The optical rotatory dispersion of MS2 bacteriophage

The optical rotatory dispersion of MS2 bacteriophage

ARCHIVES OF BIOCHEMISTRY AND The Optical BIOPHYSICS Rotatory 127, 274-282 (1968) Dispersion of MS2 Bacteriophage P. J. ORIEL Biochemical ...

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ARCHIVES

OF

BIOCHEMISTRY

AND

The Optical

BIOPHYSICS

Rotatory

127,

274-282

(1968)

Dispersion

of MS2

Bacteriophage

P. J. ORIEL Biochemical

Research

Laboratory,

The Dow Chemical

Company,

Midland,

Michigan

48640

Midland,

Michigan

48640

J. A. KOENIG Computation

Research

Laboratory,

Received

December

The Dow Chemical

Company,

12, 1967; accepted

March

6, 1968

The optical rotatory dispersions of MS2 bacteriophage, its constituent RNA’, and coat protein subunit are described, revealing little change in RNA secondary structure upon assembly. It is concluded that the unusual MS2 protein subunit rotatory dispersion probably reflects tertiary rather than secondary structure, and that this structure persists upon assembly. Analysis of heated bacteriophage revealed an increase in protein levorotation concomitant with splitting of the particles into degraded nucleic acid and protein shells.

RNA bacteriophages (1) have been the objects of intense study following their discovery (2) as they present one of the simplest nucleic acid systems for the correlation of biological structure and function. In addition it has been anticipated that derived principles will be important in the elucidation of the more structurally complex animal RNA viruses. Although the mechanism of replication of these bacteriophages is becoming known in detail (see, for example, (l)), little is known concerning the forces stabilizing the assembled structures of the intact bacteriophage. In this communication rotatory dispersion optical studies of the RNA bacteriophage MS2 will be presented. These studies indicate that MS2 protein, both as subunits and assembled, has an unusual optical rotatory dispersion. In addition a structural change in the phage coat concomitant with RNA release is described. ’ Abbreviations used: RNA, ribonucleic acid; ORD, optical rotatory dispersion; OD, optical density; PGA, poly-r-glutamic acid; CD, circular dichroism .

MATERIALS

AND

METHODS

Bacteriophage and host. MS2 bacteriophage (ATCC 15597-B) and its host E. coli C3000 (ATCC 15597) were grown and purified as described by Strauss and Sinsheimer (3) except for the inclusion of a dialysis step after the ammonium sulfate precipitation and the omission of the freon extraction. ConcentratipFri of the bacteriophage was determined of 80.3 at 260 rnr (3). Sedimentation using an E)q velocity studies were routinely made of preparations as a criterion of purity. These were in agreement with the published values of S20.w = 79-81 (3, 4). MS2 RNA. MS2 RNA was prepared as described by Strauss and Sinsheimer (3). In later preparations a bentonite step was included (5). Concentration was determined using an Elgrn of 251 at 260 rnp (3). MS2 protein. MS2 protein was prepared as described by Overby et al. (6). The sedimentation value obtained in 0.1 M carbonate buffer, pH 10.5, was 2.3 at a concentration of 0.31 mg/ml, 20’. Concentration was determined by the micmKjeldah1 method. Poly-L-glutamic acid and poly-L-lysme HCl. The poly-L-glutamic acid used was purchased from Pilot Chemicals (Lot L-49, DP 540). The poly-Llysine HCI used was a commercial Pilot preparation of poly-L-lysine HBr exchanged to the HCl salt using a Dowex 1 column. @-structures were prepared using the method of Sarkar and Doty (7). Optical 21 4

OPTICAL

ROTATORY

DISPERSION

rotatory dispersion results were in agreement with the values of these investigators. Concentration of polypeptides was determined by microKjeldah1 analysis. Den&y gradients. Cesium chloride density gradients were generated with an SW39 rotor at 37,000 rpm on a Spinco Model L ultracentrifuge. These were fractionated from the top with a Buchler pump, tube puncturing device and microfractionator. Both ODZso and refractive index were read for each tube. Refractive indices were converted to densities using the relationship of Ifft et al. (81. Spectropolarimeter. Optical rotatory dispersion measurements were obtained on a Cary 60 spectropolarimeter thermostated at 27”. Measurements were obtained using Opticell cuvettes with lengths varying between 1 and 10-l dm. Millipore filtration was routinely used prior to measurements, which were confined to samples with an optical density of less than 2. Temperature studies were carried out with an Opticell l-cm jacketed cuvette and Yellow Springs Instrument Telethermometer. The thermistor probe was maintained in the cuvette just above the light path. Analytical ultracentrifuge. Analytical ultracentrifuge measurements utilized the Beckman Spinco Model E ultracentrifuge equipped with the photoelectric scanning accessory. Infmred measurements. The MS2 protein subunits were exhaustively dialyzed against 0.5 N acetic acid, lyophilized and measured as a microKBr pellet with a Beckman IR9 spectrophotometer. ORD computations. For this and other problems requiring analysis of mixed structures (9) by optical rotatory dispersion a computational method has been implemented. Although this method is just as dependent upon model structure rotatory dispersions as those used previously (10, 11, 12), it utilizes all the rotatory information, and can readily be used for mixtures of more than two conformations. Since it appears likely that this program will also prove useful in other applications, a general mathematical description of the program is given, followed by a description of application in ORD. Alternative computational methods for conformational analysis by ORD and CD have recently been described (13, 14, 15). The experimental data was fitted with a model of the form Y = f(x;B)

+

c

where I is the error or difference between the observed (experimental) value Y and the value predicted by the postulated models. B is the independent variable describing the model parameters, and x is the independent variable of measurement (e.g., time or wavelength). Letting the error of the sum of

OF squares

MS2

275

BACTERIOPHAGE

be S(B)

= 2
~ f[x;

B])’

and since Y, and x, are fixed, the sum of squares becomes a function of B alone. The least squares method is then applied to obtain an estimate of the B that will minimize S(R). The computer program used to process the experimental data is written in ALGBL 60 for the Burrough’s B5500 computer system. The program applies when S(B) is linear in the parameters as well as when S(B) is nonlinear with respect to B. The method used is essentially an iterative approach utilizing a Taylor series expansion of the given function or model. By retaining only the linear terms of the expansion it is possible to apply the general linear least squares technique at this point. The partial derivative used in the series expansion can be either numerical approximations or the analytical form of the derivatives. Input to the program consists of n sets of values for the dependent values Y, and the independent variables x, Initial estimates for the parameters B must be part of the input. Each iteration gives a new corrective vector such that

where a, is the correction for the jth parameter and interactions continue until the corrections become smaller than a predetermined sum or the sum of squares the residuals change only slightly from one interaction to the next. When the system is nonlinear in the parameters B, the initial parameter estimates may significantly affect the rate or ability to converge. In this application Y refers to experimental rotatory dispersion, x to the wavelengths of measurement and f(B) to the set of model coefficients to be determined; i.e.,

[ffl&&Lbli. where n persions rotatory sisted of

+ eh

denotes the number of model rotatory distested for contributions to the unknown dispersion. The output of the program conthe best fitting rotatory points

B, values, and c values. The B, values were then corrected for the differences between mean residue weights of the models and the material under investigation. It should be noted that this analysis is flexible and can be used for systems where interactions with subsequent changes in constituent rotatory dispersions occur. The criteria for good fits were both the size of c compared with experimental error and the model coefficients correspondence with

276

ORIEL

AND

KOENIG

The principal question we wished to ask was to what extent are the conformations of MS2 RNA and subunit protein altered RESULTS in the assembly process? The ORD of Test analysis of PGA ORD. As a test purified MS2 is seen in Fig. 2. This curve of the regression method, the ORD of and the RNA values below are in good poly-L-glutamic acid was measured at agreement with those recently reported pH 4.5, 6.0 and 8.0. At the intermediate by Maestre and Tinoco (18). Comparison pH the rotatory dispersion should resolve with the ORD of isolated MS2 RNA in into weighted sums of those of the largely Fig. 3 suggests a trough due to the coat a-helical form at pH 4.5 and the random protein near 230 mr. coil form at pH 8.0 (16). Rotatory disperAn initial attempt was made to analyze sions and best computer fit are seen in the conformation of the bacteriophage by Fig. 1. The analysis predicted 18% (Y- determining if the MS2 ORD could be helicity, assuming that the pH 4.5 ORD divided into ORD components of protein reflected 100% a-helix. This is in agree- a-helix, protein random coil and RNA as ment with the l&-20% a-helical value reflected by PGA at pH 4.5, PGA at pH determined from linear interpolation of 8 and MS2 RNA at 27” under the condi[(~I233or [CX]IXL tions shown in Fig. 3. A satisfactory fit MS2 ORD. The MS2 bacteriophage could not be obtained, the main errors (3.6 X lo6 MW) is comprised of a single in fitting being below 250 mp. This indistrand of RNA (1.05 X lo6 MW) and an cated that the principal failure in model outer protein coat (3). The coat consists selection might be the simple assumption of a polyhedral assembly of 14,000 MW of an a-helix: random coil mixture for the subunits (6). In addition, a small quantity bacteriophage protein. of noncoat protein may be present (17). MS2 Subunit protein. To determine if the protein subunits exhibit an unusual rotatory dispersion that might be retained reality protein

(i.e., all protein residues : nucleic acid ratios correct

accounted for and for nucleoproteins).

1600

,

1200

-

600

-

400

-

[aI

5,000 t

o-400

-

-600

-

-1200

-

-1600

-

-2000

-

-2400

-

-2600

-

I

-20,000

FIG.

0 160

200

220

240 260 WAVELENGTH

260

300 (my)

320

1. Computational fit of poly-L-glutamic (---), poly-L-glutamic acid at pH 4.5. (- -), glutamic acid at pH 8. (--), poly-L-glutamic at pH 6. ( o 0 o ), best fit of poly-L-glutamic at pH 6.

340

1 \ \

220

acid. poly-Lacid acid

240

260

260

WAVELENGTH

300

320

340

(m/b)

FIG. 2. (-), optical rotatory dispersion of MS2 bacteriophage in 0.05 M sodium phosphate buffer, 0.05 M NaCl, pH 7.2. (---), predicted rotatory dispersion of MS2 bacteriophage (see text).

OPTICAL

ROTATORY

DISPERSION

277

OF MS2 BACTERIOPHAGE

5000,

6000

4000 5000

t 3000 k

4000

1L 27'C

2000 1000 [.I

0,

,'

, '--~,~O-C

3000 [al

2000

-1000 0 - 1000

-3000

-

-2000

-4ooo-3ooo-5000

-

220

240

260

260

WAVELENGTH

300

320

I60

200

tmp)

FIG. 3. Optical rotatory dispersion of MS2 RNA in 0.1 M NaCl, 0.05 M sodium phosphate buffer, pH 7.2.

by the assembled virus, the ORD of subin 0.1 M carbonate buffer at pH 10.5 was measured. A rather unusual rotatory dispersion is apparent in Fig. 4 with a shallow minimum at 229 rnp and increasing rotation below this wavelength. The shoulder near 210 rnp appeared reproducible but must be regdrded as tentative as measurements below 220 mp were made under conditions of high buffer absorption. Though the trough magnitude is very small, no random coil trough is apparent at 206 mp (compare PGA at pH 6 in Fig. 1). A Moffitt-Yang plot of the ORD from 495 to 270 rnp is shown in Fig. 5. Although nonlinear above 350 rnp, the linear region from 350 to 270 rnp yields a b. value of near zero. In view of the small size of Cotton effects in the observable region it is possible that the nonlinearity results from transitions below 200 rnp. The size and placement of the trough at 229 rnp and the b. value indicate that little or no a-helix is present in the subunit under these conditions. Shallow Cotton effects with troughs near 230 rnr and low b. values have been shown to be characteristic of antiparallel P-structures (7, 19). Although no characteristic P-structure maximum near 205

220

240

260

WAVELENGTH

340

280

300

320

340

(nip1

FIG. 4. Optical rotatory dispersion of MS2 protein subunits, 0.1 M sodium carbonate buffer, pH

units

1

‘I123456763 x0’

P-x’, FIG. 5. Moffitt-Yang X0 was 212 mp.

x 10-4

plot of MS2 protein subunits.

rnp (7, 19) was apparent in the subunit protein ORD, a test was made to determine P-structure presence as part of a mixture of conformations. The subunit ORD was regressed against models of a-helix, random coil and antiparallel pstructure. A good fit could not be obtained, as the sum of the model coefficients was far below 1. This resulted from the fact that the low rotation of the subunit near 230 and 200 rnp was not compatible with the curve shape in terms of a mixture of currently known secondary structures. As a further test of p-structure presence, the infrared spectrum of purified MS2 protein was measured (Fig. 6). The place-

278

OFUEL

2000

1800

AND

KOENIG

1600

1400

1200

lo60

cm-' FIG. 6. Infrared

spectrum

of MS2

ment of the carbonyl stretching frequency at 1655 cm-’ is well above the value near 1630 cm-’ expected for p-structures of both the cross and antiparallel type (20). Although this spectrum was taken in a different phase than the ORD measurements, it suggests that p-structures do not play a large role in the MS2 protein structure. In preliminary experiments to test the effect of denaturing conditions, the protein subunits were dissolved in 0.1 M carbonate, 8 M urea. Although measurements could not be extended below 220 rnp, it was apparent that the trough near 230 rnp was decreased in levorotation, but was still present. Heating the subunit solution to 88’ in 0.1 M carbonate buffer, pH 10.5, did not significantly alter the trough magnitude. CHANGESUPON MS2 ASSEMBLY An attempt was made to determine to what extent the protein subunit rotatory features persisted upon assembly. The MS2 ORD was regressed against rotatory dispersions of RNA.(Fig. 3) and MS2 subunit protein (Fig. 4). As seen in Fig. 1 a fair fit was achieved with the model coefficients corresponding to the correct values of 32% RNA and 68% protein. The agreement is very good in the region of the large nucleic acid rotation but a

protein.

See text

for conditions.

variation is seen in the region of the coat protein trough. Temperature studies. To further investigate the structure of intact MS2, studies of rotational temperature dependence were made. Fig. 7 shows the variation of [(~I285 of the bacteriophage with temperature. At this wavelength the rotation is primarily that of the RNA component (9OYC at 25”). A rather gradual decrease is seen followed by an abrupt decrease for all ionic strengths. In addition, in Curve 1 a reversal of rotation is seen starting at 64”. A shoulder is also seen in Curve 2. Curve 1 could not be extended above the temperatures shown because of scattering caused by time-dependent aggregation. As degradation of RNA viruses into nucleic acid and protein components have been previously observed (see, for example, 21), it seemed likely that the reversal and shoulder at higher ionic strengths was due to restructuring of the nucleic acid upon release from the bacteriophage. To test this idea, MS2 bacteriophage under the conditions of Curve 2 were heated to 55 and 70’ in separate experiments, cooled and banded by cesium chloride equilibration centrifugation. It is seen in Fig. 8 that the RNA banded at the density of intact MS2 when heated to 55”, but after heating to 70’ was found at the position of free MS2 RNA (tube bot-

OPTICAL

‘--

I

1000

-

600

-

600

-

400

-

200

-

ROTATORY

DISPERSION

I

co1 295

0. ‘\ -200

I 30

-4ooLJ--LJ -400

FIG.

teriophage 1, 0.05 7.2. (---), 0.05

M

sodium

‘.

I I I 40 50 60 TEMPERATURE,

10 “C

60

7. Temperature dependence of MS2 bacoptical rotation at 285 mp. (- -), Curve M sodium phosphate buffer, 1 M NaCl, pH Curve 2, 0.05 M sodium phosphate buffer, NaCl, pH 7.2. (-), Curve 3, 0.02 M phosphate buffer, pH 7.2.

A

II

es9

1.400

MS2

279

BACTERIOPHAGE

tom) (22), confirming that cleavage had occurred. To determine the extent of association of the MS2 protein after nucleic acid release, samples of MS2 in 0.02 M phosphate, pH 7.2, were heated to 70”, cooled, and observed using the analytical ultracentrifuge. Photoelectric scanning measurements at 260 rnp revealed that the RNA consisted of heterogeneous pieces of approximately 2s. In addition, observation at lower speed with both the Schlieren optical system and photoelectric scanning system at 280 rnp revealed 49-50s protein particles (Fig. 9). Since the MS2 has a sedimentation coefficient of 79 and MS2 RNA has a sedimentation coefficient of 27, it appears probable that the protein coat stayed largely associated upon nucleic acid release. Drees and Borna (23) have reported a similar heat splitting of poliovirus into degraded RNA and protein shells.

(x

I I

MS 2 from

I

55°C

1.500

OF

0.2)

1.600 APPARENT

1.700 DENSITY

1.800

FIG. 8. Cesium chloride equilibrium centrifugation of MS2 and MS2 RNA, 0.05 sodium phosphate buffer. The density of 1.8 corresponds to tube bottom. Absolute values have been scaled as shown for ease of comparison.

M

M

FIG. 9. Ultraviolet scan at 280 mp of MS2 heated to 70°, cooled, and rpm. The scan, taken 33 minutes after reaching speed, shows the protein absorbance background from degraded RNA.

centrifuged sedimenting

NaCl, 0.05 absorbance

at 35,800 with an

280

ORIEL

AND

KOENIG

-2500 - 1400

[aI 25s

-2600

-

-2700

-

-2600

-

-2900

-

-3000

-

-3100

-

-3200

-

-3300

-

-3400

-

-

1200

-

1000

-

000

-

600

-

400

-

200

La1285

- -200

I 30

-3500 20

I 40

I 50

TEMPERATURE,

I 60

I 70

I 00

I 90

‘C

FIG. 10. (0 a), [a]~ of MS2 bacteriophage as a function of temperature phosphate buffer, pH 7.2. [a]z,, from Fig. 7 is also shown for comparison (0 nate.

in 0.02 M sodium 0), use right ordi-

50 and 60”. The rotatory dispersion of the product upon cooling is seen in Fig. 11. The increase in levorotation is accentuated even further with cooling, indicating the irreversibility of the change.

*Ooo I

DISCUSSION

I

I

I

I

I

I

220

240

260

280

300

320

WAVELENGTH

I

(mp)

FIG. 11. Optical rotatory dispersion of MS2 teriophage in 0.02 M sodium phosphate buffer, 7.2. (---I, before heating; (-1, heated to cooled to 20” and measured.

bacpH 75”,

To determine protein coat conformation changes during heating, [CX]~~~of MS2 was measured as a function of temperature in 0.02 M phosphate buffer, pH 7.2. The results are seen in Fig. 10. A sharp increase in levorotation is apparent between

Since viral proteins are known for their unusual physical properties (24)) it is of great interest to investigate their structure. It has been repeatedly pointed out, however, that ORD conformational analysis of proteins must be approached with caution (see, for example, 25, 26). Some of the obstacles that hinder assessment of secondary structure are side-chain rotational contributions (27, 28) and difficulties in obtaining unambiguous ORD values for model structures (29). Although estimates of cY-helicity have been reasonably good for proteins and polypeptides with a high content of this structure (12, 30, 31), interpretation of the ORD data from proteins with low degrees of secondary structure has been more difficult (see, for example, 32, 33). Schellman and Schellman (34) have pointed out that regions of proteins with no secondary

OPTICAL

ROTATORY

DISPERSION

structure, termed amorphous, may not have the same distribution probability in conformational angles cp and # that do the random coil polypeptides used as models. It was suggested by these workers that rotational freedom might be particularly restricted in highly hydrophobic amorphous regions. Since almost half of the MS2 protein residues can be termed hydrophobic, it may be anticipated that use of a random coil model for amorphous regions in this protein may be particularly dangerous. In spite of the above, we felt that it was worthwhile to attempt computer resolution of protein conformation using polypeptide models. Since the ORD analysis method described allows observation of fit over a wide wavelength region, it seemed possible that useful information could be obtained from the examination of deviations between the protein ORD and curves of best fit using models. As described, the MS2 protein ORD data did not allow a good fit using a mixture of a-helix, random coil, and antiparallel P-structure as models. This was substantiated by the fact that although the shape of the subunit protein ORD differs from that expected for a random coil, evidence for P-structures and o-helix were not found using the more established methods of analysis. Three possibilities can be raised for explaining the data: (1) presence of side-chain rotational contributions, (2) presence of a new secondary structure, or alteration of the ORD of a known secondary structure by structure distortion or low dielectric constant, (3) rotational contributions from amorph-us regions unlike those found for random coil polypeptides. The first possibility appears unlikely. The MS2 subunit protein has two tryptophan residues, two tyrosine residues, two phenylalanine residues and no cystine linkages per 133 residues (4). We are aware of no investigators who have found large aromatic side-chain contributions below 250 mr in the absence of Cotton effects above 250 rnp. It is estimated that Cotton effect peaks and troughs com-

OF

MS2

BACTERIOPHAGE

281

prising more than 2%; of the observed rotation would have been observed. The second possibility cannot, of course, be ruled out at this time. We feel that in view of the relatively small rotational values found that the third possibility discussed below is more likely. Schellman and Nielsen (35) have shown that electronic coupling in diamides results in ORD similar in many respects to that which arises in proteins and polypeptides. They find that both the Kirkwood (36) and one-electron type (37) couplings are strongly dependent upon and solvent conformational angles polarity. On the basis of their work with acetyl-L-alanine amide, for instance, they suggest that a negative n-r* Cotton effect in the interior of a nonhelical protein might arise without a high degree of conformational restriction. Static or dynamic interpeptide coupling in the absence of secondary structure is a plausible origin of the MS2 subunit protein rotatory dispersion. The models of Schellman and Nielsen are not yet sufficiently elaborated to assess the relative importance of interior dielectric constant and conformational restriction in the origin of the MS2 protein ORD. In either case, however, it seems probable that a stable tertiary structure is important. Further measurements of the MS2 subunit are being carried out under denaturing conditions to test this suggestion. It is probable that MS2 RNA under the conditions measured is highly base paired (3, 15). The agreement of the experimental and predicted MS2 ORD in the region of large RNA rotation using MS2 protein and MS2 RNA models indicates that unlike the DNA bacteriophage (18) and foot and mouth disease virus (38), the RNA does not possess unusual secondary structure in the virus relative to that of isolated RNA. The ORD of the MS2 protein when assembled is best fitted using the MS2 subunit protein ORD rather than those of polypeptide model structures. It seems reasonable to conclude that features of the assembled coat have essential simi-

282

ORIEL

AND

larities to those found for the subunit. These are low amounts of a-helix or antiparallel /?-structure and possible peptide coupling from a stable tertiary structure. The difference between the experimental and predicted curves of the assembled virus cannot necessarily be attributed to the assembly process, as the subunit was measured at a different pH than the intact virus. The large change in protein rotatory dispersion concomitant with nucleic acid release is puzzling in view of the rather small difference m rotatory dispersion between the subunit and assembled coat. It may be that the most stable conformation of the assembled subunits without nucleic acid interaction differs from that found when the RNA is present. ACKNOWLEDGMENT We gratefully acknowledge the expert technical assistance of Mrs. Janice Knop. We are also grateful to Dr. William Potts for assistance with the infrared measurements and Miss Carole Kleeman for assistance with the ultracentrifuge measurements. REFERENCES 1. HOFFMAN-BERLING, H., KAERNER, H. C., AND KNIPPERS, R., Advances in Virus Research 12,329 (1966). 2. LOEB, T., AND ZINDER, N. D., Proc. Nat. Acad. Sci., U.S. 47, 282 (1961). 3. STRAUSS, J. H., JR., AND SINSHEIMER, R. L., J. Mol. Biol. 7, 43 (1963). 4. OVERBY, L. R., BARLOW, G. H., DOI, R. H., JACOB, M., AND SPIEGELMAN, S., J. Bacterial. 91, 442 (1966). 5. FRAENKEL-CONRAT, H., SINGER, B., AND TSJGITA, A., Virology 14, 54 (1961). 6. OVERBY, L. R., BARLOW, G. H., DOI, R. H., JACOB, M., AND SPIEGELMAN, S., J. Bacterial. 92, 739 (1966). 7. SARKAR, P. K., AND DOTY, P., Proc. Nat. Acad. Sci., U. S. 55, 981 (1966). 8. IFFT, J. B., VOET, D. H., AND VINOGRAD, J., J. Phys. Chem. 65, 1138 (1961). 9. DRAPER, N. R., AND SMITH, H., “Applied Regression Analysis,” John Wiley and Sons (1966). 10. MOFFITT, W., AND YANG, J. T., Proc. Nut. Acad. Sci., U. S. 42, 596 (1956). 11. SHECHTER, E., AND BLOUT, E. R., Proc. Nat. Acad. Sci., U. S. 51, 695 (1964).

KOENIG 12. SIMMONS, N. S., COHEN, C., SZENT-GYORGM, A. G., WETLAUFER, D. G., AND BLOLJT, E. R., J. Am. Chem. Sot. 83,4766 (1961). 7, 617 (1968). 13. MAGAR, M. E., Biochemistry 14. GREENFIELD, N., DAVIDSON, B., AND FASMAN, G. D., Biochemistry 6, 1630 (1967). 15. CANTOR, C. R., JASKUNAS, S. R., AND TINOCO, I., JR., J. Mol. Biol. 20, 39 (1966). 16. BLOUT, E. R., SCHMIER, I., AND SIMMONS, N. S., J. Am. Chem. Sot. 84, 3193 (1962). 17. NATHANS, D., OESCHGER, M. P., EGGEN, K., AND SHIMURA, Y., PI-W. Nat. Acad. Sci., U. S. 56, 1844 (1966). 18. MAESTRE, M., AND TINOCO, I., JR., J. Mol. Biol. 23, 323 (1967). 19. DAVIDSON, B., TOONEY, N., AND FASMAN, G. D., Biochem. Biophys. Res. Commun. 23, 156 (1966). T., AND BLOUT, E. R., J. Am. Chen. 20. MIYAZAWA, Sot. 87, 712 (1961). 21. COHEN, S., AND STANLEY, W. M., J. Biol. Chem. 144, 589 (1942). 22. ARGETSINGER, J. E., AND GUSSIN, G. N., J. Mol. Biol. 21, 423 (1966). 23. DREES, O., AND BORNA, C., Z. Naturforschg. 2Ob. 870 (1965). Quant. 24. CASPAR, D. L. D., AND KLUG, A., Symp. Biol. 28, 1 (1962). in Protein 25. URNES, P., AND DOTY, P., Advances Chemistry, 16,401(1961). 26. CARVER, J. P., SCHECHTER, E., AND BLOUT, E. R., J. Am. Chem. Sot. 88, 2562 (1966). E., AND LINDBLOW, 27. FASMAN, G. D., BODENHEIMER, C., Biochemistry 3, 1665 (1964). 28. FASMAN, G. D., LANDSBERG, M., AND BUCHWALD, M., Can. J. Chem. 43, 1588 (1965). 29. YANG, J. T., AND MCCABE, W. J., Biopolymers 3, 209 (1965). 30. URNES, P. J., IMAHORI, K.. AND DOTY, P., Proc. Nat. Acad. Sci., U. S. 47, 1635 (1961). 31. BEYCHOCK, S., AND BLOUT, E. R., J. Mol. Biol. 3, 769 (1961) 32. STEINER, L. A., AND LOWEY, S., J. Biol. Chem. 241, 231 (1966). 33. JIRGENSONS, B., J. Biol. Chem. 241, 147 (1966). 34. SCHELLMAN, J. A., AND SCHELLMAN, C., “The Proteins” (H. Neurath, ed.), Second Edition. Academic Press, New York (1964). J. A., AND NIELSEN, E. B., “Con35. SCHELLMAN, formation of Biopolymers” (G. N. Ramachandran, ed.), Vol. 1. Academic Press, New York (1967). J. G., J. Chem. Phys. 5, 479 (1937). 36. KIRKWOOD, 37. CONDON, E. V., ALTAR, W., AND EYRING, H., J. Chem. Phys. 5, 753 (1937). 38. BACHRACH, H. L., J. Mol. Biol. 8, 348 (1964).