Joan A. Wozniak

Cumulative site-directed charge-change replacements in bacteriophage T4 lysozyme suggest that long-range electrostatic interactions contribute little to protein stability.

Bacteriophage T4 lysozyme is a basic molecule with an isoelectric point above 9.0, and an excess of nine positive charges at neutral pH. It might be expected that it would be energetically costly to bring these out-of-balance charges from the extended, unfolded, form of the protein into the compact folded state. To determine the contribution of such long-range electrostatic interactions to the stability of the protein, five positively charged surface residues, Lys16, Arg119, Lys135, Lys147 and Arg154, were individually replaced with glutamic acid. Eight selected double, triple and quadruple mutants were also constructed so as to sequentially reduce the out-of-balance formal charge on the molecule from +9 to +1 units. Each of the five single variant proteins was crystallized and high-resolution X-ray analysis confirmed that each mutant structure was, in general, very similar to the wild-type. In the case of R154E, however, the Arg154 to Glu replacement caused a rearrangement in which Asp127 replaced Glu128 as the capping residue of a nearby alpha-helix. The thermal stabilities of all 13 variant proteins were found to be fairly similar, ranging from 0.5 kcal/mol more stable than wild-type to 1.7 kcal/mol less stable than wild-type. In the case of the five single charge-change variants, for which the structures were determined, the changes in stability can be rationalized in terms of changes in local interactions at the site of the replacement. There is no evidence that the reduction in the out-of-balance charge on the molecule increases the stability of the folded relative to the unfolded form, either at pH 2.8 or at pH 5.3. This indicates that long-range electrostatic interactions between the substituted amino acid residues and other charged groups on the surface of the molecule are weak or non-existent. Furthermore, the relative stabilities of the multiple charge replacement mutant proteins were found to be almost exactly equal to the sums of the relative stabilities of the constituent single mutant proteins. This also clearly indicates that the electrostatic interactions between the replaced charges are negligibly small. The activities of the charge-change mutant lysozymes, as measured by the rate of hydrolysis of cell wall suspensions, are essentially equal to that of the wild-type lysozyme, but on a lysoplate assay the mutant enzymes appear to have higher activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Structural and thermodynamic analysis of the packing of two α-helices in bacteriophage T4 lysozyme

Packing interactions in bacteriophage T4 lysozyme were explored by determining the structural and thermodynamic effects of substitutions for Ala98 and neighboring residues. Ala98 is buried in the core of T4 lysozyme in the interface between two alpha-helices. The Ala98 to Val (A98V) replacement is a temperature-sensitive lesion that lowers the denaturation temperature of the protein by 15 degrees C (pH 3.0, delta delta G = -4.9 kcal/mol) and causes atoms within the two helices to move apart by up to 0.7 A. Additional structural shifts also occur throughout the C-terminal domain. In an attempt to compensate for the A98V replacement, substitutions were made for Val149 and Thr152, which make contact with residue 98. Site-directed mutagenesis was used to construct the multiple mutants A98V/T152S, A98V/V149C/T152S and the control mutants T152S, V149C and A98V/V149I/T152S. These proteins were crystallized, and their high-resolution X-ray crystal structures were determined. None of the second-site substitutions completely alleviates the destabilization or the structural changes caused by A98V. The changes in stability caused by the different mutations are not additive, reflecting both direct interactions between the sites and structural differences among the mutants. As an example, when Thr152 in wild-type lysozyme is replaced with serine, the protein is destabilized by 2.6 kcal/mol. Except for a small movement of Val94 toward the cavity created by removal of the methyl group, the structure of the T152S mutant is very similar to wild-type T4 lysozyme. In contrast, the same Thr152 to Ser replacement in the A98V background causes almost no change in stability. Although the structure of A98V/T152S remains similar to A98V, the combination of T152S with A98V allows relaxation of some of the strain introduced by the Ala98 to Val replacement. These studies show that removal of methyl groups by mutation can be stabilizing (Val98----Ala), neutral (Thr152----Ser in A98V) or destabilizing (Val149----Cys, Thr152----Ser). Such diverse thermodynamic effects are not accounted for by changes in buried surface area or free energies of transfer of wild-type and mutant side-chains. In general, the changes in protein stability caused by a mutation depend not only on changes in the free energy of transfer associated with the substitution, but also on the structural context within which the mutation occurs and on the ability of the surrounding structure to relax in response to the substitution.(ABSTRACT TRUNCATED AT 400 WORDS)

The consequences of eye removal for the circadian rhythm of behavioral activity inAplysia

SummaryTheAplysia eye is capable of both photoreception and circadian oscillation. We have tried to find out if either of these ocular functions plays an important role in controlling the circadian rhythm of locomotor behavior. Eyeless, sham-operated and intactAplysia were tested in LD, DD and pseudo-LD. The behavioral rhythm was measured and analyzed by event recorder techniques supplemented with periodogram analysis.1.In agreement with others we found that (i) intactAplysia were diurnal, (ii) activity onsets frequently occurred slightly before dawn inLD 12∶12 and (iii) a majority of intact animals gave convincing freeruns for up to 2 weeks following release into DD or pseudo-LD (Figs. 1 and 2).2.In intact animals the phase angle difference was more positive in LD 8∶16 than in LD 12∶12 (compare Figs. 1 and 5).3.EyelessAplysia reliably responded to light onset (Figs. 3, 4, and 5). The response persisted for more than 90 days after eye removal (Figs. 3 and 8) and at lighttime intensities of 3 lux or lower (Figs. 7, 8, and 10). Photic responses in eyelessAplysia did not depend on social communication with intact animals (Figs. 9 and 10).4.Even though eye removal did not prevent photic responses, it could have several effects on the daily pattern of activity in LD. These could include (i) an increase in scattered activity during the darktime, (ii) a decrease in activity during the final two-thirds of the lighttime, and (iii) attenuation or elimination of predawn anticipatory activity (Figs. 3, 4, and 5). The third of these effects was by far the most reliable consequence of eye removal in LD.5.Only a minority of eyelessAplysia gave convincing freeruns in DD or pseudo-LD. Nonetheless a few eyeless animals freeran as vigorously as the most vigorous intact ones (compare Fig. 2 with Figs. 3, 4, and 6). We saw eyeless animals in neighboring apparatuses which freeran with distinctly different periods (Fig. 4). These facts indicate that the eye is neither the only photoreceptor nor the only circadian oscillator coupled to the locomotor rhythm. The eye, however, does participate in the generation of temporal patterns of activity in the absence of h-to-h sensory guidance. It is therefore reasonable to believe that the eye is one among several endogenous oscillators coupled to locomotion. The only role so far demonstrated for the ocular photoreceptors is their previously known capacity to entrain the ocular clock.

Circadian organization inAplysia explored with red light, eye removal and behavioral recording

SummaryWe tested the sensitivity of theAplysia activity rhythm to red light in three ways: first, by measuring the response of the rhythm to 24 h cycles of red light (RD), second, by measuring the effect of constant red background light (RR) on the rhythm driven by superimposed white LD, third, by measuring the effect of RR on freerunning. In several experiments we removed the eyes in order to assess their role in mediating the action of red light. Results for individuals and groups were analyzed by calculating form estimates and periodograms.1.When tested with RD, intact animals were predominantly, though not exclusively, diurnal. In RD 8∶1G activity typically began several hours before dawn (Figs. 1, 3).2.Eye removal did not prevent diurnal behavior but caused (i) a prominent decrease in activity prior to dawn, (ii) an increase in activity immediately after dawn, (iii) a small decrease of activity during the late lighttime and (iv) a small increase of activity during the mid darktime (Figs. 2, 3). The contribution of the eyes to the entrained locomotor rhythm is thus facilitory at some phases and suppressive at other phases.3.Continuous red background light reduced the amplitude of the diurnal rhythm driven by superimposed white LD 12∶12. Activity during the white-on time was suppressed and activity during the white-off time was enhanced by continuous red background. This effect was greater in eyeless than in intact animals (Figs. 4, 5).4.In contrast to its effects on the driven rhythm, continuous red background light strengthened freerunning (Figs. 6, 7, Table 1). These results show thatAplysia have photoreceptors outside the eyes that are sensitive to red light and that are coupled to locomotion. It is unlikely that the eyes themselves are red sensitive photoreceptors that can reflexively drive locomotion in red lightcycles. On the other hand, it is quite likely that the eyes are a part of a circadian pacemaking system that can be entrained by extraocular red sensitive photoreceptors.We have drawn a hypothetical flow diagram that relates the known facts about circadian organization inAplysia (Fig. 8). In the diagram rhythmic behavior arises from the convergence and integration of temporal information from several classes of photoreceptors. Internally timed signals from the eyes both facilitate locomotion and uncouple locomotion from extraocular mechanisms.

Crystallization of mutant lysozymes from bacteriophage T4

Abstract The crystallization properties of a large number of T4 lysozyme mutant proteins have been analyzed. Approximately 80% of the mutant proteins crystallize under conditions very similar to those used for the wild-type protein, regardless of the type of amino acid substitution or its location. Of the mutants that crystallize all but two, A146F (tetragonal) and M6I (orthorhombic), are isomorphous with wild-type lysozyme (trigonal). The two nonisomorphous crystals are obtained in cases where internal residues are changed. Substitution of a large side chain for a small one, as in A146F, appears to exceed a critical internal packing volume above which nonisomorphism is induced. The nonisomorphism of M6I may be due to structural changes induced by the introduction of a β-branched side chain.

Crystallographic and Genetic Approaches Toward the Design of Proteins of Enhanced Thermostability

The advent of directed mutagenesis has made it possible to alter protein structures at will. For the first time it is possible to design and to introduce modifications into a protein that are intended to change its behavior in predictable ways.

Selected and Directed Mutants of T4 Phage Lysozyme

The lysozyme from bacteriophage T4 is being used as a model system to determine the factors that influence the folding and stability of proteins. The three-dimensional structure of the protein is known and lysozymes with modified properties arising from single amino acid substitutions have been obtained by classical selection techniques as well as by site-directed mutagenesis. By rationalizing the stabilities of the mutant proteins in terms of their observed three-dimensional structures we are attempting to quantitate the contributions that single amino acids make to protein stability. Our studies to date lead to the following conclusions. (1) Thermal stability is global. (2) Different types of interaction contribute to stability. (3) Changes in stability need not be associated with large structural changes. (4) An increase in the stability of an α-helix may enhance the stability of the protein as a whole. (5) Hydrogen bonds contribute to stability. (6) Protein structures can readily adjust to changes in amino acid sequence.