Distribution of amino acids in a lipid bilayer from computer simulations

We have calculated the distribution in a lipid bilayer of small molecules mimicking 17 natural amino acids in atomistic detail by molecular dynamics simulation. We considered both charged and uncharged forms for Lys, Arg, Glu, and Asp. The results give detailed insight in the molecular basis of the preferred location and orientation of each side chain as well the preferred charge state for ionizable residues. Partitioning of charged and polar side chains is accompanied by water defects connecting the side chains to bulk water. These water defects dominate the energetic of partitioning, rather than simple partitioning between water and a hydrophobic phase. Lys, Glu, and Asp become uncharged well before reaching the center of the membrane, but Arg may be either charged or uncharged at the center of the membrane. Phe has a broad distribution in the membrane but Trp and Tyr localize strongly to the interfacial region. The distributions are useful for the development of coarse-grained and implicit membrane potentials for simulation and structure prediction. We discuss the relationship between the distribution in membranes, bulk partitioning to cyclohexane, and several amino acid hydrophobicity scales.     

The effects of hydrophobic mismatch between phosphatidylcholine bilayers and transmembrane alpha-helical peptides depend on the nature of interfacially exposed aromatic and charged residues

In this study, we investigated the extent to which different aromatic and positively charged side chains, which often flank transmembrane segments of proteins, can influence lipid-peptide interactions. Model systems consisting of phosphatidylcholine and hydrophobic alpha-helical peptides with different flanking residues were investigated. The peptides were incorporated in relatively thick and in relatively thin lipid bilayers to create a peptide-bilayer hydrophobic mismatch, and the compensating effects on lipid structure were analyzed. When relatively long with respect to the thickness of the bilayer, the peptides that are flanked by the aromatic side chains, Trp, Tyr, and Phe, all induce a significant ordering of the lipid acyl chains, while the peptides flanked by the charged residues Lys, Arg, and His do not. However, when the peptides are relatively short with respect to the thickness of the bilayer, their effect on lipid organization does not depend primarily on their aromatic or charged character. Peptides flanked by Trp, Tyr, Lys, or (at low pH) His residues are effective in inducing mismatch-relieving cubic and inverted hexagonal phases, while analogues flanked by Phe, Arg, or (at neutral pH) His residues cannot induce an inverted hexagonal phase. The different responses to mismatch might reflect the different interfacial affinities of the residues that were investigated.     

Synthetic copoly(Lys/Phe) and poly(Lys) translocate through lipid bilayer membranes

Several membrane-transporting peptides (MTP) containing basic amino acid residues such as Lys and Arg that carry macromolecules such as DNA and proteins across cell plasma membranes by an unknown mechanism have been actively studied. On the basis of these results, we have been investigating the translocation ability of synthetic polypeptides, copoly(Lys/Phe) and poly(Lys), through negatively charged phospholipid (soybean phospholipid (SBPL)) bilayer membranes by zeta potential analysis, circular dichroism (CD) spectroscopy, fluorescence spectroscopy, an electrophysiology technique, and confocal laser scanning microscopy (CLSM). The binding of these polypeptides to the membrane, which is the first step for translocation across the membrane, resulted in the conformational transition of the polypeptide from a random coil form or helix-poor form to a helix-rich form. The fluorescence studies demonstrated that the time-dependent decrease in the fluorescence intensities of the FITC-labeled polypeptides bound to the SBPL liposome reflected translocation of the polypeptide across the lipid bilayer with the low dielectric constant. Both the rate constant and the efficiency of the polypeptide translocation across the lipid bilayer were greater for copoly(Lys/Phe) than for poly(Lys). These results suggest that the random incorporation of the hydrophobic Phe residue into the positively charged Lys chain results in a lowering of the potential barrier for passage of the polypeptide in the hydrophobic core portion of the lipid bilayer. We presented the first direct observation that the positively charged polypeptides, copoly(Lys/Phe) (MW: 41,500) and poly(Lys) (MW: 23,400), could translocate across the lipid bilayer membrane.     

Synthetic copoly(Lys/Phe) and poly(Lys) translocate through lipid bilayer membranes

Several membrane-transporting peptides (MTP) containing basic amino acid residues such as Lys and Arg that carry macromolecules such as DNA and proteins across cell plasma membranes by an unknown mechanism have been actively studied. On the basis of these results, we have been investigating the translocation ability of synthetic polypeptides, copoly(Lys/Phe) and poly(Lys), through negatively charged phospholipid (soybean phospholipid (SBPL)) bilayer membranes by zeta potential analysis, circular dichroism (CD) spectroscopy, fluorescence spectroscopy, an electrophysiology technique, and confocal laser scanning microscopy (CLSM). The binding of these polypeptides to the membrane, which is the first step for translocation across the membrane, resulted in the conformational transition of the polypeptide from a random coil form or helix-poor form to a helix-rich form. The fluorescence studies demonstrated that the time-dependent decrease in the fluorescence intensities of the FITC-labeled polypeptides bound to the SBPL liposome reflected translocation of the polypeptide across the lipid bilayer with the low dielectric constant. Both the rate constant and the efficiency of the polypeptide translocation across the lipid bilayer were greater for copoly(Lys/Phe) than for poly(Lys). These results suggest that the random incorporation of the hydrophobic Phe residue into the positively charged Lys chain results in a lowering of the potential barrier for passage of the polypeptide in the hydrophobic core portion of the lipid bilayer. We presented the first direct observation that the positively charged polypeptides, copoly(Lys/Phe) (MW: 41,500) and poly(Lys) (MW: 23,400), could translocate across the lipid bilayer membrane.     

A fluorescently labeled undecapeptide derived from a protein in royal jelly of the honeybee—royalisin—for specific detection of oxidized low‐density lipoprotein

The probes for detection of oxidized low‐density lipoprotein (ox‐LDL) in plasma and in atherosclerotic plaques are expected to facilitate the diagnosis, prevention, and treatment of atherosclerosis. Recently, we have reported that a heptapeptide (Lys‐Trp‐Tyr‐Lys‐Asp‐Gly‐Asp, KP6) coupled through the ε‐amino group of N‐terminal Lys to fluorescein isothiocyanate (FITC), (FITC)KP6, can be useful as a fluorescent probe for specific detection of ox‐LDL. In the present study, to develop a novel fluorescent peptide for specific detection of ox‐LDL, we investigated the interaction (with ox‐LDL) of an undecapeptide corresponding to positions 41 to 51 of a potent antimicrobial protein (royalisin, which consists of 51 residues; from royal jelly of honeybees), conjugated at the N‐terminus to FITC in the presence of 6‐amino‐n‐caproic acid (AC) linker, (FITC‐AC)‐royalisin P11, which contains both sequences, Phe‐Lys‐Asp and Asp‐Lys‐Tyr, similar to Tyr‐Lys‐Asp in (FITC)KP6. The (FITC‐AC)‐royalisin P11 bound with high specificity to ox‐LDL in a dose‐dependent manner, through the binding to major lipid components in ox‐LDL (lysophosphatidylcholine and oxidized phosphatidylcholine). In contrast, a (FITC‐AC)‐shuffled royalisin P11 peptide, in which sequences Phe‐Lys‐Asp and Asp‐Lys‐Tyr were modified to Lys‐Phe‐Asp and Asp‐Tyr‐Lys, respectively, hardly bound to LDL and ox‐LDL. These findings strongly suggest that (FITC‐AC)‐royalisin P11 may be an effective fluorescent probe for specific detection of ox‐LDL and that royalisin from the royal jelly of honeybees may play a role in the treatment of atherosclerosis through the specific binding of the region at positions 41 to 51 to ox‐LDL.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

Empirical lipid propensities of amino acid residues in multispan alpha helical membrane proteins

Characterizing the interactions between amino acid residues and lipid molecules is important for understanding the assembly of transmembrane helices and for studying membrane protein folding. In this study we develop TMLIP (TransMembrane helix-LIPid), an empirically derived propensity of individual residue types to face lipid membrane based on statistical analysis of high-resolution structures of membrane proteins. Lipid accessibilities of amino acid residues within the transmembrane (TM) region of 29 structures of helical membrane proteins are studied with a spherical probe of radius of 1.9 A. Our results show that there are characteristic preferences for residues to face the headgroup region and the hydrocarbon core region of lipid membrane. Amino acid residues Lys, Arg, Trp, Phe, and Leu are often found exposed at the headgroup regions of the membrane, where they have high propensity to face phospholipid headgroups and glycerol backbones. In the hydrocarbon core region, the strongest preference for interacting with lipids is observed for Ile, Leu, Phe and Val. Small and polar amino acid residues are usually buried inside helical bundles and are strongly lipophobic. There is a strong correlation between various hydrophobicity scales and the propensity of a given residue to face the lipids in the hydrocarbon region of the bilayer. Our data suggest a possibly significant contribution of the lipophobic effect to the folding of membrane proteins. This study shows that membrane proteins have exceedingly apolar exteriors rather than highly polar interiors. Prediction of lipid-facing surfaces of boundary helices using TMLIP1 results in a 54% accuracy, which is significantly better than random (25% accuracy). We also compare performance of TMLIP with another lipid propensity scale, kPROT, and with several hydrophobicity scales using hydrophobic moment analysis.     

Membrane topography of ColE1 gene products: the hydrophobic anchor of the colicin E1 channel is a helical hairpin.

The paucity of crystallographic data on the structure of intrinsic membrane proteins necessitates the development of additional techniques to probe their structures. The colicin E1 ion channel domain contains one prominent hydrophobic region near its COOH terminus that has been proposed to be an anchor for the assembly of the channel. Saturation site-directed mutagenesis of the hydrophobic anchor region of the colicin E1 ion channel was used to probe whether it spanned the bilayer once or twice. A nonpolar amino acid was replaced by a charged residue in 29 mutations made at 26 positions in the channel domain. Substitution of the charged amino acid at all positions except those in the center of the hydrophobic region and the periphery of the hydrophobic region caused a large decrease in the cytotoxicity of the purified mutant colicin E1 protein. This result implies that the hydrophobic domain spans the membrane bilayer twice in a helical hairpin loop, with the center of this domain residing in an aqueous or polar phase. The lengths of the trans-membrane helices appear to be approximately 18 and 16 residues. The absence of significant changes in ion selectivity in five of nine mutants indicated that these mutations did not cause a large change in the channel structure. The ion selectivity changes in four mutants and those previously documented for the flanking Lys residues imply that the hydrophobic hairpin is part of the channel lumen. Water may "abhor" the hydrophobic side of the channel, explaining the small effects of residue charge changes on ion selectivity.     

Amino acid sequence of seminalplasmin, an antimicrobial protein from bull semen

Analytical ultracentrifugation of highly purified seminalplasmin revealed a molecular mass of 6300. Amino acid analysis of the protein preparation indicated the absence of sulfur-containing amino acids cysteine and methionine. The amino acid sequence of seminalplasmin was determined by manual Edman degradation of peptides obtained by proteolytic enzymes trypsin, chymotrypsin and thermolysin: NH₂-Ser Asp Glu Lys Ala Ser Pro Asp Lys His His Arg Phe Ser Leu Ser Arg Tyr Ala Lys Leu Ala Asn Arg Leu Ser Lys Trp Ile Gly Asn Arg Gly Asn Arg Leu Ala Asn Pro Lys Leu Leu Glu Thr Phe Lys Ser Val-COOH. The number of amino acids according to the sequence were 48, the molecular mass 6385. As predicted from the sequence, seminalplasmin very likely contains two α-helical domains in which residues 8-17 and 40-48 are involved. No evidence for the existence of β-sheet structures was obtained. Treatment of seminalplasmin with the above proteases as well as with amino peptidase M and carboxypeptidase Y completely eliminated biological activity.     

Topography of the prostaglandin endoperoxide H2 synthase-2 in membranes

The topology of association of the monotopic protein cyclooxygenase-2 (COX-2) with membranes has been examined using EPR spectroscopy of spin-labeled recombinant human COX-2. Twenty-four mutants, each containing a single free cysteine substituted for an amino acid in the COX-2 membrane binding domain were expressed using the baculovirus system and purified, then conjugated with a nitroxide spin label and reconstituted into liposomes. Determining the relative accessibility of the nitroxide-tagged amino acid side chains for the solubilized COX-2 mutants, or COX-2 reconstituted into liposomes to nonpolar (oxygen) and polar (NiEDDA or CrOx) paramagnetic reagents allowed us to map the topology of COX-2 interaction with the lipid bilayer. When spin-labeled COX-2 was reconstituted into liposomes, EPR power saturation curves showed that side chains for all but two of the 24 mutants tested had limited accessibility to both polar and nonpolar paramagnetic relaxation agents, indicating that COX-2 associates primarily with the interfacial membrane region near the glycerol backbone and phospholipid head groups. Two amino acids, Phe(66) and Leu(67), were readily accessible to the non-polar relaxation agent oxygen, and thus likely inserted into the hydrophobic core of the lipid bilayer. However these residues are co-linear with amino acids in the interfacial region, so their extension into the hydrophobic core must be relatively shallow. EPR and structural data suggest that membrane interaction of COX-2 is also aided by partitioning of 4 aromatic amino acids, Phe(59), Phe(66), Tyr(76), and Phe(84) to the interfacial region, and by the electrostatic interactions of two basic amino acids, Arg(62) and Lys(64), with the phospholipid head groups.     

The effects of polar and/or ionizable residues in the core and flanking regions of hydrophobic helices on transmembrane conformation and oligomerization

To explore the influence of amino acid composition on the behavior of membrane-inserted alpha-helices, we examined the behavior of Lys-flanked polyleucyl (pLeu) helices containing a single polar/ionizable residue within their hydrophobic core. To evaluate the location of the helices within the membrane by fluorescence, each contained a Trp residue at the center of the sequence. When incorporated into dioleoylphosphatidylcholine (DOPC) model membrane vesicles, pLeu helices with or without a single Ser, Asn, Lys, or Asp residue in the hydrophobic core maintained a transmembrane state (named the N state) at neutral and acidic pH. In this state, the central Trp exhibited highly blue-shifted fluorescence, and fluorescence quenching by nitroxide-labeled lipids showed it located at the bilayer center. A state in which Trp fluorescence red-shifted by several nanometers (named the B state) was observed above pH 10-11. B state formation appears to result from deprotonation of the flanking Lys residues. Despite the red shift in Trp emission, fluorescence quenching showed that in the B state the Trp at most is only slightly shallower than in the N state, suggesting the B state also is a transmembrane or near-transmembrane structure. The B state is characterized by increased helix oligomerization, as shown by the dependence of Trp lambda(max) on the concentration of the peptide within the bilayer at high pH. The pLeu peptide with a Asp residue in the core underwent a pH-dependent transition at a lower pH than the other peptides (pH 8-9). At high pH, it exhibited both a more highly red-shifted fluorescence and shallower Trp location than the other peptides. This state (named the S state) did not exhibit a concentration-dependent Trp lambda(max). We attribute S state behavior to the formation of a charged Asp residue at high pH, and a consequent movement of the Asp toward the membrane surface, resulting in the formation of a nontransmembrane state. We conclude that a polar or ionizable residue can readily be tolerated in a single transmembrane helix, but that the charges on ionizable residues in the core and regions flanking the helix significantly modulate the stability of transmembrane insertion and/or helix-helix association.     

Fluorescence studies on a membrane-embedded peptide from the carboxy terminus of lipophilin

Fluorescence of an intramembranous polypeptide (T-3) derived from the carboxy-terminal sequence of lipophilin was studied in aqueous solution, detergent micelles, and lipid vesicles. In all cases, the fluorescence of the only Trp (211) was indicative of a hydrophobic, buried residue. Addition of lysophosphatidylcholine (LPC) or phosphatidylcholine (PC) gave Trp-211 a more hydrophobic, less quenching environment as compared to that in aqueous solution. Energy transfer between Trp and Tyr observed in aqueous solution was decreased by the addition of lipid or detergent. There was limited quenching by acrylamide both in the aqueous and in the lipid or detergent environments. However, PC or LPC further decreased this quenching. Cs+ and I- were even less accessible than acrylamide to Trp, further proving that the Trp was located inside the lipid bilayer. The quenching indicated that I- binds to positive charges of the protein located on the surface of the membrane. This, combined with knowledge of the sequence of lipophilin, suggested that Trp-211 was located within the membrane but was close to amino acid residues that are external to the bilayer.     

Structure-based statistical analysis of transmembrane helices

Recent advances in determination of the high-resolution structure of membrane proteins now enable analysis of the main features of amino acids in transmembrane (TM) segments in comparison with amino acids in water-soluble helices. In this work, we conducted a large-scale analysis of the prevalent locations of amino acids by using a data set of 170 structures of integral membrane proteins obtained from the MPtopo database and 930 structures of water-soluble helical proteins obtained from the protein data bank. Large hydrophobic amino acids (Leu, Val, Ile, and Phe) plus Gly were clearly prevalent in TM helices whereas polar amino acids (Glu, Lys, Asp, Arg, and Gln) were less frequent in this type of helix. The distribution of amino acids along TM helices was also examined. As expected, hydrophobic and slightly polar amino acids are commonly found in the hydrophobic core of the membrane whereas aromatic (Trp and Tyr), Pro, and the hydrophilic amino acids (Asn, His, and Gln) occur more frequently in the interface regions. Charged amino acids are also statistically prevalent outside the hydrophobic core of the membrane, and whereas acidic amino acids are frequently found at both cytoplasmic and extra-cytoplasmic interfaces, basic amino acids cluster at the cytoplasmic interface. These results strongly support the experimentally demonstrated biased distribution of positively charged amino acids (that is, the so-called the positive-inside rule) with structural data.     

Roles of the P1, P2, and P3 residues in determining inhibitory specificity of kallistatin toward human tissue kallikrein

Kallistatin is a serpin with a unique P1 Phe, which confers an excellent inhibitory specificity toward tissue kallikrein. In this study, we investigated the P3-P2-P1 residues (residues 386-388) of human kallistatin in determining inhibitory specificity toward human tissue kallikrein by site-directed mutagenesis and molecular modeling. Human kallistatin mutants with 19 different amino acid substitutions at each P1, P2, or P3 residue were created and purified to compare their kallikrein binding activity. Complex formation assay showed that P1 Arg, P1 Phe (wild type), P1 Lys, P1 Tyr, P1 Met, and P1 Leu display significant binding activity with tissue kallikrein among the P1 variants. Kinetic analysis showed the inhibitory activities of the P1 mutants toward tissue kallikrein in the order of P1 Arg > P1 Phe > P1 Lys >/= P1 Tyr > P1 Leu >/= P1 Met. P1 Phe displays a better selectivity for human tissue kallikrein than P1 Arg, since P1 Arg also inhibits several other serine proteinases. Heparin distinguishes the inhibitory specificity of kallistatin toward kallikrein versus chymotrypsin. For the P2 and P3 variants, the mutants with hydrophobic and bulky amino acids at P2 and basic amino acids at P3 display better binding activity with tissue kallikrein. The inhibitory activities of these mutants toward tissue kallikrein are in the order of P2 Phe (wild type) > P2 Leu > P2 Trp > P2 Met and P3 Arg > P3 Lys (wild type). Molecular modeling of the reactive center loop of kallistatin bound to the reactive crevice of tissue kallikrein indicated that the P2 residue required a long and bulky hydrophobic side chain to reach and fill the hydrophobic S2 cleft generated by Tyr(99) and Trp(219) of tissue kallikrein. Basic amino acids at P3 could stabilize complex formation by forming electrostatic interaction with Asp(98J) and hydrogen bond with Gln(174) of tissue kallikrein. Our results indicate that tissue kallikrein is a specific target proteinase for kallistatin.     

Analysis of the NH2-Terminal 83rd Amino Acid of Escherichia coli GyrA in Quinolone-Resistance

Artificial mutations of Gyrase A protein (GyrA) in Escherichia coli by site-directed mutagenesis were generated to analyze quinolone-resistant mechanisms. By genetic analysis of gyrA genes in a gyrA temperature sensitive (Ts) background, exchange of Ser at the NH₂-terminal 83rd position of GyrA to Trp, Leu, Phe, Tyr, Ala, Val, and Ile caused bacterial resistance to the quinolones, while exchange to Gly, Asn, Lys, Arg and Asp did not confer resistance. These results indicate that it is the most important for the 83rd amino acid residue to be hydrophobic in expressing the phenotype of resistance to the quinolones. These findings also suggest that the hydroxyl group of Ser would not play a major role in the quinolone-gyrase interaction and Ser83 would not interact directly with other amino acid residues.     

Relative reactivity of lysine and other peptide-bound amino acids to oxidation by hypochlorite

Antibacterial and inflammatory responses of neutrophils and macrophages produce hypochlorite as a major oxidant. Numerous side chains of amino acids found in extracellular proteins can be modified by hypochlorite, including His, Arg, Tyr, Lys, Trp, and Met. We studied the relative reactivity of each of these amino acid residues in short N-blocked peptides, where other residues in the peptide were highly resistant to hypochlorite attack. Hypochlorite treatment led to modified peptides in each case, which were detected by changes in retention on reversed-phase HPLC. A distinct single product, consuming two equivalents of hypochlorite per equivalent of peptide, was obtained from the Lys-containing peptides. UV spectroscopy, nuclear magnetic resonance (NMR), and electrospray/mass spectroscopy identified this product as the dichloramine at the epsilon-amino group of the Lys side chain. The dichloramine at Lys did not decompose to form a detectable amount of carbonyl reactive with dinitrophenylhydrazine. The dichloramine at Lys did however quantitatively revert back to Lys during HCl digestion of the tetrapeptide for amino acid analysis, with simultaneous modification of the adjacent Phe residue. The formation of the dichloramine at Lys was not blocked by peptides or acetylated amino acids that contained Tyr, His, or Arg. In contrast, the presence of equimolar Met-containing peptide, or N-Acetyl-Trp, both inhibited the formation of the dichloramine at Lys. Thus, Met and Trp side chains of proteins might be able to protect Lys from chloramine formation under some circumstances, but this interpretation must consider that Met and Trp are typically found in relatively inaccessible hydrophobic sites, whereas lysine is typically exposed on the protein surface. The hierarchy of amino acid reactivities examined here will aid in the prediction of residues in biological samples most likely to be modified by hypochlorite.     

Caspase-3 binds diverse P4 residues in peptides as revealed by crystallography and structural modeling

Caspase-3 recognition of various P4 residues in its numerous protein substrates was investigated by crystallography, kinetics, and calculations on model complexes. Asp is the most frequent P4 residue in peptide substrates, although a wide variety of P4 residues are found in the cellular proteins cleaved by caspase-3. The binding of peptidic inhibitors with hydrophobic P4 residues, or no P4 residue, is illustrated by crystal structures of caspase-3 complexes with Ac-IEPD-Cho, Ac-WEHD-Cho, Ac-YVAD-Cho, and Boc-D(OMe)-Fmk at resolutions of 1.9–2.6 Å. The P4 residues formed favorable hydrophobic interactions in two separate hydrophobic regions of the binding site. The side chains of P4 Ile and Tyr form hydrophobic interactions with caspase-3 residues Trp206 and Trp214 within a non-polar pocket of the S4 subsite, while P4 Trp interacts with Phe250 and Phe252 that can also form the S5 subsite. These interactions of hydrophobic P4 residues are distinct from those for polar P4 Asp, which indicates the adaptability of caspase-3 for binding diverse P4 residues. The predicted trends in peptide binding from molecular models had high correlation with experimental values for peptide inhibitors. Analysis of structural models for the binding of 20 different amino acids at P4 in the aldehyde peptide Ac-XEVD-Cho suggested that the majority of hydrophilic P4 residues interact with Phe250, while hydrophobic residues interact with Trp206, Phe250, and Trp214. Overall, the S4 pocket of caspase-3 exhibits flexible adaptation for different residues and the new structures and models, especially for hydrophobic P4 residues, will be helpful for the design of caspase-3 based drugs.     

Mutational analysis and membrane-interactions of the beta-sheet-like N-terminal domain of the pediocin-like antimicrobial peptide sakacin P

To gain insight into how the N-terminal three-stranded beta-sheet-like domain in pediocin-like antimicrobial peptides positions itself on membranes, residues in the well-conserved (Y)YGNGV-motif in the domain were substituted and the effect of the substitutions on antimicrobial activity and binding of peptides to liposomes was determined. Peptide-liposome interactions were detected by measuring tryptophan-fluorescence upon exposing liposomes to peptides in which a tryptophan residue had been introduced in the N-terminal domain. The results revealed that the N-terminal domain associates readily with anionic liposomes, but not with neutral liposomes. The electrostatic interactions between peptides and liposomes facilitated the penetration of some of the peptide residues into the liposomes. Measuring the antimicrobial activity of the mutated peptides revealed that the Tyr2Leu and Tyr3Leu mutations resulted in about a 10-fold reduction in activity, whereas the Tyr2Trp, Tyr2Phe, Tyr3Trp and Tyr3Phe mutations were tolerated fairly well, especially the mutations in position 3. The Val7Ile mutation did not have a marked detrimental effect on the activity. The Gly6Ala mutation was highly detrimental, consistent with Gly6 being in one of the turns in the beta-sheet-like N-terminal domain, whereas the Gly4Ala mutation was tolerated fairly well. All mutations involving Asn5, including the conservative mutations Asn5Gln and Asn5Asp, were very deleterious. Thus, both the polar amide group on the side chain of Asn5 and its exact position in space were crucial for the peptides to be fully active. Taken together, the results are consistent with Val7 positioning itself in the hydrophobic core of target membranes, thus forcing most of the other residues in the N-terminal domain into the membrane interface region: Tyr3 and Asn5 in the lower half with their side chains pointing downward and approaching the hydrophobic core, Tyr2, Gly4 and His8 and 12 in the upper half, Lys1 near the middle of the interface region, and the side chain of Lys11 pointing out toward the membrane surface.     

Comparison of CD spectra in the aromatic region on a series of variant proteins substituted at a unique position of tryptophan synthase alpha-subunit

CD spectra in the aromatic region of a series of the mutant alpha-subunits of tryptophan synthase from Escherichia coli, substituted at position 49 buried in the interior of the molecule, were measured at pH 7.0 and 25 degrees C. These measurements were taken to gain information on conformational change produced by single amino acid substitutions. The CD spectra of the mutant proteins, substituted by Tyr or Trp residue in place of Glu residue at position 49, showed more intense positive bands due to one additional Tyr or Trp residue at position 49. The CD spectra of other mutant proteins also differed from that of the wild-type protein, despite the fact that the substituted residues at position 49 were not aromatic. Using the spectrum of the wild-type protein (Glu49) as a standard, the spectra of the other mutants were classified into three major groups. For 10 mutant proteins substituted by Ile, Ala, Leu, Met, Val, Cys, Pro, Ser, His, or Gly, their CD values of bands (due to Tyr residues) decreased in comparison with those of the wild-type protein. The mutant protein substituted by Phe also belonged to this group. These substituted amino acid residues are more hydrophobic than the original residue, Glu. In the second group, three mutant proteins were substituted by Lys, Gln, or Asn, and the CD values of tyrosyl bands increased compared to those of the wild-type proteins. These residues are polar. In the third group, the CD values of tyrosyl bands of two mutant proteins substituted by Asp or Thr were similar to those of the wild-type protein, except for one band at 276.5 nm. These results suggested that the changes in the CD spectra for the mutant proteins were affected by the hydrophobicity of the residues at position 49.     

Sequences of tryptic peptides containing the five cysteinyl residues of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Tryptic peptides which account for all five cysteinyl residues in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been purified and sequenced. Collectively, these peptides contain 94 of the approximately 500 amino acid residues per molecule of subunit. Due to one incomplete cleavage at a site for trypsin and two incomplete chymotryptic-like cleavages, eight major radioactive peptides (rather than five as predicted) were recovered from tryptic digests of the enzyme that had been carboxymethylated with [³H]iodoacetate. The established sequences are: GlyTyrThrAlaPheValHisCys¹Lys TyrValAspLeuAlaLeuLysGluGluAspLeuIleAla GlyGlyGluHisValLeuCys¹AlaTyr AlaGlyTyrGlyTyrValAlaThrAlaAlaHisPheAla AlaGluSerSerThrGlyThrAspValGluValCys¹ ThrThrAsxAsxPheThrArg AlaCys¹ThrProIleIleSerGlyGlyMetAsnAla LeuArg ProPheAlaGluAlaCys¹HisAlaPheTrpLeuGly GlyAsnPheIleLys In these peptides, radioactive carboxymethylcysteinyl residues are denoted with asterisks and the sites of incomplete cleavage with vertical wavy lines. None of the peptides appear homologous with either of two cysteinyl-containing, active-site peptides previously isolated from spinach ribulosebisphosphate carboxylase/oxygenase.