Revisiting the Phylogenetic Position of Synchroma grande

ABSTRACT. Two new classes Synchromophyceae and Picophagea, belonging to the heterokonts, have been proposed recently in separate studies of 18S rRNA phylogenies. Here we revise the 18S phylogeny of these classes by including all available sequenced species and applying Bayesian and maximum likelihood methods; Synchroma grande groups with the photophagotrophic Chlamydomyxa labyrinthuloides with high statistical support. This clade is sister to Chrysophyceae, together they share a common ancestry. Our results show that the creation of class Synchromophyceae by Horn et al. was premature, because they did not include data from the closely related C. labyrinthuloides and Picophagus flagellatus species. A revision of these classes should include additional species and most likely multigene phylogenies.     

Packing is a key selection factor in the evolution of protein hydrophobic cores.

The energy derived from optimized van der Waals interactions in closely packed, folded proteins has been proposed to be of similar energetic magnitude to hydrophobicity in stabilizing the native state. If packing is this energetically important, it should influence the evolution of protein core sequences. To test this hypothesis, the occurrence of various amino acid side chains in the major hydrophobic core of staphylococcal nuclease and 42 homologous proteins was determined. Most such positions in this protein family are usually isoleucine, leucine, or valine. Previously we have constructed and measured the stabilities of 12 single, 44 double, 64 triple, and 32 quadruple mutants, representing all possible permutations of these three side chains at two overlapping sets of four positions in the core of staphylococcal nuclease. The stabilities and interaction energies of those mutants with various combinations of the most common, or consensus, sequence were compared to the stabilities of all other mutants. Mutants which had the consensus side-chain combinations were not necessarily the most stable, but usually were found to have the best interaction energies. In other words, these proteins were far more stable than would be predicted from simply summing the observed energetic effects of the component single mutations, apparently reflecting particularly favorable packing interactions that are possible for the most common side chains. An additional 12 mutants which tested possible alternate explanations of the results were constructed. The stabilities and interaction energies of these mutants also support the conclusion that packing is a crucial determinant guiding the sequence evolution of protein cores.     

Proteins with simplified hydrophobic cores compared to other packing mutants

Efforts to design proteins with greatly reduced sequence diversity have often resulted in proteins with so-called molten globule properties. Substitutions were made at six neighboring sites in the major hydrophobic core of staphylococcal nuclease to create variants with all leucine, all isoleucine or all valine at these sites. The mutant proteins with simplified cores constructed here are quite unstable and have poorly packed cores, attested to by interaction energies. Eight related mutants with greater sequence diversity were also constructed. Comparison to these mutants and 159 other permutations of these 3 aliphatic side chains at these same 6 sites previously constructed shows that the simplified cores are not unusual in their stabilities or interaction energies. Further, crystal structures of the two mutants with the worst packing, as measured by interaction energies, showed no unusual disorder in the core. Therefore, reduction of sequence diversity is not necessarily incompatible with a single stable native structure. Other factors must also contribute to previous protein design failures.     

High apparent dielectric constants in the interior of a protein reflect water penetration

A glutamic acid was buried in the hydrophobic core of staphylococcal nuclease by replacement of Val-66. Its pK(a) was measured with equilibrium thermodynamic methods. It was 4.3 units higher than the pK(a) of Glu in water. This increase was comparable to the DeltapK(a) of 4.9 units measured previously for a lysine buried at the same location. According to the Born formalism these DeltapK(a) are energetically equivalent to the transfer of a charged group from water to a medium of dielectric constant of 12. In contrast, the static dielectric constants of dry protein powders range from 2 to 4. In the crystallographic structure of the V66E mutant, a chain of water molecules was seen that hydrates the buried Glu-66 and links it with bulk solvent. The buried water molecules have never previously been detected in >20 structures of nuclease. The structure and the measured energetics constitute compelling and unprecedented experimental evidence that solvent penetration can contribute significantly to the high apparent polarizability inside proteins. To improve structure-based calculations of electrostatic effects with continuum methods, it will be necessary to learn to account quantitatively for the contributions by solvent penetration to dielectric effects in the protein interior.     

Energetics of side chain packing in staphylococcal nuclease assessed by exchange of valines, isoleucines, and leucines

To examine the importance of side chain packing to protein stability, each of the 11 leucines in staphylococcal nuclease was substituted with isoleucine and valine. The nine valines were substituted with leucine and isoleucine, while the five isoleucines, previously substituted with valine, were substituted with leucine and methionine. These substitutions conserve the hydrophobic character of these side chains but alter side chain geometry and, in some cases, size. In addition, eight threonine residues, previously substituted with valine, were substituted with isoleucine to test the importance of packing at sites normally not occupied by a hydrophobic residue. The stabilities of these 58 mutant proteins were measured by guanidine hydrochloride denaturation. To the best of our knowledge, this is the largest library of single packing mutants yet characterized. As expected, repacking stability effects are tied to the degree of side chain burial. The average energetic cost of moving a single buried methyl group was 0.9 kcal/mol, albeit with a standard deviation of 0.8 kcal/mol. This average is actually slightly greater than the value of 0.7-0.8 kcal/mol estimated for the hydrophobic transfer energy of a methylene from octanol to water. These results appear to indicate that van der Waals interactions gained from optimal packing are at least as important in stabilizing the native state of proteins as hydrophobic transfer effects.     

Higher-order packing interactions in triple and quadruple mutants of staphylococcal nuclease

Sixty-four triple and 32 quadruple mutants were constructed in the core of staphylococcal nuclease. This is the first time that a large number of multiple mutants with all possible variations and all possible lower-order mutants has been systematically constructed in any protein core. Stabilities were determined by solvent denaturation. The energetic effects of these multiple mutants have been analyzed in combination with the stability data from the component single and double mutants. It was found that most of the stability changes in triple and quadruple mutants cannot be correctly predicted from stability effects of component single mutants. However, if the interaction energy between pairs of side chains in the component double mutants is taken into account, correct stability prediction can be made for most triple and quadruple mutants. The data further show that while packing interactions unique to triple and quadruple mutations do occur, they are of much less energetic significance than interactions between pairs of residues. The results presented here show that the packing of a protein interior can be closely approximated in most cases as a series of short-range, nearest-neighbor interactions. This has profound implications for rational protein design and structure prediction.     

Temporal response of desmin and dystrophin proteins to progressive resistance exercise in human skeletal muscle.

We have investigated the adaptations of the cytoskeletal proteins desmin and dystrophin in relationship to known muscular adaptations of resistance exercise. We measured desmin, dystrophin, and actin protein contents, myosin heavy chain (MHC) isoform distribution, muscle strength, and muscle cross-sectional area (CSA) during 8 wk of progressive resistance training or after a single bout of unaccustomed resistance exercise. Muscle biopsies were taken from the vastus lateralis of 12 untrained men. For the single-bout group (n=6) biopsies were taken 1 wk before the single bout of exercise (week 0) and 1, 2, 4, and 8 wk after this single bout of exercise. For the training group (n=6), biopsies were taken 1 wk before the beginning of the program (week 0) and at weeks 1, 2, 4, and 8 of the progressive resistance training program. Desmin, dystrophin, and actin protein levels were determined with immunoblotting, and MHC isoform distribution was determined using SDS-PAGE at each time point for each group. In the training group, desmin was significantly increased compared with week 0 beginning at week 4 (182% of week 0; P<0.0001) and remained elevated through week 8 (172% of week 0; P<0.0001). Desmin did not change at any time point for the single-bout group. Actin and dystrophin protein contents were not changed in either group at any time point. The percentage of MHC type IIa increased and MHC type IIx decreased at week 8 in the training group with no changes occurring in the single-bout group. Strength was significantly increased by week 2 (knee extension) and week 4 (leg press), and it further increased at week 8 for both these exercises in the training group only. Muscle CSA was significantly increased at week 4 for type II fibers in the training group only (5,719+/-382 and 6,582+/-640 microm2, weeks 0 and 4, respectively; P<0.05). Finally, a significant negative correlation was observed between the desmin-to-actin ratio and the percentage of MHC IIx (R=-0.31; P<0.05, all time points from both groups). These data demonstrate a time course for muscular adaptation to resistance training in which desmin increases shortly after strength gains and in conjunction with hypertrophy, but before changes in MHC isoforms, whereas dystrophin remains unchanged.     

The fluorescence detected guanidine hydrochloride equilibrium denaturation of wild-type staphylococcal nuclease does not fit a three-state unfolding model

A three-state equilibrium unfolding of a protein can be difficult to detect if two of the states fail to differ in some easily measurable way. It has been unclear whether staphylococcal nuclease unfolds in a two-state fashion, with only the native and denatured states significantly populated at equilibrium, or in a three-state manner, with a well-populated intermediate. Since equilibrium unfolding experiments are commonly used to determine protein stability and the course of denaturation are followed by changes in the fluorescence which has difficulty in distinguishing various states, this is a potential problem for many proteins. Over the course of twenty years we have performed more than one hundred guanidine hydrochloride equilibrium denaturations of wild-type staphylococcal nuclease; to our knowledge, a number of denaturations unrivaled in any other protein system. A careful examination of the data from these experiments shows no sign of the behavior predicted by a three-state unfolding model. Specifically, a three-state unfolding should introduce a slight, but characteristic, non-linearity to the plot of stability versus denaturant concentration. The average residuals from this large number of repeated experiments do not show the predicted behavior, casting considerable doubt on the likelihood of a three-state unfolding for the wild-type protein. The methods used for analysis here could be applied to other protein systems to distinguish a two-state from a three-state denaturation.     

Empirical evaluation of the influence of side chains on the conformational entropy of the polypeptide backbone

Changes in amino acid side chains have long been recognized to alterthe range and distribution of ?, ψ angles found in the main chain of polypeptides. Altering the range and distribution of ?, ψ angles also alters the conformational entropy of the flexible denatured state and may thus stabilize or destabilize it relative to the comparatively conformationally rigid native state. A database of 12,320 residues from 61 nonhomologous, high resolution crystal structures was examined to determine the ?, ψ conformational preferences of each of the 20 amino acids. These observed distributions in the native state of proteins are assumed to also reflect the distributions found in the denatured state. The distributionswere used to approximate the energy surface for each residue, allowing the calculation of relative conformational entropies for each residue relative to glycine. In the most extreme case, replacement of glycine by proline, conformational entropy changes will stabilize the native state relative to the denatured state by ?0.82 ± 0.08 kcal/mol at 20°C. Surprisingly, alanine is found to be the most ordered residue other than proline. This unexpected result is a result of the high percentage of alanines found in helical conformations. This either indicates that the observed distributions in the native state do not reflect the distributions in the denatured state, or that alanine is much more likely to adopt a helical conformation in the denatured state than residues with longer side chains. Among those residues with ?, ψ angles compatible with helix incorporation the percentage of alanines actually in helices is very similar to other residues. This and the consistent ordering of alanine relative to other residues regardless of secondary structure are evidence that ?, ψ distributions in native states reflect those in the denatured states. © 1995 Wiley-Liss, Inc.