It is demonstrated that high resolution confocal laser scanning microscopy (CLSM) is a powerful tool for in situ observation and analysis of protein crystal growth. CLSM is used to study the early crystallization stage of Des-ThrB30 human insulin in aqueous solution, under conditions known to lead to monoclinic crystals. A modified batch crystallization method for CLSM purposes is applied which allows the growth behavior of crystallites to be studied in reflected light. A few hours after the start of the experiment, microcrystallites of characteristic shapes (mainly prismatic and pyramidal) are observed, the number of which strongly depends on the concentration of higher insulin aggregates in the initial solution. From direct observation as well as from model calculations we conclude that for solute concentrations up to about 3.5-times the saturation value, growth starts from few active insulin precipitate particles while 3D nucleation is neglegible for observation times up to 24 h. The anisotropic growth rates of monoclinic, prismatic crystallites are measured along the long edge of the cover face and perpendicular to the latter. A simultaneous crossover to signifcantly higher growth rates is found when the crystallite size reaches about 2 μm. The higher growth rates are connected with the appearence of striations. We argue that this growth rate crossover is caused by an increased 2D nucleation rate at the edges and corners, which finally results in bunching of steps simultaneously spreading over adjacent crystallite faces.

Diabetes affects more than 142 million people worldwide; however, little structural information is currently available for the intact insulin receptor and its complex with the hormone. Recently, cryoelectron microscopy of the whole complex and X-ray crystallography of sections have been combined to tackle the problem (see picture; © American Association for the Advancement of Science, 1999). Here we review the latest approaches in this direction.

BACKGROUND:The traditional picture of charged amino acids in globular proteins is that they are almost exclusively on the outside exposed to the solvent. Buried charges, when they do occur, are assumed to play an essential role in catalysis and ligand binding, or in stabilizing structure as, for instance, helix caps.RESULTS:By analyzing the amount and distribution of buried charged surface and charges in proteins over a broad range of protein sizes, we show that buried charge is much more common than is generally believed. We also show that the amount of buried charge rises with protein size in a manner which differs from other types of surfaces, especially aromatic and polar uncharged surfaces. In large proteins such as hemocyanin, 35% of all charges are greater than 75% buried. Furthermore, at all sizes few charged groups are fully exposed. As an experimental test, we show that replacement of the buried D178 of muconate lactonizing enzyme by N stabilizes the enzyme by 4.2 degrees C without any change in crystallographic structure. In addition, free energy calculations of stability support the experimental results.CONCLUSIONS:Nature may use charge burial to reduce protein stability; not all buried charges are fully stabilized by a prearranged protein environment. Consistent with this view, thermophilic proteins often have less buried charge. Modifying the amount of buried charge at carefully chosen sites may thus provide a general route for changing the thermophilicity or psychrophilicity of proteins.

We have refined to 2.3 A resolution two muconate cycloisomerase (MCIase) variant structures, F329I and I54V, that differ from each other and from wild-type in their activity toward cis,cis-muconate (CCM) and substituted CCMs. The working and free R-factors for F329I are 17.4/21.6% and for I54V, 17.6/22.3% with good stereochemistry. Except for the mutated residue, there are no significant changes in structure. To understand the differences in enzymatic properties we docked substituted CCMs and CCM into the active sites of the variants and wild type. The extra space the mutations create appears to account for most of the enzymatic differences. The lack of other structural changes explains why, although structurally equivalent changes occur in chloromuconate cycloisomerase (CMCIase), the changes in themselves do not convert a MCIase into a dehalogenating CMCIase. Reanalysis of the CMCIase structure revealed only one general acid/base, K169. The structural implication is that, in 2-chloro-CCM conversion by CMCIase, the lactone ring of 5-chloromuconolactone rotates before dehalogenation to bring the acidic C4 proton next to K169. Therefore, K169 alone performs both required protonation and deprotonation steps, the first at C5 as in MCIase, and the second, after ring rotation, at C4. This distinguishes CMCIase from alpha/beta barrel isomerases and racemases, which use two different bases.

The chloromuconate cycloisomerase of Pseudomonas sp. B13 was purified from 3-chlorobenzoate-grown wild-type cells while the chloromuconate cycloisomerases of Ralstonia eutropha JMP134 (pJP4) and Pseudomonas sp. P51 (pP51) were purified from Escherichia coli strains expressing the corresponding gene. Kinetic studies were performed with various chloro-, fluoro-, and methylsubstituted cis , cis -muconates. 2,4-Dichloro- cis , cis -muconate proved to be the best substrate for all three chloromuconate cycloisomerases. Of the three enzymes, TfdD of Ralstonia eutropha JMP134 (pJP4) was most specific, since its specificity constant for 2,4-dichloro- cis , cis -muconate was the highest, while the constants for cis , cis -muconate, 2-chloro- and 2,5-dichloro- cis , cis -muconate were especially poor. The sequence of ClcB of the 3-chlorobenzoate-utilizing strain Pseudomonas sp. B13 was determined and turned out to be identical to that of the corresponding enzyme of pAC27 (though slightly different from the published sequences). Corresponding to 2-chloro- cis , cis -muconate being a major metabolite of 3-chlorobenzoate degradation, the k cat / K m with 2-chloro- cis , cis -muconate was relatively high, while that with the still preferred substrate 2,4-dichloro- cis , cis -muconate was relatively low. This enzyme was thus the least specific and the least active among the three compared enzymes. TcbD of Pseudomonas sp. P51 (pP51) took an intermediate position with respect to both the degree of specificity and the activity with the preferred substrate.

Since the immune response to Des-[Phe(B1)] porcine insulin is weaker than for native insulin, this mutant is of interest for diabetes therapy. Additionally, Des-[Phe(B1)] insulin showed an interesting crystallisation behaviour. It was crystallised in the presence of Zn and phenol, which is added as a preservative in pharmaceutical insulin preparations. It has been shown previously that phenolic compounds bind to specific sites on the insulin hexamer and act as allosteric effectors, inducing a transformation of the T6 hexamer (in rhombohedral spacegroup R3) to the R6 hexamer (mostly in monoclinic spacegroup P21) [1,2]. However, mutant Des-[Phe(B1)], in the presence of phenol, exclusively crystallised in the rhombohedral spacegroup R3. This indicates that residue B1 is obviously essential for interhexameric contacts in monoclinic crystal forms.

ABSTRACT Muconate cycloisomerases play a crucial role in the bacterial degradation of aromatic compounds by convertingcis,cis-muconate, the product of catechol ring cleavage, to (4S)-muconolactone. Chloromuconate cycloisomerases catalyze both the corresponding reaction and a dehalogenation reaction in the transformation of chloroaromatic compounds. This study reports the first thorough examination of the substrate specificity of the muconate cycloisomerases from Pseudomonas putidaPRS2000 and Acinetobacter “calcoaceticus” ADP1. We show that they transform, in addition tocis,cis-muconate, 3-fluoro-, 2-methyl-, and 3-methyl-cis,cis-muconate with high specificity constants but not 2-fluoro-, 2-chloro-, 3-chloro-, or 2,4-dichloro-cis,cis-muconate. Based on known three-dimensional structures, variants of P. putidamuconate cycloisomerase were constructed by site-directed mutagenesis to contain amino acids found in equivalent positions in chloromuconate cycloisomerases. Some of the variants had significantly increased specificity constants for 3-chloro- or 2,4-dichloromuconate (e.g., A271S and I54V showed 27- and 22-fold increases, respectively, for the former substrate). These kinetic improvements were not accompanied by a change from protoanemonin to cis,cis-dienelactone as the product of 3-chloro-cis,cis-muconate conversion. The rate of 2-chloro-cis,cis-muconate turnover was not significantly improved, nor was this compound dehalogenated to any significant extent. However, the direction of 2-chloro-cis,cis-muconate cycloisomerization could be influenced by amino acid exchange. While the wild-type enzyme discriminated only slightly between the two possible cycloisomerization directions, some of the enzyme variants showed a strong preference for either (+)-2-chloro- or (+)-5-chloromuconolactone formation. These results show that the different catalytic characteristics of muconate and chloromuconate cycloisomerases are due to a number of features that can be changed independently of each other.