RUFUSLUMRYAND HENRYEYRING
110
Vol. 58
CONFORMATION CHANGES OF PROTEINS’ BY RUFUSLUMRY AND HENRYEYRING Department of Chemistry, Universitv of Utah, Salt Lake City, Utah Received July 8.4, 1959
Recent developments in protein structure make one model for globular proteins especially attractive. This model consists of polypeptide chains folded on themselves to give hydrogen-bonded secondary structures which are in turn folded in a rigid tertiary arrangement through the interaction of amino acid side chains. The implications of the model are examined in terms of possible energy states available to proteins and possible denaturation reactions. Denaturation is defined as change in conformation. The folding process is considered to be spontaneous and directed by the composition and order of peptide amino acid residues. Reversible thermal denaturation processes are presented as changes in tertiary structure, irreversible processes as changes in secondary structure. The activated complex for denaturation and for enzymic function is discussed and the wssibility_ presented that enzymic activity, reversible denaturation and ion-binding are all aspects _ of the same protein property.
Introduction The term protein denaturation even in its original meaning included all those reactions destroying the solubility of native proteins and has since acquired so many other meanings as to become virtually useless. It is now generally realized that peptide and other primary bonds are not destroyed under the mild conditions of most denaturation reactions unless specific reagents or catalysts for these bonds are available. Hess and Sullivan2were able to preserve intact sulfhydryl and primary sulfur-sulfur groups during careful hydrolysis of several proteins but sulfur-sulfur bonds are easily destroyed catalytically and their exact state in any given denaturation reaction may be uncertain. I n any event the bulk of denaturation changes consist of changes in secondary bonds: ion-dipole, hydrogen and van der Waals; and in the rotational positions about single bonds which are controlled by the secondary bond structure. Such rearrangements of the secondary-bonded structure are defined as changes in conformation3 and are exemplified by the conversion of cyclohexane from “chair” to “boat” form or the melting of a crystal. We shall thus speak of transconformation reactions rather than denaturation reactions. Certain transconformation reactions involve only a few secondary bonds; others are extenOH sive and similar to second and higher order phase H AH? H changes in solids. These last are of particular in- -c-k-Lc-~terest and we shall designate them cooperative transconformations. Numerous attempts have been made to describe such reactions in terms of changes in ionization, hydration or degree of folding of polypeptide chain^.^^^ In this paper we shall examine conformation changes in terms of the newer pictures of protein structure. The Structure of Globular Proteins 1. Primary Structure.-Proteins will be destroyed by breaking a single primary bond in the
1
8
(1) This work was supported by the National Science Foundation.
The paper was originally presented a t a Symposium on Protein Denaturation held during the Spring meeting of the American Chemical Society at Los Angeles, 1953. (2) W. C. Hess and M. X. Sullivan, J . B i d . Chem., 161, 635 (1943). (3) W.J. Kausmann, J. E. Walter and H. Eyring, Chem. Revs., 26,
339 (1940). (4) H. Neurath, J. P. Greenstein, F. W. P u t n a m and J. 0. Erickson, ibid., 34, 157 (1944). ( 4 ) M. L. Anson, ddvances in Protein Chem., 2 , 361 (1945).
skeleton of a constituent polypeptide chain if the broken bond can be rearranged before the break is healed. Cathode ray,6 ultrasonic,’ ultravioletS and ionizing radiation in general breaks or weakens primary bonds but the structure of the molecule in native form, especially when held in the solid, does not allow reorganization and subsequent reaction a t the broken ends. At low temperatures which favor the recombination process of low activation energy or in the absence of oxygen which is frequently able to cauterize the stumps or to react with strained bonds, the break may recover or persist in high-energy state.g At normal temperatures in solution unfolding rapidly permits destructive rearrangement. Rubber and other large polymeric substances behave in a similar fashion following breakage or strain of primary bonds.’O Enzyme attack on peptide linkages will also introduce fatal weaknesses” though in the conversion of proenzymes to active enzymes simple conversion from one native protein to another occurs. Recently Elliott12 has established another important source of weakness in the primary structure previously suggested by Bergmann, et u E . ’ ~ Where serine and threonine residues exist in the chain, “acyl shifts” may occur in acid media, about pH 3 in model compounds
O-CHz
-c/ \N-
I
C-C-N-
I ll
H 0
H
1
z-C-O-CH2-
+ HzO
N
H
Hf
8
AI
--C-N-
I
II
NHZ+ 0
The reaction is in effect an internal hydrolysis to increase the chain by one bond length and to introduce an easily hydrolyzed ester bond. Du Vigneaud14 actually observed the formation of Nmethyl groups in insulin in a reversible reaction (6) H.Snietana, J . E z p t l . M e d . , 73, 223 (1941). (7) P. Grabar and R. Prudhomme, J . chim. phys., 4 4 , 145 (1948); Bull. doc. chim. B i d . , 29, 122 (1947). (8) A. D. McLaren and S. Pearson, J . Polymer. Sci., 4 , 45 (1949). (9) S. Fiala, Biochem. Z., 318, 67 (1947). (10) W. Kausrnann and H. Eyring, J . Am. Chem. Sac., 62, 3113 (1940). (11) K. V. LinderstrZm-Lang, Lane Medical Lectures: “Proteins and Enzymes,” Stanford University Press, Stanford, California, 1952. (12) D. F. Ellibtt, Biochem. J . , 6 0 , 542 (1952). (13) R I . Bergmann and J. S. Fruton, J . BioE. Chem., 118, 405 (1937). (14) V. du Vigneaud, Cold Sprine Harbor Sumposia Quant. Biol., 6 , 275 (1938).
Feb., 1954
CONFORMATION CHANGES OF PROTEINS
and Elliott has observed their occurrence in silk fibroin. The reaction is normally slow and its pH of occurrence will undoubtedly vary with the type of bonds the hydroxy or amino forms can make. The well-known lability of peptide chains a t serine and threonine residue^'^^^^ is thus explained and we shall see indications of acyl shift involvement in several transconformation reactions. Acid titration experiments will almost universally need reevaluation in light of this finding. A somewhat similar set of reactions leading to thiazolidine formation between cysteine, sulfhydryl and aldehyde groups has been suggested by Linderstrgm-Lang and Jacobsen. l7 Sulfhydryl groups of proteins frequently become available to external attack only after extreme treatment of the protein and well after the exposure of disulfide groups. I n view of the demonstration of N to 0 rearrangement in the acyl shift, a similar N to S shift may possibly be an important reaction of -SH groups thoughit is probably not energetically favored. At present only the serine and threonine reaction is established as a source of alternation in primary structure during denaturation. We shall thus be primarily concerned with secondary bonds to the exclusion of the hydrolyzed states of proteins. Oomplete peptide hydrolysate is probably the ultimate state of lowest free energy for proteins though some question as to this conclusion exists in view of the high stabilization energies available from the secondary bonds of the folded structure.” The free energy difference between native (folded) protein and hydrolyzed protein (amino acids) is probably small and, in any event, the peptide chain is protected by high free energies of activation from hydrolysis. Structural Features Due to Secondary Bonds.Physical and immunological studies demonstrate unequivocally that globular proteins are rigid bodies undoubtedly a result of cross linking between peptide chains. X-Ray diffraction studies show considerably more internal order than is consistent with random cross linking. Pauling and cohave pointed out that the key to protein structure lies in the considerable strength (8 kcal.) of hydrogen bonds which can exist between the carbonyl of one peptide link and the amino group of another. Only this type of bond can be made in quantity and in a way to produce extensive regularity. It would be favored in competition with other secondary bonds even though a considerable entropy decrease was concomitant. Fibrous rods have been reported to exist in insulin,21 hemoglobin,22 myoglobin,2 3 ribonuclea~e,~~ and a (15) Ovalbumin, for example, has a total of 59serineand threonine residues, 56 of which are very susceptible to acid hydrolysis. Lysozyme shows a similar susceptibility to hydrolysis in acid solution. (16) Reviewed b y H. L. Fevold, Advances Protein Chem., 6,205 (1951). (17) I
