Conformation and activity of delta-lysin and its analogs

Delta-Lysin is a 26-residue hemolytic peptide secreted by Staphylococcus aureus. Unlike the bee venom peptide melittin, delta-lysin does not exhibit antibacterial activity. We have synthesized delta-lysin and several analogs wherein the N-terminal residues of the toxin were sequentially deleted. The toxin has three aspartic acids, four lysines and no prolines. Analogs were also generated in which all the aspartic acids were replaced with lysines. A proline residue was introduced in the native sequences as well as in the analogs where aspartic acids were replaced with lysines. We observed that 20- and 22-residue peptides corresponding to residues 7-26 and 5-26 of delta-lysin, respectively, had greater hemolytic activity than the parent peptide. These shorter peptides, unlike delta-lysin, did not self-associate to adopt alpha-helical conformation in water, at lytic concentrations. Introduction of proline or substitution of aspartic acids by lysines resulted in loss in propensity to adopt helical conformation in water. When proline was introduced in the peptides corresponding to the native toxin sequence, loss of hemolytic activity was observed. Substitution of all the aspartic acids with lysines resulted in enhanced hemolytic activity in all the analogs. However, when both proline and aspartic acid to lysine changes were made, only antibacterial activity was observed in the shorter peptides. Our investigations on delta-lysin and its analogs provide insights into the positioning of anionic, cationic residues and proline in determining hemolytic and antibacterial activities.     

Epr Evidence for the Oxidation-Induced Formation of Negatively Charged Species on the Low-Density Lipoprotein Surface

Oxidation-induced increase of the net negative charge on low-density lipoprotein was studied by electro-phoretic mobility and by electron paramagnetic resonance. The negative-charge increase is associated not only with neutralization of the lysine residues of apoprotein B, but also with the exposition of the excessive negatively charged residues on the lipoprotein surface. The accumulation of the negatively charged residues is believed to be brought about by the conformational change of apoprotein B, triggered by neutralization of lysines and cleavage of peptide bonds. Alternatively, reactive oxygen species could also convert histidine to aspartic acid and proline to glutamic acid.     

Epr Evidence for the Oxidation-Induced Formation of Negatively Charged Species on the Low-Density Lipoprotein Surface

Oxidation-induced increase of the net negative charge on low-density lipoprotein was studied by electro-phoretic mobility and by electron paramagnetic resonance. The negative-charge increase is associated not only with neutralization of the lysine residues of apoprotein B, but also with the exposition of the excessive negatively charged residues on the lipoprotein surface. The accumulation of the negatively charged residues is believed to be brought about by the conformational change of apoprotein B, triggered by neutralization of lysines and cleavage of peptide bonds. Alternatively, reactive oxygen species could also convert histidine to aspartic acid and proline to glutamic acid.     

Identification of a consensus motif for retention of transmembrane proteins in the endoplasmic reticulum.

Several families of transmembrane endoplasmic reticulum (ER) proteins contain retention motifs in their cytoplasmically exposed tails. Mutational analyses demonstrated that two lysines positioned three and four or five residues from the C-terminus represent the retention motif. The introduction of a lysine preceding the lysine that occurs three residues from the terminus of Lyt2 renders this cell surface protein a resident of the ER. Likewise, the appropriate positioning of two lysine residues in a poly-serine sequence confines marker proteins to the ER. Arginines or histidines cannot replace lysines, suggesting that simple charge interactions are not sufficient to explain the retention. The identified consensus motif may serve as a retrieval signal that brings proteins back from a sorting compartment adjacent to the ER.     

Unique amino acid composition of proteins in halophilic bacteria

The amino acid compositions of proteins from halophilic archaea were compared with those from non-halophilic mesophiles and thermophiles, in terms of the protein surface and interior, on a genome-wide scale. As we previously reported for proteins from thermophiles, a biased amino acid composition also exists in halophiles, in which an abundance of acidic residues was found on the protein surface as compared to the interior. This general feature did not seem to depend on the individual protein structures, but was applicable to all proteins encoded within the entire genome. Unique protein surface compositions are common in both halophiles and thermophiles. Statistical tests have shown that significant surface compositional differences exist among halophiles, non-halophiles, and thermophiles, while the interior composition within each of the three types of organisms does not significantly differ. Although thermophilic proteins have an almost equal abundance of both acidic and basic residues, a large excess of acidic residues in halophilic proteins seems to be compensated by fewer basic residues. Aspartic acid, lysine, asparagine, alanine, and threonine significantly contributed to the compositional differences of halophiles from meso- and thermophiles. Among them, however, only aspartic acid deviated largely from the expected amount estimated from the dinucleotide composition of the genomic DNA sequence of the halophile, which has an extremely high G+C content (68%). Thus, the other residues with large deviations (Lys, Ala, etc.) from their non-halophilic frequencies could have arisen merely as "dragging effects" caused by the compositional shift of the DNA, which would have changed to increase principally the fraction of aspartic acid alone.     

Entropic stabilization of proteins and its proteomic consequences

Evolutionary traces of thermophilic adaptation are manifest, on the whole-genome level, in compositional biases toward certain types of amino acids. However, it is sometimes difficult to discern their causes without a clear understanding of underlying physical mechanisms of thermal stabilization of proteins. For example, it is well-known that hyperthermophiles feature a greater proportion of charged residues, but, surprisingly, the excess of positively charged residues is almost entirely due to lysines but not arginines in the majority of hyperthermophilic genomes. All-atom simulations show that lysines have a much greater number of accessible rotamers than arginines of similar degree of burial in folded states of proteins. This finding suggests that lysines would preferentially entropically stabilize the native state. Indeed, we show in computational experiments that arginine-to-lysine amino acid substitutions result in noticeable stabilization of proteins. We then hypothesize that if evolution uses this physical mechanism as a complement to electrostatic stabilization in its strategies of thermophilic adaptation, then hyperthermostable organisms would have much greater content of lysines in their proteomes than comparably sized and similarly charged arginines. Consistent with that, high-throughput comparative analysis of complete proteomes shows extremely strong bias toward arginine-to-lysine replacement in hyperthermophilic organisms and overall much greater content of lysines than arginines in hyperthermophiles. This finding cannot be explained by genomic GC compositional biases or by the universal trend of amino acid gain and loss in protein evolution. We discovered here a novel entropic mechanism of protein thermostability due to residual dynamics of rotamer isomerization in native state and demonstrated its immediate proteomic implications. Our study provides an example of how analysis of a fundamental physical mechanism of thermostability helps to resolve a puzzle in comparative genomics as to why amino acid compositions of hyperthermophilic proteomes are significantly biased toward lysines but not similarly charged arginines.     

τ Protein from Alzheimer's Disease Patients Is Glycated at Its Tubulin-Binding Domain

Abstract: Glycated residues of τ protein from paired helical filaments isolated from the brains of Alzheimer's disease patients were localized by doing a proteolytic cleavage of the protein, fractionation of the resulting peptides, and identification of those peptides using specific antibodies. The most suitable residues for glycation, lysines, present at the tubulin-binding motif of τ protein, seem to be preferentially modified compared with those lysines present at other regions. Among these modified lysines, those located in the sequence comprising residues 318–336 (in the largest human τ isoform) were found to be glycated, as determined by the reaction with an antibody that recognizes a glycated peptide containing this sequence. Because those lysines are present in a tubulin binding motif of τ protein, its modification could result in a decrease in the interaction of τ with tubulin.     

Determination of Β-isomerized aspartic acid as the corresponding alcohol

The age-related formation of succinimides in proteins, through spontaneous deamidation of asparagine, and through cyclization of aspartic acid, is thought to be followed by the hydrolysis of the succinimide ring, yielding a mixture of normal aspartic acid sites and-isomerized aspartic acid sites (isoaspartic acid). The chemical reduction of an isoaspartyl site to the corresponding amino acid alcohol, isohomoserine, has now been investigated as a general approach to measuring the accumulation of isomerized residues in aging proteins. The methods employed were based on conditions previously found to be successful in reducing protein aspartic acid to homoserine. Borane was employed as the reducing agent, and was found to produce the expected amino acid alcohols in reactions with model peptides. In addition, amino acid analysis revealed a complex pattern of unknown products of these reduction reactions, some of which were also evident when a much stronger reducing agent, lithium aluminum hydride, was used. The correlation of some of these side-products with the isomerization of the peptide suggests, unexpectedly, that the reactivity of reducing agents toward aspartyl residues and perhaps other sites in the peptide may be influenced by steric factors related to aspartyl isomerization. The borane reduction method was also applied to proteins. No detectable isohomoserine was formed either in ovalbumin, a model aged protein, or in human lens proteins of advanced age, with conditions that fully reduced normal aspartyl residues to homoserine. These tests thus indicate that the percentage of aspartic acid in the isomerized form in these proteins is below the limit of detectability (below 5%). These results complement previous experimental results that have indicated a low bulk isoaspartyl content in most natural proteins.     

Tropomyosin lysine reactivities and relationship to coiled-coil structure

We have carried out a detailed analysis of tropomyosin structure using lysines as specific probes for the protein surface in regions of the molecule that have not been investigated by other methods. We have measured the relative reactivities of lysines in rabbit skeletal muscle alpha, alpha-tropomyosin with acetic anhydride using a competitive labeling procedure. We have identified 37 of 39 lysines and find that they range 20-fold in reactivity. The observed reactivities are related to the coiled-coil model of the tropomyosin molecule [Crick, F.H.C. (1953) Acta Crystallogr. 6, 689-697; McLachlan, A.D., Stewart, M., & Smillie, L.B. (1975) J. Mol. Biol. 98, 281-291] and other available chemical and physical information about the structure. In most cases, the observed lysine reactivities can be explained by allowable interactions with neighboring amino acid side chains on the same or facing alpha-helix. However, we found no correlation between reactivity and helical position of a given lysine. For example, lysines in the outer helical positions included lysines of low as well as high reactivity, indicating that they vary widely in their accessibility to solvent and that the coiled coil is heterogeneous along its length. Furthermore, the middle of the molecule (residues 126-182) that is susceptible to proteolysis and known to be the least stable region of the protein also contains some of the least and most reactive lysines. We have discussed the implications of our results on our understanding the structures of tropomyosin and other coiled-coil proteins as well as globular proteins containing helical regions.     

Cytochrome c and cytochrome c oxidase interactions: the effects of ionic strength and hydrostatic pressure studied with site-specific modifications of cytochrome c.

Seven cytochromes c, in which individual lysines have been modified to the propylthiobimane derivatives, have been prepared. These derivatives were also converted to the porphyrin cytochromes c by treatment with HF. The properties of both types of modified proteins were studied in their reactions with cytochrome c oxidase. The results show that lysines 25, 27, 60, 72, and 87 do not contribute a full charge to the binding interaction with the oxidase. These five residues, with the exception of the lysine-60 derivative, on the front surface of the protein and contain the solvent-accessible edge of the heme prosthetic group. By contrast, lysines 8 and 13 at the top of the front surface do contribute a full charge to the binding interaction with the oxidase. The removal of the positive charge on any one lysine weakens the binding to cytochrome c oxidase by at least 1 kcal (1 cal = 4.1868 J). The presence of bimane at lysines 13 and 87 clearly forces the separation of the cytochrome c and oxidase, but this does not occur with the other complexes. The bimane-modified lysine-13 protein, and to a lesser extent that modified at lysine 8, show the interesting effect of enhanced complex formation with cytochrome c oxidase when subjected to pressure, possibly because of entrapment of water at the newly created interface of the complex. Our observations indicate that the two proteins of the cytochrome c - cytochrome oxidase complex have preferred, but not obligatory, spatial orientations and that interaction occurs without either protein losing significant portions of its hydration shell.     

Ionization behaviour of native apolipoproteins and of their complexes with lecithin. 1. Calorimetric and potentiometric titration of the native apoA-I protein and of the apoA-I protein-dimyristoyl lecithin complex.

The ionization behaviour of native apoA-I protein is compare to that of its complex with synthetic dimyristoyl lecithin in studies using calorimetric, potentiometric and spectrophotometric titration. In the presence of phospholipids, 10 out of 21 lysines together with 22 acidic residues are masked in the complex. All tyrosines remain accessible to titration below pH 13. The apparent ionization enthalpy of the 11 lysine residues is not affected by the presence of phospholipids. These data are consistent with discrete binding sites located in the apoprotein helical segments as suggested by the model of Segrest et al. [FEBS Lett. 38, 247-253 (1974)]. A tentative localisation of lysine, arginine, aspartic acid and glutamic acid residues directly involved in phospholipid binding is suggested, assuming that such helical regions are involved in apoprotein-phospholipid association.     

Structural Basis for the Aminoacid Composition of Proteins from Halophilic Archea

Proteins from halophilic organisms, which live in extreme saline conditions, have evolved to remain folded at very high ionic strengths. The surfaces of halophilic proteins show a biased amino acid composition with a high prevalence of aspartic and glutamic acids, a low frequency of lysine, and a high occurrence of amino acids with a low hydrophobic character. Using extensive mutational studies on the protein surfaces, we show that it is possible to decrease the salt dependence of a typical halophilic protein to the level of a mesophilic form and engineer a protein from a mesophilic organism into an obligate halophilic form. NMR studies demonstrate complete preservation of the three-dimensional structure of extreme mutants and confirm that salt dependency is conferred exclusively by surface residues. In spite of the statistically established fact that most halophilic proteins are strongly acidic, analysis of a very large number of mutants showed that the effect of salt on protein stability is largely independent of the total protein charge. Conversely, we quantitatively demonstrate that halophilicity is directly related to a decrease in the accessible surface area.     

Addition of negatively charged residues can reverse the decrease in the solubility of an acidic protein caused by an artificially introduced non-polar surface patch

A non-polar patch on the surface of a protein can cause a reduction in the solubility and stability of the protein, and thereby induce aggregation. However, a non-polar patch may be required so that the protein can bind to another molecule. The mutant 6L—derived from the acidic, dimeric α-helical protein sulerythrin and containing six additional leucines arranged to form a non-polar patch on its surface when properly folded—has a substantially reduced solubility in comparison with that of wild-type sulerythrin. This reduced solubility appears to cause 6L to aggregate. To reverse this aggregation, we mutated 6L so that it contained three to six additional glutamates or aspartates that we predicted would surround the non-polar leucine patch on natively folded 6L. Although the introduction of three glutamates or aspartates increased solubility, the mutants still aggregate and have a reduced α-helical content. Conversely, mutants with six additional glutamates or aspartates appear to exist mostly as dimers and to have the same α-helical content as that of wild-type sulerythrin. Notably, the introduction of five lysines or five arginines at the positions held by the glutamates or aspartates did not recover solubility as effectively as did the negatively charged residues. These results demonstrate that negatively charged residues, but not positively charged ones, surrounding a non-polar patch on an acidic protein can completely reverse the decrease in its solubility caused by the patch of non-polar surface residues.     

In silico study on the effect of surface lysines and arginines on the electrostatic interactions and protein stability

Charged amino acids are mostly exposed on a protein surface, thereby forming a network of interactions with the surrounding amino acids as well as with water. In particular, positively charged arginine and lysine have different side chain geometries and provide a different number of potential electrostatic interactions. This study reports a comparative analysis of the difference in the number of two representative electrostatic interactions, such as salt-bridges and hydrogen bonds, contributed by surface arginine and lysine, as well as their effect on protein stability using molecular modeling and dynamics simulation techniques. Two in silico variants, the R variant with all arginines and the K variant with all lysines on the protein surface, were modeled by mutating all the surface lysines to arginines and the surface arginines to lysines, respectively, for each of the 10 model proteins. A structural comparison of the respective two variants showed that the majority of R variants possessed more salt-bridges and hydrogen bond interactions than the K variants, indicating that arginine provides a higher probability of electrostatic interactions than lysine owing to its side chain geometry. Molecular dynamics simulations of these variants revealed the R variants to be more stable than the K variants at room temperature but this effect was not prominent under protein denaturating conditions, such as 353 and 333 K with 8 M urea. These results suggest that the arginine residues on a protein surface contribute to the protein stability slightly more than lysine by enhancing the electrostatic interactions.     

C-terminal lysines determine phospholipid interaction of sarcomeric mitochondrial creatine kinase

High affinity interaction between octameric mitochondrial creatine kinase (MtCK) and the phospholipid cardiolipin in the inner mitochondrial membrane plays an important role in metabolite channeling between MtCK and inner membrane adenylate translocator, which itself is tightly bound to cardiolipin. Three C-terminal basic residues revealed as putative cardiolipin anchors in the x-ray structures of MtCK and corresponding to lysines in human sarcomeric MtCK (sMtCK) were exchanged by in vitro mutagenesis (K369A/E, K379Q/A/E, K380Q/A/E) to yield double and triple mutants. sMtCK proteins were bacterially expressed, purified to homogeneity, and verified for structural integrity by enzymatic activity, gel filtration chromatography, and CD spectroscopy. Interaction with cardiolipin and other acidic phospholipids was quantitatively analyzed by light scattering, surface plasmon resonance, and fluorescence spectroscopy. All mutant sMtCKs showed a strong decrease in vesicle cross-linking, membrane affinity, binding capacity, membrane ordering capability, and binding-induced changes in protein structure as compared with wild type. These effects did not depend on the nature of the replacing amino acid but on the number of exchanged lysines. They were moderate for Lys-379/Lys-380 double mutants but pronounced for triple mutants, with a 30-fold lower membrane affinity and an entire lack of alterations in protein structure compared with wild-type sMtCK. However, even triple mutants partially maintained an increased order of cardiolipin-containing membranes. Thus, the three C-terminal lysines determine high affinity sMtCK/cardiolipin interaction and its effects on MtCK structure, whereas low level binding and some effect on membrane fluidity depend on other structural components. These results are discussed in regard to MtCK microcompartments and evolution.     

Interference with Ubiquitination in CFTR Modifies Stability of Core Glycosylated and Cell Surface Pools

It is recognized that both wild-type and mutant CFTR proteins undergo ubiquitination at multiple lysines in the proteins and in one or more subcellular locations. We hypothesized that ubiquitin is added to specific sites in wild-type CFTR to stabilize it and at other sites to signal for proteolysis. Mass spectrometric analysis of wild-type CFTR identified ubiquitinated lysines 68, 710, 716, 1041, and 1080. We demonstrate that the ubiquitinated K710, K716, and K1041 residues stabilize wild-type CFTR, protecting it from proteolysis. The polyubiquitin linkage is predominantly K63. N-tail mutants, K14R and K68R, lead to increased mature band C CFTR, which can be augmented by proteasomal (but not lysosomal) inhibition, allowing trafficking to the surface. The amount of CFTR in the K1041R mutant was drastically reduced and consisted of bands A/B, suggesting that the site in transmembrane 10 (TM10) was critical to further processing beyond the proteasome. The K1218R mutant increases total and cell surface CFTR, which is further accumulated by proteasomal and lysosomal inhibition. Thus, ubiquitination at residue 1218 may destabilize wild-type CFTR in both the endoplasmic reticulum (ER) and recycling pools. Small molecules targeting the region of residue 1218 to block ubiquitination or to preserving structure at residues 710 to 716 might be protein sparing for some forms of cystic fibrosis.     

Experimental evaluation of the effective dielectric constant of proteins

Chemical modifications that alter the net charge of residues in reduction-oxidation proteins influence the redox potential of the protein by changing the electrostatic potential at the redox center. If the locations of the modified charges are known, the shift in redox potential may be used to determine the effective dielectric constant for the interactions between the redox center and modified residues. From the shift in redox potential upon charge neutralization of specific lysines in the hemoprotein cytochrome c, an effective dielectric constant of approximately 50 is calculated for the electrostatic interaction between the modified lysines and heme iron in the native protein.     

Molecular bases of protein halotolerance

Halophilic proteins are stable and function at high salt concentration. Understanding how these molecules maintain their fold stable and avoid aggregation under harsh conditions is of great interest for biotechnological applications. This mini-review describes what is known about the molecular determinants of protein halotolerance. Comparisons between the sequences of halophilic/non-halophilic homologous protein pairs indicated that Asp and Glu are significantly more frequent, while Lys, Ile and Leu are less frequent in halophilic proteins. Homologous halophilic and non-halophilic proteins have similar overall structure, secondary structure content, and number of residues involved in the formation of H-bonds. On the other hand, on the halophilic protein surface, a decrease of nonpolar residues and an increase of charged residues are observed. Particularly, halophilic adaptation correlates with an increase of Asp and Glu, compensated by a decrease of basic residues, mainly Lys, on protein surface. A thermodynamic model, that provides a reliable explanation of the salt effect on the conformational stability of globular proteins, is presented.     

Acetylation of the yeast histone H4 N terminus regulates its binding to heterochromatin protein SIR3.

Heterochromatin at yeast telomeres and silent mating (HM) loci represses adjacent genes and is formed by the binding and spreading of silencing information regulators (SIR proteins) along histones. This involves the interaction between the C terminus of SIR3 and the N terminus of histone H4. Since H4 is hypoacetylated in heterochromatin we wished to determine whether acetylation is involved in regulating the contacts between SIR3 and H4. Binding of H4 peptide (residues 1-34) acetylated at lysines Lys-5, Lys-8, Lys-12, and Lys-16 to an immobilized SIR3 protein fragment (residues 510-970) was investigated using surface plasmon resonance. We find that acetylation of H4 lysines reduces binding (K(a)) of H4 to SIR3 in a cumulative manner so that the fully acetylated peptide binding is decreased approximately 50-fold relative to unacetylated peptide. Thus, by affecting SIR3-H4 binding, acetylation may regulate the formation of heterochromatin. These data help explain the hypoacetylated state of histone H4 in heterochromatin of eukaryotes.     

Unraveling the Role of the C-terminal Helix Turn Helix of the Coat-binding Domain of Bacteriophage P22 Scaffolding Protein

Many viruses encode scaffolding and coat proteins that co-assemble to form procapsids, which are transient precursor structures leading to progeny virions. In bacteriophage P22, the association of scaffolding and coat proteins is mediated mainly by ionic interactions. The coat protein-binding domain of scaffolding protein is a helix turn helix structure near the C terminus with a high number of charged surface residues. Residues Arg-293 and Lys-296 are particularly important for coat protein binding. The two helices contact each other through hydrophobic side chains. In this study, substitution of the residues of the interface between the helices, and the residues in the β-turn, by aspartic acid was used examine the importance of the conformation of the domain in coat binding. These replacements strongly affected the ability of the scaffolding protein to interact with coat protein. The severity of the defect in the association of scaffolding protein to coat protein was dependent on location, with substitutions at residues in the turn and helix 2 causing the most significant effects. Substituting aspartic acid for hydrophobic interface residues dramatically perturbs the stability of the structure, but similar substitutions in the turn had much less effect on the integrity of this domain, as determined by circular dichroism. We propose that the binding of scaffolding protein to coat protein is dependent on angle of the β-turn and the orientation of the charged surface on helix 2. Surprisingly, formation of the highly complex procapsid structure depends on a relatively simple interaction.