LIST OF SYMBOLS FOR CHAPTER 8

[oc] specific rotation λ wavelength Κ y a o » parameters Μ molecular weight Μτ mean residue weight η refractive index n & y n b principal refractive indices Ρ specific polarizability

> P\> principal specific polarizabilities piypt longitudinal and transverse bond polarizabilities p x , p 2 longitudinal and transverse molecular polarizabilities iV Avogadro's number / orientation function (see Chapter 5) ρ macroscopic density

166

Chapter 8

Other Methods of Studying Conformation

In the foregoing chapters methods which have proved to be especially valuable in the elucidation of conformation in fibrous proteins have been discussed in some detail. There are, in addition, a number of other methods which, although inherently incapable of yielding detailed information about conformation, nevertheless provide a means of measuring and detecting changes in a small number of molecular parameters. Concerning such methods, Schellman and Schellman (1964) remark: "The limitation is inescapable: If all the groups of a molecule contribute to a physical property, then the interpretation must invoke simple (and presumably oversimplified) models. If only a few groups contribute, then only a small part of the desired structure is under experimental attack" [p. 4]. However, many of these methods provide information which may be of assistance in devising models of fibrous protein structures and the most profitable, in this respect, will be reviewed briefly in this chapter.

Information on molecular shape and size can be obtained from studies of solutions of fibrous proteins by the techniques of sedimentation analysis (Coates, 1970), gel filtration (Andrews, 1970), viscosity mea­surement (Bradbury, 1970), light scattering (Timasheff and Townend, 1970), and low-angle X-ray scattering (Brumberger, 1967; Pilz, 1973). In addition, ultraviolet absorption (Gratzer, 1967), optical rotatory dispersion, and circular dichroism spectra (Yang, 1967, 1969; Beychok, 1967; Jirgensons, 1969; Tinoco and Cantor, 1970) exhibit effects which are associated with the spatial arrangement of the amide-group chro-mophores of the main chain and have been widely used to study

167

168 8. OTHER METHODS OF STUDYING CONFORMATION

conformation in solution. Nuclear magnetic resonance has also been used in studies of conformation and conformational change in solution (Metcalfe, 1970; Lui and Anderson, 1970; Roberts and Jardetzky, 1970; Sheard and Bradbury, 1971) but its main value in the study of fibrous proteins at present appears to be in investigations of the role of water in native structures (Clifford and Sheard, 1966; Berendsen, 1968; Cope, 1969; Dehl and Hoeve, 1969; Hazlewood et al, 1969; Katz, 1970; Tait and Franks, 1971; Khanagov, 1971).

The foregoing methods of studying conformation in solution have been very adequately described in the references cited. In the following sections attention will be confined to certain methods which are applicable or potentially applicable to the study of solid fibrous protein structures.

A. ULTRAVIOLET ABSORPTION SPECTRA

The ultraviolet absorption spectra of proteins and synthetic poly­peptides have been studied in considerable detail (Donovan, 1969) and the absorption bands near 200 nm associated with the main-chain amide chromophore have been found to be conformation-sensitive (Gratzer, 1967). This sensitivity to conformational change is a result of differences in the nature of the interactions between electronic transitions in different amide groups.

Exciton splitting, resulting from the coupling of the πχ -> π* transi­tions, has been observed in oriented films of poly(y-methyl-L-glutamate) in the α-helical form by Gratzer et al. (1961) and Momii and Urry (1968) and the directions of polarization were found to be as predicted by Moffitt (1956). Exciton splitting has also been observed in the α form of poly(L-alanine) (Gratzer et al, 1961; Momii and Urry, 1968; Bensing and Pysh, 1971) and in other regular structures such as the pleated-sheet β conformations (Pysh, 1966; Rosenheck and Sommer, 1967; Woody, 1969) and the polyproline I and II conformations (Rosenheck et al., 1969). Measurements of this type on fibrous proteins are likely to yield information similar to that provided by observations of infrared dichroism. However, specimen preparation is more exacting, measure­ment of spectra is more difficult, band overlap is considerable, and the prospect of obtaining reliable quantitative estimates of the contents of different conformations in a specimen seem remote at the present time. An important feature of linear dichroism measurements, however, is the possibility of distinguishing between the parallel-chain and antiparallel-chain pleated sheets (Woody, 1969).

Β. OPTICAL ROTATORY DISPERSION AND CIRCULAR DICHROISM 169

Hypochromism, resulting from interactions between electronic transi­tions of different energies in adjacent amide groups, has been observed in polypeptides in the α-helical form in solution (Imahori and Tanaka, 1959; Rosenheck and Doty, 1961; Tinoco et aL, 1962; Goodman and Listowsky, 1962; Goodman et ah, 1963c; Goodman and Rosen, 1964; McDiarmid and Doty, 1966) and used as a basis for estimating α-helix content of various proteins in solution (Rosenheck and Doty, 1961; Gratzer, 1967). The results obtained were not encouraging, however, and the possibility of applying this method to solid specimens of fibrous proteins seems slight in view of the difficulties of making quantitative intensity measurements on solid specimens.

Extensive studies of the optical rotatory dispersion and circular dichroism of synthetic polypeptides in solution have shown that these properties, which are closely related (Moffitt and Moscowitz, 1959; Moscowitz, 1960), are sensitive to conformation and thus a potentially useful means of investigating protein structure. Valuable accounts of experimental methods and reviews of the topic have been given by Yang (1967, 1969), Beychok (1967), Jirgensons (1969), and Tinoco and Cantor (1970). Attempts have been made to assign characteristic spectra to different conformations (Moffitt and Yang, 1956; Pysh, 1966; Rosenheck and Sommer, 1967; Greenfield et aL, 1967; Greenfield and Fasman, 1969; Woody, 1969; Chen and Yang, 1971; Saxena and Wetlaufer, 1971), thus providing a basis for estimating the contents of different conformations from observed spectra. There are, however, a number of theoretical and experimental problems which remain to be solved before the method can be made quantitative (Yang, 1969; Fasman et aL, 1970; Jirgensons, 1970; Chen et aL, 1972; Dalgleish, 1972; Madison and Schellman, 1972).

Measurements of optical rotatory dispersion in the visible and near-ultraviolet regions are generally fitted to an equation proposed by Moffitt and Yang (1956)

B. OPTICAL ROTATORY DISPERSION

AND CIRCULAR DICHROISM

3 MT

n2 + 2 100 [a] = a0 λ2 - λ0* 2

(8.1)

where [a] is the specific rotation, η is the refractive index of the medium, Mr is the mean residue weight, λ0 is a constant which has a value close

170 8. OTHER METHODS OF STUDYING CONFORMATION

to 212 nm in many cases, and aQ and b0 are adjustable parameters. The value of a0 has been found to be highly variable and to depend upon conformation, composition, and the molecular environment. The value of b0 , on the other hand, exhibits a strong dependence on the conformation of the main chain. Estimates of b0 for synthetic polypeptides in the α-helical conformations yield values of about —630 deg cm2 dmole"1

and the quantity — 100ft0/630 has been used widely as an empirical estimate of α-helix content. The value so obtained can only be regarded as approximate, however, if the α-helix content is low or regular conformations other than the α-helix are present (Jirgensons, 1969; Yang, 1969). Chen and Yang (1971) have analyzed optical rotatory dispersion data for a number of globular proteins of known structure and suggest an alternative value of —580 deg cm2 dmole -1 for the α-helix. In certain polymers the quantitative correlation between b0 and α-helix content breaks down completely due to a contribution to b0 from a regular arrangement of electronically coupled side-chain chromophores (Goodman et al, 1963b; Fraser et al, 1967b).

Attempts have been made to assign a value of b0 to the β conformation (Fasman and Blout, 1960; Imahori, 1960; Wada et al, 1961; Bradbury et al, 1962b; Imahori and Yahara, 1964; Davidson et al, 1966; Iizuka and Yang, 1966; Sakar and Doty, 1966) but the results obtained so far are inconclusive. It seems certain, however, that the magnitude of b0

is very much less for the β conformation than for the α-helix. From a study of globular proteins of known structure Chen and Yang (1971) suggest a value of 60 ± 30 deg cm2 dmole -1 for the β conformation. Chirgadze et al (1971) have pointed out that the optical rotatory dispersion of polypeptides and proteins in the near-infrared can be represented by an expression similar to that given in Eq. (8.1) provided that a constant term c0 is added. The value of c0 is conformation-sensitive and has zero value for the random-coil conformation. Its magnitude for the β conformation is an order of magnitude greater than for the α-helix and so the method has potentialities for the quantitative estimation of β contents.

Elliott et al (1957b, 1958) have stressed the difficulties associated with the measurement of optical rotatory dispersion spectra of solids. These arise from birefringence due to local preferred orientation and to residual strain. Visible and near-ultraviolet spectra were obtained from synthetic polypeptides and from films cast from solubilized silk fibroin. The results, however, were less accurate than those obtained from solution studies. In the case of poly(y-benzyl-L-glutamate) the b0

values in the solid state were similar to those measured in solution, but with poly(jS-benzyl-L-aspartate) anomalous results were obtained

C. BIREFRINGENCE 171

and this was attributed to a regular array of interacting side-chain chromophores (Bradbury et aL, 1959; Elliott et aL, 1962).

Conformation-dependent Cotton effects, associated with the electronic transitions discussed in Section A, are observed below 250 nm in the optical rotatory dispersion spectra of polypeptides in solution and these have been used as a basis for conformation analysis (see for example Greenfield et aL, 1967; Yang, 1969). The spectra of solid films of a number of synthetic polypeptides in this region have been reported by Fasman and Potter (1967). Very little work has been done on native fibrous protein structures, due to the difficulties of obtaining optically homogeneous specimens and estimating the mass per unit area (McGavin, 1964).

The measurement of circular dichroism spectra rather than optical rotatory dispersion spectra offers certain advantages and the two types of spectra can be related to one another by means of the Kronig-Kramers relationships (Moscowitz, 1960). Circular dichroism studies of poly­peptides and proteins in solution have been reviewed by Beychok (1967), Jirgensons (1969), Yang (1969), and Tinoco and Cantor (1970). As noted earlier, attempts to use circular dichroism spectra for the estimation of the contents of different conformations in a molecule have not been completely successful. Measurements have been made on solid films of both synthetic polypeptides and globular proteins (Fasman et aL, 1965, 1970; Fasman and Potter, 1967; Stevens et aL, 1968) but very little work has been done on fibrous proteins. Spectra for powdered elastin have been reported by Mammi et aL (1970).

A practical problem encountered in obtaining spectra from oriented specimens is the presence of linear birefringence and dichroism, which make it difficult to extract the required circular dichroism spectrum (Tinoco and Cantor, 1970). There are also a number of unresolved problems in the interpretation of the circular dichroism spectra of solid films (Fasman et aL, 1970).

C. BIREFRINGENCE

The phenomenon of birefringence arises from anisotropy of polar-izability, which is in turn related to molecular and textural anisotropy. General accounts of the theory of birefringence in polymer specimens and methods of measurement have been given by Hermans (1946), Bunn (1961), Morton and Hearle (1962), Stein (1964, 1969), and Wilkes (1971). Most naturally occurring fibrous protein structures are bire-fringent and the observed values are, in general, the sum of an intrinsic

172 8. OTHER METHODS OF STUDYING CONFORMATION

component, which is related to the anisotropy of polarizability of the individual molecules, and a second component, arising from the texture (Wiener, 1912; Schmitt, 1939), which is termed form birefringence.

An approximate value for the intrinsic component of the birefringence of a material of known structure can be calculated by assigning longi­tudinal and transverse polarizabilities to individual chemical bonds and summing their contributions to the polarizabilities parallel to the principal directions of the refractive index ellipsoid using the formula

P = £ ^ c o s 2 0 + £ /> ts in 20 (8.2)

where Ρ is the intrinsic component of the specific polarizability, p l and p t are the longitudinal and transverse bond polarizabilities, respectively, and θ is the angle between the bond and the principal direction. The summation extends over unit volume, and provided the birefringence is small, the difference between the refractive indices for two principal directions can be shown to be (Stein, 1964)

M a_ W b = ^ l ± 2 ) ! ( P a_ P b ) ( 8 - 3)

where P a and P b are the corresponding specific polarizabilities and η is the mean value of the refractive index. Values of p l and p t for various bonds have been given by Wang (1939), Denbigh (1940), Bunn and Daubeny (1954), Bunn (1961), and Nagatoshi and Arakawa (1970). The bond polarizabilities given by these authors can only be regarded as average values and the true value is expected to vary with the electronic structure of the bond. Under these circumstances, birefringence values calculated using Eq. (8.3) can only be regarded as approximate and are likely to be seriously in error if the birefringence is small. Ingwall and Flory (1972) have investigated the anisotropy of the polarizability of amides and derived values for a glycyl peptide unit.

An alternative approach is to use Eq. (8.2) to calculate the polar­izability p x parallel to the axis of an elongated molecule and a mean polarizability p 2 perpendicular to that axis and to use the concept of an orientation parameter / as defined in Chapter 5. In this case the intrinsic component of the macroscopic anisotropy of specific polarizability will be given by

Pa-Pb=(NfPIM)(Pl-p2) (8.4)

where Ν is Avogadro's number, Μ is the molecular weight, and ρ is the macroscopic density. If a number of phases are present, the right-hand side of Eq. (8.4) must be replaced by an appropriate summation. Since the intrinsic birefringence is a function of conformation and

D. LIGHT SCATTERING 173

orientation, it provides a means of detecting changes in these molecular properties. A complication in quantitative studies is the existence of form birefringence, which arises from the anistropic shape of the molecular aggregates. This effect has been treated theoretically (Wiener, 1912, 1927) but the contribution to the observed birefringence must, in general, be determined experimentally (Hermans, 1946; Haly and Swanepoel, 1961; Stein, 1964; Cassim et al., 1968).

Measurements of birefringence provide a useful means of comparing the degree of orientation in different specimens of the same material and have also been used to detect changes in orientation and conforma­tion in, for example, collagen fibers (Ramanathan and Nayudamma, 1962; Basu et aL, 1962), muscle fibers (Fischer, 1947; Mommaerts, 1950; Takahashi et aL, 1962), and keratin fibers (Fraser, 1953b; Feughelman et aL, 1962; Feughelman, 1966, 1968).

D. LIGHT SCATTERING

Many fibrous proteins are laid down in ordered aggregates which extend over distances comparable with the wavelength of visible light and the accompanying fluctuations in polarizability give rise to light scattering. The angular distribution of the scattered light depends upon the nature of the fluctuations and two approaches to the interpretation of light-scattering results on solids have been used. In the first a model for the fluctuations is assumed, for example, optically anisotropic spheres embedded in an isotropic medium, and the observed data are used to test the model and to estimate values of the parameters describing the model. In the second approach the problem is dealt with in terms of a set of statistics which defines the correlation between fluctuations in polarizability in different parts of the specimen. Details of the experimental arrangement used to measure light scattering from solids and a discussion of the theory are given by Stein (1964).

Moritani et al. (1971) have recorded light scattering from several types of collagen films prepared from acid-soluble collagen and collagens solubilized by treatment with proteolytic enzymes. An optical model for the microscopic texture of collagen was assumed and changes in the light scattering with denaturation were interpreted in terms of this model. Although it is difficult to relate the optical model to the texture of the molecular aggregates in collagen, these experiments are important because they point the way to making similar measurements in vivo on fibrous protein assemblies such as those found in connective tissue and muscle.

174 8. OTHER METHODS OF STUDYING CONFORMATION

E. RAMAN SPECTRA

Raman spectra arise from a scattering process in which a change takes place in the vibrational energy of the molecule. The frequency of the scattered light differs from that of the incident light by an amount corresponding to the change in vibrational energy, and information which is in many ways complementary to that given by infrared spectra can be obtained on the normal modes of vibration of a molecule (Colthup et al.y 1964; Koenig, 1971). Raman scattering is in general much weaker than the Rayleigh type of scattering, discussed in Section D, in which there is no change in frequency on scattering. Experimental aspects have been discussed by Hawes et al. (1966), Beattie (1967), Tobin (1968, 1971), Cornell and Koenig (1968), and Fanconi et al. (1969) and valuable reviews of the application of Raman spectra to polymers have been given by Hendra (1969) and Gall et al. (1971).

The selection rules for Raman activity of vibrations require that the polarizability must change during the vibration, and the intensity of the Raman band is a function of the magnitude of the change in polarizability. The selection rules for Raman scattering from helical molecules have been given by Higgs (1953) and a detailed treatment of the polarized spectra of poly(L-alanine) has been given by Fanconi et al. (1969). Partially ordered systems have been considered by Snyder (1971). The interpretation of polarized Raman spectra is more complex than with polarized infrared spectra and the results are less readily interpretable in terms of molecular conformation. However, observations of Raman spectra are of value in cases where vibrations are infrared-inactive, such as the v(0, 0) modes of the antiparallel-chain pleated sheet (Chapter 5, Section A.II.b), and these modes, which are Raman-active, have been observed in the spectra of synthetic polypeptides (Koenig and Sutton, 1971).

Most vibrational modes of proteins are both infrared- and Raman-active and the relative intensities in the two types of spectra are markedly different. The Raman spectrum is not dominated by the main-chain amide bands, as is the case with the infrared spectrum, and side-chain bands are prominent. The aromatic side-chain groups of phenylalanine, tryptophan, and tyrosine give rise to sharp and intense bands and bands associated with the —C—S—S —C— grouping are prominent in cystine-containing proteins (Lord and Yu, 1970a,b). Thus it is in the finer details of side-chain conformation that Raman spectra seem likely to yield information, not available from other methods, on the structure of fibrous proteins. The other property of Raman spectra which seems likely to be exploited in the future is that

F. MECHANICAL AND THERMAL PROPERTIES 175

the bands associated with water are relatively very much less intense than in infrared spectra, thus permitting observations on hydrated materials. One of the problems which must be overcome before satisfactory Raman spectra can be obtained is the elimination of fluo­rescence and the reduction, as far as possible, of turbidity. These present difficult problems with naturally occurring fibrous protein structures and most of the studies of polypeptides so far reported (Fanconi et al, 1969; Koenig and Sutton, 1969, 1970, 1971; Rippon et al, 1970; Deveney et al, 1971) have been made on synthetic materials, from which fluorescent impurities can be removed by appropriate chemical treatment. Other problems encountered in obtaining spectra from solid films are overheating and photodegradation.

F. MECHANICAL AND THERMAL PROPERTIES

Since fibrous proteins have a primarily structural function in living tissue, the mechanical properties and thermal stability are topics of considerable interest and have been studied extensively. The classical investigations of Astbury and co-workers (Astbury and Street, 1931; Astbury and Woods, 1933; Astbury and Sisson, 1935) for example, provide a wealth of data against which model structures can be tested. While studies of this type still have a place in investigations of con­formation, there is a growing realization that the direct correlation of detailed conformation with mechanical and thermal data is fraught with difficulties.

The value of mechanical studies and the difficulties associated with their interpretation in terms of molecular structure have been elegantly summarized by Chapman (1969a):

A mechanical model of fiber structure may be found useful in a number of ways: firstly, merely as a convenient method of describing structure and predicting mechanical behavior in a fairly compact way; secondly, as a means of testing structures that are proposed on the basis of other work; and lastly, it may be possible from experimental results to infer, on the basis of a particular model, some general structural features. In the last case, however, the conclusions drawn concerning the structure are usually highly dependent on the particular model system chosen to describe the behavior.... Of necessity, the model must generally be an oversimplification of the actual structure, to allow any degree of mechanical analysis [p. 1102].

176 8. OTHER METHODS OF STUDYING CONFORMATION

There are a number of instances, however, where the use of a simple model has served to give a very clear understanding of the mechanical properties in terms of the molecular texture, for example, in the filament-matrix model used to explain the behavior of the elastic moduli of α-keratin fibers on hydration (Feughelman, 1959).

The literature on the mechanical properties of fibrous proteins, their dependence on time, temperature, hydration, and other parameters, and attempts to interpret these properties in terms of molecular structure is voluminous. Valuable reviews of these topics and of experimental techniques have been given by Morton and Hearle (1962), Crewther et al (1965), Harkness (1968), Chapman (1969b), Hearle and Greer (1970), and by various authors in the Encyclopedia of Polymer Science and Technology (Bikales, 1966-70).

Structural transitions occur when fibrous proteins and other polymers are heated or deformed and these can be studied by differential thermal analysis (Ke, 1964a,b; Morita, 1970). This technique has been applied to collagen (Witnauer and Wisnewski, 1964; Naghski et al, 1966), silk (Schwenker and Dusenbury, 1960; Pande, 1965; Crighton and Happey, 1968), and α-keratin fibers (Schwenker and Dusenbury, 1960; Felix et al, 1963; Menefee and Yee, 1965; Pande, 1965; Haly and Snaith, 1967; Crighton et al, 1967; Crighton and Happey, 1968).