

scale segmental disorder. The Fc part which has a mol. wt of 50,000 and represents the stem of the Y-shaped molecule, showed no significant electron density while the Fab arms were well ordered [28]. ‘Recently the same phenomenon was discovered in a different intact IgG molecule Zie. No detailed structural analysis of the Zie protein is yet available but, as crystals of the intact molecule and its (Fab)z fragments are isomorphous and the diffraction pattern is very similar, the Fc part must be disordered [29]. The situation is different with the IgG molecule Dob, where the Fc part is ordered. But Dob is chemically abnormal, as it lacks the hinge region [30]. These data suggest a correlation between the presence of a hinge region and Fc disorder. Alternatively, crystallizability and disorder might be correlated in the cases of chemically normal IgG molecules; here also the type of disorder is unclear, but it is well known from spectroscopic experiments that antibodies are flexible in solution [31]. Apart from this variability in the relative arrangement of Fab arms and Fc stem, allowed by the long, extended hinge segment, structural analyses revealed other flexible joints. Fig. 6 demonstrates variability in the relative arrangement of the C and V modules in the Fab parts. Kol Fab arms are compared as seen inthe intact molecule and in the Fab fragment. These show a difference in elbow bend of a few degrees [32]. However, the Fab fragment McPC603 [333 has a very different elbow angle. Freedom to move is provided by the ‘switch’ peptides and is certainly more restricted than the relative Fab-Fc movement which is allowed by a far longer and

1979

more extended hinge segment. Restricted variability of about 5” in angle is also observed between CH3 and CH2 domains when we compare the two chemically identical chains in the Fc fragment crystals or when we compare the Fc conformations before and after complex formation with protein A [34,353. The scheme shown in Fig. 7 summarizes the various hinges displayed by an IgG antibody. The variability discussed so far concerns the relative arrangement of the domains, which appear as rigid building blocks: especially the variable domains and those constant domains which associate tightly in a lateral fashion, CBl-Q, &3-C~3. The CH~ domain, however, shows exceptional features. It is a single domain with no lateral association; it was not surprising to find that this domain is less rigidly folded than other domains. Flexibility is documented by the vanishing electron density of part of the CH2 domain as observed in the Fc-protein A complex [35] (Fig. 8). These segments are flexible in the crystal structure of the complex, but ?re defined and rigid in crystals of free Fc [341. In the latter instance, crystal packing obviously stabilizes a particular conformation. As these segments are not involved in crystal packing in the complex, this structure is likely to resemble more closely the structure in solution. The functional significance of independent arm and stem movement may lie in the ability to reach antigenic determinants in different arrangements. Electron micrographs of antibody trimers and tetramers cross-linked with bivalent haptens suggest this [15]. Does antigen binding induce a conformational change at the Fc part which influences its binding properties for the proteins of complement? The question is controversial, and no indisputable evidence for such

Fig. 7. Scheme of an IgG antibody and its various hinges.

275

TIBS - December 1979

linking agents or to mediate transfer of enzymatic intermediates. A different func-

tional aspect of disorder and flexibility appears in the case of viral proteins [ 16,17a,b, 181 where flexibility allows adaptation and bonding to differently arranged RNA strands. Possibly complement binding to the flexible part of the IgG CH~ domain falls into the same category. Fig. 9 is a schematic summary of these various functional aspects of flexibility. I believe that more functional aspects of disorder will emerge in the future. Unfortunately, disorder evades exact study by structural determinations: an uncertainty principle in biology. Acknowledgement

Fig. 8. Stereo drawing of the Ca positions on the Fe-protein drawn in thin lines [35].

A complex. Segments disordered

in the Fe part are

Prof. R. L. Baldwin’s help with the final version of the manuscript is gratefully acknowledged. References

a conformational change has been presented [ 14,36,37]. Transmission of a signal from one part of a molecule to another which is flexibly connected appears impos-

VeriableUVSS-

linkii agent

d 0

0

Fig. 9. Schematic representation of various examples of functional flexibility discussed: antibodies as variable cross-linking agents. A flexible domain as a shuttle between two active sites in c&a& synthnre. D&orderorder transition to regulate enzymatic activiry in hypsinogen. Adaptable binding surface in viral protein and antibody Fc parts.

sible, except by postulating exotic signals, such as changes in the anisotropy of vibrations. Cooperative interaction between domains, if they occur at all, probably require a rigid conformer. This remains to be tested. As discussed earlier, CH~ is a ‘soft’ domain. The flexible segments in CH2 may form the binding site for the complement protein Clq, which interacts with the Fc components of antigen-antibody complexes [35]. The pronounced dependence of this interaction on salts argues for the importance of salt linkages, as do chemical modification studies of charged groups [38a,b]. Interaction of flexible components is energetically less favourable than interaction of rigid, complementary partners, but it is less demanding with respect to the exact arrangement. This might be important when the multivalent Clq binds to a multivalent antigen-antibody complex [391.

1

Watenpaugh, K. D., Sieker, L. C., Heniott, J. R. and Jensen, L. H. (1973)Acro Crystallogr. Sect. B 29,943-956

2

3

Huber, R., Kukla, D., Bode, W., Schwager, P., Bartels, K., Deisenhofer, J. and Steigemann, W. (1974)J. Mol. Biol. 89,73-101 McCammon, J. A. and Karplus, M. (1977) Nature (London) 268,765-766; McCammon, J. A., Gelin, B. R. and Karplus, M. (1977) Nature (London) 267,585-590 Karplus, M. and McCammon, J. A. (1979) Nature (London) 277,578 Deisenhofer, J. and Steigemann, W. (1975) Acta Crystallogr. Sect. B 31,238-250

Snyder, G. H., Rowan, R., Karplus, S. and Sykes, B. D. , (1975) Biochemistry 14, 3765-3777

Wagner, G., DeMarco, A. and Wiithrich, K. (1976) Biophys. Struct. Mechandm 2,139-158 WI Cochran, W. (1964) in Phonons and Phonon Interaction (Bat, T. A., ed.), W. A. Benjamin Inc., New York and Amsterdam, pp. 102-180 [bl Laue, M. von (1960) Akademische Verlagsgesellschaft, Frankfurt a.M. 9bl Bode, W., Schwager, P. and Huber, R. (1978)

7

J. Mol. Biol. 118,99-l

12

PI Douzou, P., Hui Bon Hoa, G. and Petsko, G. A. (1974)J. Mol. Biol. 96,367-380

Conclusion

Functionality and rigidity are believed to be intimately related. This is undoubtedly correct and the trypsinogen-trypsin system appears to be a particularily good example. The rigid enzyme is the active species. The flexible pro-enzyme is unable to bind substrate with sufficient strength. Rigidification, which is necessary for the enzyme-substrate complex to form requires too much energy. The order-disorder transition serves as a regulatingprinciple here. Flexibility and disorder in the case of antibodies and citrate synthase are directly required for the molecular functions in order to serve as effective cross-

[cl Fink, A. L. (1979) Trends B&hem.

Sci. 4,

8-10

10bl Singh, T. and Bode, W. (in preparation) @I Frauenfelder, H., Petsko, G. A. and TsemogIOUD., Nature (London) (submitted)

[cl Artymiuk, P. J., Blake, C. C. F., Grace, D. E. P., Oatley, S. J., Phillips, D. C. and Stemberg, M. J. E., Nature (London) (submitted) Il[al Parak, F. and Formanek, H. (1971) Acta Crystallogr. Sect. A 27,573-578

[b] Steigemann, W. and Weber, E. (1979) J. Mol. Biol. 1127,30%-338 12[a] Huber, R. and Bode, W. (1978) Act. Res. 11,11&122

Chem.

[b] Fehlhammer, H., Bode, W. and Huber, R. (1977)J. Mol. Biol. 111,415438 13

Wiegand, G., Kukla, D., Scholze, H., Jones, T. A. and Huber, R. (1979) Eur. .I. Biochem. 93.41-50

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TIBS -December Huber, R., Deisenhofer, J., Colman, P. M., Mathushima, M. and Palm, W. (1976) Nature (London) 264,415-420

24 25

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Seegam, G. W., Smith, C. A., Schumaker, V. N. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 907-911 16 Bloomer, A. C., Campness, J. N., Bricogne, G., Staden, R. and Klug, A. (1978) Nature (London) 276,362-368 17[al Stubbs, G., Warren, S. and Holmes, K.‘(1977) Nature (London) 267,216221 [bl Jardetzky, O., Akasaka, K., Vogel, D., Morris, S. and Holmes, K. C. (1978) Nature (London) 273,564-566 18 Harrison, S. C., Olsen, A. J., Schiitt, C. E., Winkler, F. K. and Bricogne, G. (1978) Nature (London) 276,368-373 19 Sasaki, K., Docker& S., Adamiak, D. A., Tickle, I. J. and Blundell, T. (1975) Nature (London) 257,751-757 20 Knights, R. J. and Light, A. (1976) 1. Biol. Chem. 25 1,222-228 21 Bode, W. (1949) .I. Mol. Biol. 127,357-374 22 Bode, W. and Huber, R. (1976) FEBS Lett. 68, 231-236 23 NBlte, H. J. and Neumann, E. (1979) Biophys. Chem. (submitted)

Kerr, M. A., Walsh, K. A. and Neurath, H. (1975) Biochemistry 14,5088-5094 Sweet, R. R., Wright, H. T., Janin, J., Chothia, C. H. andBlow, D. R. (1974) Biochemistry 13, 4212-4228

Sumper, M. and Lynen, F. (1972) in 23rd Colloquium der Gesellschaji fir Biologische Chemie in MosbachlBaden. Protein-Protein Interaction (Jaenicke, R. and Hebnreich, E., eds), pp. 365-393, Springer-Verlag, Berlin, Heidelberg and New York 27[a] Jardetzky, N. W., Bray, R. P., Conover, W. W., Jardetzky, O., Geisler, N. and Weber, K. (1979)J. Mol. Biol. 128,259-264 [bl Buck, F., Riiterjans, H. and Beyreuther, K. (1978) FEBS Lett. 96,335-338 28 Colman, P. M., Deisenhofer, J., Huber, R. and Palm, W. (1976)5. Mol. Biol. 100,257-282 29 Ely, K. p., Colman, P. M., Abola, E. E., Hess,

26

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31

A C., Peabody, D. S., Parr, D. M., Connell, G. E., Laschinger, C. A. and Edmundson, A. B. (1978) Biochemktry 17,820-823 Silverton, E. W., Navia, M. A. and Davies, D. R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5140-5144 Yguerabide, J., Epstein, H. F. and Stryer, L. ( 1970) J. Mol. Biol. 5 1,573-590

31P Nuclear magnetic resonance observations in biolog;ical svstems II. Nucleic acids ana prot&ns* C. Tyler Burt and Alice M. Wyrwicz 31P nucleus has proven to be an extremely use@1 nmr probe of phosphates in biochemical systems. It provides information on their structure and environment; any changes in these two factors are reflected in 31P chemical shift perturbations. This approach has been most useful in studying the nucleic acia3 backbone conformation.

31Pat 100% natural abundance occurs in a number of biological molecules, many of which have now been investigated by phosphorus nmr. In the first part of this review we discussed studies of some of the low-molecular-weight metabolites in intact tissue. There are, however, many phosphorus-containing macromolecules such as nucleic acids, phosphoproteins and phosphorylated intermediates of enzyme reactions which are of great scientific interest. However, these must be investigated as isolated systems, since in intact cells either their concentration or physical state makes them essentially invisible. Recent instrumental advances, mentioned in the first part of this review, have made studies of these biologically important * This is part II of a two-part ‘Intact Tissue’ was published 244-246.

review. Part I, subtitled in TIBS Nov. 1979 pp.

C. Tyler Buti is at the Dept. of Biological Chemistry, University of Illktotk at the Medical Center, Chicago, Illinois, U.S.A. and Alice M. Wynvicz is at the Dept. of Chemistry, University of Illinois at Chicago Circle, Chicago, Illinoir, U.S.A.

molecules possible. Chemical shift is the most widely used nmr parameter and by itself can yield a wealth of valuable information. In particular, as shown by Gorenstein and coworkers [ 11, the 31Pchemical shift of phosphate esters is very sensitive to the ester torsional angles of the ester and can therefore provide information on the phosphodiester backbone conformation in nucleic acids in solution. This approach has been successfully applied in a number of studies, ranging from small oligo-nucleotides to DNA in chromatin. We shall discuss them in order of increasing complexity. Helix-to-coil transitions have been followed for several small oligonucleotides (tetra- to hexanucleotides [2,3]) as well as for some polynucleotides [4,5]. As predicted, the transition from helix-to-coil is accompanied by large downfield shifts resulting from changes in the phosphodiester bond geometry. For most of the tetra- and hexanucleotides studied, all the non-equivalent internucleotide phosphates were observed; however, no attempt was made to assign them.

1979

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Matsushima, M., Marquart, M., Jones, T. A., Colman, P. M., Bartels, K., Huber, R. and Palm, W. (1978)J. Mol. Biol. 121,441-459 33 Segal, D. M., Padlan, E. A., Cohen, G. H., Rudikoff, S., Potter, M. and Davies, D. R. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 42984302 34 Deisenhofer, J., Colman, P. M., Epp, 0. and Huber, R. (1976) Hoppe-Seyler’s Z. Physiol. Chem., Bd. 357,1421-1434 35 Deisenhofer, J., Jones, T. A., Huber, R., SjGdahl, J. and Sjaquist, J. (1978) HoppeSeyler’s Z. Physiol. Chem. Bd. 359,975-985 36[al Metzger, H. (1974) in Adv. Immunol., Vol. 18, (F. J. Doxon and H. G. Kunkel, eds), Academic Press, New York and London, pp. 169-207 [b] Metzger, H. (1978) Top. Mol. Zmmunol. 7, 119-152 37 Wright, J. K., Engel, J. and Jaton, J. -C. (1978) Eur. J. Immunol. 8,309-314 38[al Paques, E., Huber, R. and Priess, H. (1979) Hoppe-Seyler’s Z. Physiol. Chem. Bd. 360, 177-183 [bl Lin, T.-Y. and Fletscher, D. S. (1978) Immunochemistry 15,107-l 17 39 Porter, R. R. (1977) Fed. Proc., Fed. Am. Sot. Exp. Biol. 36,2191-2196

In contrast to the simple oligonucleotides, the majority of alp signals from tRNA is located in an unresolved cluster of peaks [6]. There is, however, a number of well-resolved resonances both to the low- and high-field side of the main peak. It has been suggested that these resonances are involved in tertiary structure and arise from phosphate groups in unusual conformations, perhaps where sharp kinks occur in the backbone. Results from the 31P relaxation studies on yeast tRNAPhe [7] support an earlier suggestion by GuCron and Shulman [6]: namely, that the high-field part of the main 31P cluster originates from phosphodiester groups involved in the formation of double-helical regions of tRNA, whereas the low-field part originates from the non-helical regions. ‘lP nmr spectroscopy has also been applied to differentiate between two extreme models proposed for the packing of DNA in nucleosome cores’ containing 140 base pairs. One [8] is that the doublehelix is kinked at regular intervals so as to permit sharp bending of the helical axis; the other [9] assumes that the helixal axis is altered gradually and uniformly. Any substantial kinking involving changes in the phosphodiester torsional angles should be accompanied by correspondingly distinctive 31P signals, such as previously mentioned for tRNA. The 3*Pspectrum of nucleosome cores as shown by Cotter and Lilly [lo] and Kallenbach et aE. [l l] has only a single symmetric peak with the chemical shift corresponding to that of B-form DNA.? The linewidths of core particles and their extracted DNA are also very similar, 2 1 v. -. _ .,._^