John A. Rupley

Environment and exposure to solvent of protein atoms. Lysozyme and insulin

Abstract A computer program is described for calculating the environment and the exposure to solvent of atoms of a protein. The computation is based on the atomic co-ordinates of the protein and on assumptions like those of Lee & Richards (1971). Results for lysozyme and insulin are presented. Changes in exposure to solvent and in the nature of contacts that develop through folding, association reactions and crystallization are described numerically. The computations suggest several generalizations. (a) Lattice contacts within the protein crystal are characterized by a significantly smaller involvement of non-polar side chains and a proportionately greater involvement of ionizable side chains than is found for protein folding or for protein association reactions important for biological function, (b) In helical regions the carbonyl oxygen of the first residue in the helix has high probability of being shielded from solvent, (c) Glycine is among the residues having exposure least affected by folding; this accords with the expectation that it lies at bends of the peptide chain on the surface of the molecule.

Protein hydration and function.

Publisher Summary Hydration can be considered a process of adding water incrementally to dry protein, until a level of hydration is reached beyond which further addition of water produces no change and only dilutes the protein. The hydration process has several stages and an end point, reflected similarly in different types of measurements. Time-average measurements appear to have a common pattern and most closely fit a single picture of the process. Dynamic measurements sometimes show a dependence on hydration level that lies outside the common pattern of the time-average results. The chapter summarizes the literature that bears on the protein hydration process and the hydration shell, categorized by type of measurement, and provides picture of the hydration process and the hydration shell and an assessment of the ways in which the hydration shell may modulate enzyme and other protein functions. The percolation model is discussed that focuses on the long-range connectivity established at a threshold coverage of a surface or volume with conducting or otherwise functional elements.

Water and globular proteins

Abstract Dynamic, thermodynamic and structural studies of the hydration of globular proteins indicate how the macromolecule-water interface can influence folding, enzymatic activity and other biological properties.

21 Vertebrate Lysozymes

Publisher Summary This chapter focuses on vertebrate lysozymes, particularly hen egg-white (HEW) lysozyme. Lysozymes of other types generally are distinguishable from HEW lysozyme in having higher molecular weights and somewhat different enzymic activities. Lysozyme is small and basic, and it separates well on weak acid resins, like Amberlite XE-64 or Bio-Rex 70 or carboxymethyl cellulose and on calcium phosphate gel. Affinity chromatography of lysozyme has been carried out using dispersed chitin or carboxymethyl (CM)-chitin. The conditions required for elution from chitin columns suggest that there are two classes of adsorbent sites that are differently affected by pH and ionic strength in their interaction with the enzyme. The preparation of lysozyme from diverse animal sources is generally achieved in four main steps, which include (1) the preparation of a lysozyme-rich extract, (2) chromatography on CM-cellulose, (3) filtration on Sephadex G-25, and (4) ion exchange chromatography on Amberlite CG-50 at 20° C with a 0.2 M phosphate buffer. The chromatography is sensitive to the pH and small variation of no more than 0.1 pH unit can involve complete retention or exclusion.

Oxidation of lysozyme by iodine: Identification and properties of an oxindolyl ester intermediate; evidence for participation of glutamic acid 35 in catalysis☆

Abstract The first product formed in the iodine oxidation of tryptophan 108 of lysozyme has a transition temperature more than 20 deg. C higher than that of native and oxindolealanine 108-lysozyme. Irreversible rapid conversion to the oxindole follows unfolding. The spectrum of the oxidized residue of the intermediate resembles that of tryptophan. The iodine oxidation of tryptophan 108 is faster than that of N -acetyltryptophan ethyl ester. These and other aspects of the lysozyme-iodine reaction are explained by the formation, possibly concerted with oxidation, of the oxindolyl ester of glutamic acid 35. The data accord with results of high-resolution crystallographic analysis (Beddell & Blake, 1970). Ester 108-lysozyme binds substrate like the native enzyme but retains less than 0.1% of the native activity. These results and the crystallographic data demonstrate catalytic function for glutamic acid 35. Oxindolealanine 108-lysozyme binds substrate only weakly. Introduction of an ester crosslink adds more than 6 kcal to the stability of lysozyme.

Oxidation of lysozyme by iodine: Isolation of an inactive product and its conversion to an oxindolealanine-lysozyme

Abstract Reaction of lysozyme with a half-molar amount of iodine at pH 5.5 gives, in 50% yield (based on iodine), an enzymatically-inactive singly-oxidized derivative. This intermediate can be converted to an oxindole-lysozyme. Analysis showed no modification other than two-electron oxidation of one tryptophan. Substrate analogs prevent the oxidative inactivation. Reaction of crystalline lysozyme with iodine gives the same inactive product found in solution.

Comparison of protein structure in the crystal and in solution: IV. Protein solubility

Changes in protein solubility can be related to the effect of crystallization on the equilibrium reaction of proteins with small molecules. Such measurements offer a simple approach to the comparison of reactivity in crystal and solution. The pH-dependence of the solubility of horse hemoglobin ( Green, 1931 ; Sorensen & Sorensen, 1933 ) is compared with titrations of crystalline and soluble protein and used to determine changes in proton binding that occur during crystallization. Out of approximately 130 ionizable groups with pK less than 9, crystallization affects about ten of them. It is possible that some and perhaps all of these are involved in lattice contacts. A phase change at pH 5·9 that had been observed in crystallographic studies ( Perutz, 1946 , Perutz, 1965 ) was also observed through solubility. The rearrangement of the lattice in this transition requires relatively little energy. Solubility data for insulin ( Frederique & Neurath, 1950 ) could not be interpreted in the same way as those for hemoglobin.

In vitro analysis of mutant LexA proteins with an increased rate of specific cleavage.

Specific cleavage of LexA repressor plays a crucial role in the SOS response of Escherichia coli. In vivo, cleavage requires an activated form of RecA protein. However, previous work has shown that the mechanism of cleavage is unusual, in that the chemistry of cleavage is probably carried out by residues in the repressor, and not those in RecA; RecA appears to facilitate this reaction, acting as a coprotease. We recently described a new type of lexA mutation, a class termed lexA (IndS) and here called IndS, that confers an increased rate of in vivo cleavage. Here, we have characterized the in vitro cleavage of these IndS mutant proteins, and of several double mutant proteins containing an IndS mutation and one of several mutations, termed Ind-, that decrease the rate of cleavage. We found, first, that the autodigestion reaction for the IndS mutant proteins had a higher maximum rate and a lower apparent pKa than wild-type LexA. Second, the IndS mutations had little or no effect on the rate of RecA-mediated cleavage, measured at low protein concentrations, implying that the value of Kcat/Km was unaffected. Third, the rate of autodigestion for the double-mutant proteins, relative to wild-type, was about that rate predicted from the product of the effects of the two single mutations. Finally, by contrast, these proteins displayed the same rate of RecA-mediated cleavage as did the single Ind- mutant protein. We interpret these data to mean that the IndS mutations mimic to some extent the effect of RecA on cleavage, perhaps by favoring a conformational change in LexA. We present and analyze a model that embodies these conclusions.

Oxidation of lysozyme by iodine: Identification of oxindolealanine 108

Abstract Examination of the tryptic peptides of the product of iodine oxidation of lysozyme demonstrated that tryptophan 108 was the residue modified. At some stage of the analysis subsequent to reduction and carboxymethylation, there was non-oxidative cleavage of the peptide bond of the α-carboxyl of oxindolealanine 108. Four oxinodolealanine peptides were observed by analysis of tryptic digests. Three of these peptides were purified and were shown to have the same amino acid composition after acid hydrolysis.

Comparison of protein structure in the crystal and in solution. VI. Volume change in the crystallization of horse methemoglobin.

The crystallization of horse methemoglobin was followed dilatometrically and found to be associated with a contraction of 21 ml. per mole of hemoglobin tetramer in 1·35 to 1·5 M -Na 2 SO 4 and with one of 42 ml. per mole in 1·8 M -(NH 4 ) 2 SO 4 . The absence of any substantial volume change suggests that both the conformation and the solvent surrounding the molecule are unaffected by crystallization.