Carlsberg Res. Commun. Vol. 49, p. 1-55, 1984 PROTEIN STRUCTURE AND FUNCTION, FROM A COLLOIDAL TO A MOLECULAR VIEW" by HAROLD A. SCHERAGA Baker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853, USA Presented as the 7th Linderstrom-Lang Lecture at the Carlsberg Laboratory, Copenhagen, on lOth May, 1983 Keywords: Hydrodynamic properties, internal interactions, ribonuclease, synthetic polypep- tides, conformational changes, protein folding, conformational energy calculations, structural elements of proteins, enzyme-substrate complexes 1. INTRODUCTION opment of theoretical methods to use these Forty years ago, proteins were described in experimental distance constraints, together with terms of ellipsoids of revolution in order to empirical potential energy functions, to gain an account for their hydrodynamic properties. understanding as to how interatomic inter- With the determination of the amino acid sequ- actions dictate the structural features of proteins ence of insulin (290) and other proteins, it and to try to compute the three-dimensional became meaningful to discuss protein structure structures of polypeptides and proteins in solu- and function in terms of the interatomic inter- tion. At the same time, both theoretical and actions within the protein molecule. Various experimental methods were used to elucidate physical chemical methods were developed and the pathway(s) of folding from a nascent poly- applied to elucidate such interactions. Many of peptide chain to the three-dimensional structure the pairwise interactions deduced from such of the native protein. The same theoretical and studies were found to exist, e.g. in bovine pan- experimental methods have also been applied to creatic ribonuclease A (303), when the X-ray study the biological function of the folded (na- crystal structure was subsequently determined tive) protein, e.g. the interaction of an enzyme (404, 405,407). The ability to identify pairwise with its substrate. The development of some of interactions by physical chemical means pro- our current views of protein structure and func- vided distance constraints with which to define tion will be described here by focussing on the the three-dimensional structure of a protein in impact of physical chemistry on the advance- solution. There then followed the rapid devel- ment of our knowledge in this field. "~The work reviewed here was supported by research grants from the National Institutes of Health, the National Science Foundation, and the National Foundation for Cancer Research Springer-Verlag 0105-1938/84/0049/0001/$11.00 H. A. SCHERAGA:Protein structure Scale I I o I00.~, No + CI- Glucose II Albumin Hemoglobin .a, - Globulin 69.000 68.000 90.000 a,- Lipoprolein y_ Globulin 200,000 156,000 Lipoprotein 1,300,000 Fibrinogen 4OO,O0O Figure 1. Early representation of protein molecules in terms of ellipsoids of revolution. Relative dimensions of several protein molecules in blood (293). 2. EARLY VIEW OF PROTEINS peptide chains) was altered to one in which the In the 1940's, proteins were thought of as protein swells (almost isotropically) (325). Thus, charged colloidal particles. A summary of their the concept that a protein is a rigid particle had acid-base equilibria and dielectric properties has to be abandoned in favor of one in which the been provided by COHN and EDSALL (47), and molecule is a dynamically flexible one (325). a description of their sizes and shapes, deter- The rigid-particle view had also been used to mined by various hydrodynamic and optical interpret titration data of proteins. LINDER- methods, has been presented by EDSALL (63). STROM-LANG (173) was the first one to exploit Figure 1 is a pictorial interpretation of such data, the newly-formulated DEBYE-HOCKEL theory in terms of ellipsoids of revolution (293). A (52, 53) for this purpose by applying it to a re-interpretation of hydrodynamic data with the spherical model of a protein, with the charge aid of shape-dependent [3- and 8-functions (325) distributed uniformly on its surface. Subse- led to an alteration of the pictorial represent- quently, the spherical model was extended to a ations of Figure 1; e.g. the computed axial ratio cylindrical one by HILL (113), and the uniform offibrinogen was reduced from ! 8:1 to 5:1 (327, distribution of charge was modified to a discrete 344), a result that is compatible with electron one (for a spherical model) by TANFORD and micrographs (103) of this protein. Further, the IORKWOOD (369, 371). In the succeeding years, view that urea denaturation of proteins leads to TANFORD (370) and KLOTZ (138) made consi- increased asymmetry (due to unfolding ofpoly- derable use of such models to provide clues that 2 Carlsberg Res. Commun. Vol. 49, p. 1-55, 1984 H. A. SCHERAGA:Protein structure interactions involving ionizable groups alter were confirmed when the a-helix was subse- their normally-observed pK's and binding con- quently found in the crystal structures of myo- stants for various ligands. globin (133) and hemoglobin (244), and the Simultaneously, the thermodynamic proper- [3-pleated sheet in the crystal structure of lyso- ties of protein solutions had also been treated zyme (18). with rigid spherical charged models. For exam- Simultaneously, the need was recognized ple, SCATCHARD and coworkers (294) ac- [even before the successful determinations of counted for the non-ideality of aqueous solu- protein structures by X-ray diffraction (18, 133, tions of proteins by interpreting the second virial 244)] for obtaining structural information about coefficient in the expression for the osmotic proteins in solution, and for determining how pressure in terms of interactions between the polypeptide chains fold into the native confor- various components of the system. mations of proteins and then interact with other Of course, it was realized that such simple molecules. Hence, methods were developed to models provided only the crudest description of synthesize homopolymers and copolymers of a protein, and efforts were made to try to probe amino acids as simple models (19, 20, 130) in the more detailed structure of the protein mole- order to elucidate the interactions present in cule. The aforementioned titration and binding proteins, and various physical chemical techni- experiments suggested that there were inter- ques [such as optical rotation and circular di- actions between functional groups that were chroism (204, 205, 298, 375, 377), infrared reflected in modified pK's and binding con- (203), Raman (381), and nuclear magnetic reso- stants. Such interactions also modified the ease nance (127, 406) spectroscopies, kinetics of of hydrolysis of peptide bonds, and LINDER- deuterium-hydrogen exchange (122, 176), and STROM-LANG (174, 175, 177), OTTESEN(234), immunochemical measures of folding equilibria RICHARDS (285) and others demonstrated this (291)] were developed to investigate these inter- very clearly by observations of limited proteo- actions. At the same time, the development of lysis by enzymes. But a detailed interpretation of thermodynamic and statistical mechanical me- such results had to await the pioneering work of thods began to emerge (150-152, 296, 297) to SANGER (290) who first determined the amino treat the interactions that determine conforma- acid sequence of a protein, insulin. tion, conformational changes, and intramolecu- lar structure in polypeptides and proteins. We shall therefore trace the parallel and symbiotic 3. TRANSITION TO A MOLECULAR elaboration of theoretical and experimental pro- APPROACH cedures to study protein structure and function With the determination of the amino acid in solution. sequence of insulin (290), and subsequently of ribonuclease (99, 114, 268, 347-349), lysozyme (32, 128) and other proteins, a new era in protein 4. INTERNAL INTERACTIONS chemistry was opened up. The protein was no Internal interactions in proteins affect their longer a colloidal particle, but an organic mole- various properties, and physical chemical stu- cule whose complete covalent structure was dies of such properties, accompanied by appro- describable. It then remained the province of the priate theoretical interpretation, provide infor- physical chemist to provide a description of its mation about such internal interactions. three-dimensional structure, of the interactions Initially, the experiments were carried out with that led to it, and of its interactions with other insulin, lysozyme and ribonuclease (301) molecules. because these were among the first proteins After a long series of trials by numerous whose amino acid sequences were determined, investigators, PAULING and COREY (241, 242) and attention was focussed on electrostatic ef- succeeded in elucidating the stereochemistry of fects and on hydrogen and hydrophobic inter- the polypeptide chain in terms of the ix-helix and actions. the I]-pleated sheet. These pioneering proposals Carlsberg Res. Commun. Vol. 49, p. 1-55, 1984 3 H. A. SCHERAGA:Protein structure ._.= -- CH2--CH2-C,o .... HO.-< )'-CH2-- Figure 2. Schematic representation of a hydrogen bond between the hydroxyl group of a tyrosyl residue and the carboxylate ion of a glutamyl residue between two rigid, helical polypeptide chains (150). 4.1. Electrostatic effects hydrogen bond will affect the observed pK's of Generalized electrostatic effects, which are the donor and acceptor groups (150). In the reflected in altered pK's observed iia acid-base illustration of Figure 2, the observed pK of the titrations of proteins, are usually treated by