Several properties of complexes of polynucleotides with aromatic amino acid amides of oligonucleotides

Residues of Phe, Tyr and Trp in the complexes of their oligonucleotide amidates and polynucleotides of A-U of G-C nucleotide composition are most likely localized in the minor groove of the Watson--Crick part of the triple helix where they interact with bases but do not intercalate into the helix. Formation of the complexes is accompanied with a change in the relative localization of amino acids and bases. The major geometrical parameters of the triple helices of the complexes are not changed by the residues of aromatic amino acids (according to CD data). A slight violation of stacking interactions between bases is observed along with an increase of the cooperativity of melting of the complexes of A-U composition (according to UV absorption data). The effect of the residues of aromatic amino acids on the stability of triple helices is determined by the nucleotide composition of the latter, i.e. complexes of A-U composition are destabilized with the Phe, Tyr and Trp residues, whereas the Trp residue does not affect the stability of the complexes of G-C composition. The hydrophobic character of aromatic amino acids and their different affinity for bases of different structure seem to account for this difference in stability. The dependence of the thermal stability of RNH-dp(An).2poly(U)-complexes on the structure of the amide radical (residues of glycin, aromatic amino acids, alkyl- and arylalkyl amines) testifies the ability of the radical to "regulate" the interaction between the oligomer and the complementary polynucleotide. This capacity for "regulation" is not observed in the system of G-C composition.     

Anchoring mechanisms of membrane-associated M13 major coat protein

Bacteriophage M13 major coat protein is extensively used as a biophysical, biochemical, and molecular biology reference system for studying membrane proteins. The protein has several elements that control its position and orientation in a lipid bilayer. The N-terminus is dominated by the presence of negatively charged amino acid residues (Glu2, Asp4, and Asp5), which will always try to extend into the aqueous phase and therefore act as a hydrophilic anchor. The amphipathic and the hydrophobic transmembrane part contain the most important hydrophobic anchoring elements. In addition there are specific aromatic and charged amino acid residues in these domains (Phe 11, Tyr21, Tyr24, Trp26, Phe42, Phe45, Lys40, Lys43, and Lys44) that fine-tune the association of the protein to the lipid bilayer. The interfacial Tyr residues are important recognition elements for precise protein positioning, a function that cannot be performed optimally by residues with an aliphatic character. The Trp26 anchor is not very strong: depending on the context, the tryptophan residue may move in or out of the membrane. On the other hand, Lys residues and Phe residues at the C-terminus of the protein act in a unique concerted action to strongly anchor the protein in the lipid bilayer.     

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.     

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.     

The art of observing rare protein species in proteomes with peptide ligand libraries

The present review deals with the use of combinatorial ligand libraries, composed by hexapeptides, in the capture and concentration of the low‐abundance proteome. This method, first reported in 2005, is compared with other current methodologies aimed at exploring the “deep proteome”, such as: depletion of high‐abundance proteins (especially in sera and cerebrospinal fluid) by individual or combined antibodies (up to 20 against the most abundant species); narrow (1‐pH‐unit) IPGs (zoom gels) and prefractionation with multicompartment electrolyzers or with Off‐Gel electrophoresis. The physico‐chemical properties of the hexapeptide library are also explored, namely in assessing the proper length of the baits and the behavior of shorter oligopeptides, down to capture elicited by single amino acids. A number of examples on the use of this library is given, such as the analysis of biological fluids (human sera, urine, bile, cerebrospinal fluid) and of cell lysates (platelets, red blood cells). In all cases, it was possible to detected from three to five times as many proteins as compared to control, untreated samples. Perhaps the most spectacular results were obtained with the erythrocyte proteome, where 1570 proteins could be identified in the “minority” proteome, representing only 2% of the total cell lysate. Another interesting area of application regards the concentration and detection of trace impurities contaminanting r‐DNA proteins meant for human consumption: several host proteins, never reported before, could be revealed for the first time. Other nonhuman samples are currently under investigation, such as egg‐white (where no less than 148 unique gene products could be identified), egg yolk (with 255 unique species) and latex from Hevea brasiliensis. It is anticipated that the ligand library could be a most useful tool for detecting biomarkers for different pathologies and for drug treatment. As a future outlook, one could envision the synthesis of specialized libraries able to capture given classes of proteins (e.g., phospho‐ and glyco‐proteins). Additionally, the library could be used in association with other techniques currently in vogue, such as zoom gels, Off‐Gels, and the like.     

Functional and structural consequences of aromatic residue substitutions within the kringle-2 domain of tissue-type plasminogen activator.

Aromatic amino acid residues within kringle domains play important roles in the structural stability and ligand-binding properties of these protein modules. In previous investigations, it has been demonstrated that the rigidly conserved Trp25 is primarily involved in stabilizing the conformation of the kringle-2 domain of tissue-type plasminogen activator (K2tpA), whereas Trp63, Trp74, and Tyr76 function in omega-amino acid ligand binding, and, to varying extents, in stabilizing the native folding of this kringle module. In the current study, the remaining aromatic residues of K2tPA, viz., Tyr2, Phe3, Tyr9, Tyr35, Tyr52, have been subjected to structure-function analysis via site-directed mutagenesis studies. Ligand binding was not significantly influenced by conservative amino acid mutations at these residues, but a radical mutation at Tyr35 destabilized the interaction of the ligand with the variant kringle. In addition, as reflected in the values of the melting temperatures, changes at Tyr9 and Tyr52 generally destabilized the native structure of K2tPA to a greater extent than changes at Tyr2, Phe3, and Tyr35. Taken together, results to date show that, in concert with predictions from the crystal structure of K2tpA, ligand binding appears to rely most on the integrity of Trp63 and Trp74, and aromaticity at Tyr76. With regard to aromatic amino acids, kringle folding is most dependent on Tyr9, Trp25, Tyr52, Trp63, and Tyr76. As yet, no obvious major roles have been uncovered for Tyr2, Phe3, or Tyr35 in K2tpA.     

EPR characterization of free radical intermediates formed during ultrasound exposure of cell culture media

Free radicals and/or hydrogen peroxide produced by exposure of cells to ultrasound are potentially cytotoxic and mutagenic. The formation and type of free radical species can be substantially modulated by the chemical composition of the media in which the ultrasound exposures of cells are carried out. In the current study, we examined the free radical intermediates formed during ultrasound exposure of a typical cell culture medium (RPMI-1640); the dominant free radicals that were identified by spin trapping were derived from the hydrophobic amino acids Trp, Leu, and Phe, and were formed by hydrogen abstraction from these amino acids. Compared to exposures in phosphate-buffered saline, the yield of *OH radicals and H2O2 was significantly reduced in the cell culture medium, glucose (the main organic component in the medium), and the hydrophobic amino acids (Trp, Phe, Tyr, Leu, Val, Met) being chiefly responsible for this effect. In contrast, other nonhydrophobic amino acids did not contribute significantly to the *OH or H2O2 decrease. These findings are consistent with the accumulation of hydrophobic solutes at the liquid-gas interface of the collapsing cavitation bubbles resulting in increased efficiency of radical scavenging.     

Effect of amino acids on X-ray-induced hydrogen peroxide and hydroxyl radical formation in water and 8-oxoguanine in DNA

Generation of hydrogen peroxide and hydroxyl radicals in L-amino acid solutions in phosphate buffer, pH 7.4, under X-ray irradiation was determined by enhanced chemiluminescence in the luminol-p-iodophenol-peroxidase system and using the fluorescent probe coumarin-3-carboxylic acid, respectively. Amino acids are divided into three groups according to their effect on the hydrogen peroxide formation under irradiation: those decreasing yield of H2O2, having no effect, and increasing its yield. All studied amino acids at 1 mM concentration decrease the yield of hydroxyl radicals in solution under X-ray irradiation. However, the highest effect is observed in the order: Cys > His > Phe = Met = Trp > Tyr. At Cys, Tyr, and His concentrations close to physiological, the yield of hydroxyl radicals decreases significantly. Immunoenzyme analysis using monoclonal antibodies to 8-oxoguanine (8-oxo-7,8-dihydroguanine) was applied to study the effect of amino acids with the most pronounced antioxidant properties (Cys, Met, Tyr, Trp, Phe, His, Lys, Arg, Pro) on 8-oxoguanine formation in vitro under X-ray irradiation. It is shown that amino acids decrease the content of 8-oxoguanine in DNA. These amino acids within DNA-binding proteins may protect intracellular DNA against oxidative damage caused by formation of reactive oxygen species in conditions of moderate oxidative stress.     

Site-directed mutagenesis of conserved C-terminal tyrosine and tryptophan residues of PsbO, the photosystem II manganese-stabilizing protein, alters its activity and fluorescence properties

The extrinsic photosystem II PsbO subunit (manganese-stabilizing protein) contains near-UV CD signals from its complement of aromatic amino acid residues (one Trp, eight Tyr, and 13 Phe residues). Acidification, N-bromosuccinimide modification of Trp, reduction or elimination of a disulfide bond, or deletion of C-terminal amino acids abolishes these signals. Site-directed mutations that substitute Phe for Trp241 and Tyr242, near the C-terminus of PsbO, were used to examine the contribution of these residues to the activity and spectral properties of the protein. Although this substitution is, in theory, conservative, neither mutant binds efficiently to PSII, even though these proteins appear to retain wild-type solution structures. Removal of six residues from the N-terminus of the W241F mutant restores activity to near-wild-type levels. The near-UV CD spectra of the mutants are modified; well-defined Tyr and Trp peaks are lost. Characterizations of the fluorescence spectra of the full-length WF and YF mutants indicate that Y242 contributes significantly to PsbO's Tyr fluorescence emission and that an excited-state tyrosinate could be present in PsbO. Deletion of W241 shows that this residue is a major contributor to PsbO's fluorescence emission. Loss of function is consistent with the proposal that a native C-terminal domain is required for PsbO binding and activity, and restoration of activity by deletion of N-terminal amino acids may provide some insights into the evolution of this important photosynthetic protein.     

Optimization of aromatic side chain size complementarity in the hydrophobic core of a designed coiled-coil.

The coiled-coil structure plays an important roles, especially in protein assembly. Previously we constructed AAB-type heterotrimeric coiled-coils by manipulating the packing in the hydrophobic core using Trp and Ala residues, where one Trp and two Ala residues were placed in the hydrophobic core instead of three Ile residues. To optimize the packing complementarity in the hydrophobic core, we investigated the effects of introducing various aromatic amino acids on the formation of an AAB-type heterotrimeric coiled-coil, by circular dichroism, thermal stability, and nuclear magnetic resonance (NMR) studies. We found that the Phe residue was more suitable for heterotrimeric coiled-coil formation than the Trp residue, when combined with two Ala residues, whereas the Tyr and His residues did not induce the coiled-coil structure efficiently.     

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.     

A cluster of aromatic amino acids in the i2 loop plays a key role for Gs coupling in prostaglandin EP2 and EP3 receptors

To assess the structural requirements for G(s) coupling by prostaglandin E receptors (EPs), the G(s)-coupled EP2 and G(i)-coupled EP3beta receptors were used to generate hybrid receptors. Interchanging of the whole i2 loop and its N-terminal half (i2N) had no effect on the binding of both receptors expressed in HEK293 cells. Agonist-induced cAMP formation was observed in wild type EP2 but not in the i2 loop- or i2N-substituted EP2. Wild type EP3beta left cAMP levels unaffected, whereas i2 loop- and i2N-substituted EP3 gained agonist-induced adenylyl cyclase stimulation. In EP2, the ability to stimulate cAMP formation was lost by mutation of Tyr(143) into Ala but retained by mutations into Phe, Trp, and Leu. Consistent with this observation, substitution of the equivalent His(140) enabled EP3beta to stimulate cAMP formation with the rank order of Phe > Tyr > Trp > Leu. The point mutation of His(140) into Phe was effective in another EP3 variant in which its C-terminal tail is different or lacking. Simultaneous mutation of the adjacent Trp(141) to Ala but not at the following Tyr(142) weakened the acquired ability to stimulate cAMP levels in the EP3 mutant. Mutation of EP2 at adjacent Phe(144) to Ala but not at Tyr(145) reduced the efficiency of agonist-induced cAMP formation. In Chinese hamster ovary cells stably expressing G(s)-acquired EP3 mutant, an agonist-dependent cAMP formation was observed, and pertussis toxin markedly augmented cAMP formation. These results suggest that a cluster of hydrophobic aromatic amino acids in the i2 loop plays a key role for G(s) coupling.     

Amino acid- and purine ribonucleoside-induced germination of Bacillus anthracis DeltaSterne endospores: gerS mediates responses to aromatic ring structures

Specific combinations of amino acids or purine ribonucleosides and amino acids are required for efficient germination of endospores of Bacillus anthracis DeltaSterne, a plasmidless strain, at ligand concentrations in the low-micromolar range. The amino acid L-alanine was the only independent germinant in B. anthracis and then only at concentrations of >10 mM. Inosine and L-alanine both play major roles as cogerminants with several other amino acids acting as efficient cogerminants (His, Pro, Trp, and Tyr combining with L-alanine and Ala, Cys, His, Met, Phe, Pro, Ser, Trp, Tyr, and Val combining with inosine). An ortholog to the B. subtilis tricistronic germination receptor operon gerA was located on the B. anthracis chromosome and named gerS. Disruption of gerS completely eliminated the ability of B. anthracis endospores to respond to amino-acid and inosine-dependent germination responses. The gerS mutation also produced a significant microlag in the aromatic-amino-acid-enhanced-alanine germination pathways. The gerS disruption appeared to specifically affect use of aromatic chemicals as cogerminants with alanine and inosine. We conclude that efficient germination of B. anthracis endospores requires multipartite signals and that gerS-encoded proteins act as an aromatic-responsive germination receptor.     

烧伤患者血浆和红细胞游离氨基酸变化及输注氨基酸对其变化的影响

动态测定烧伤患者血浆及红细胞内游离氨基酸的含量 ,探讨输入外源性氨基酸后对血及红细胞内游离氨基酸的影响。以日立 835— 5 0型氨基酸自动分析仪测定烧伤患者血浆及红细胞内游离氨基酸含量。结果发现烧伤患者血浆总游离氨基酸浓度从伤后到 2 1天均显著降低 (P <0 .0 5～ 0 .0 1) ;赖、苯丙和苯丙 酪氨酸比值显著升高 (P <0 .0 5～ 0 .0 1) ;色、组、精、丙、甘、苏、脯和丝氨酸比值显著降低 (P <0 .0 5～ 0 .0 1) ;缬、亮、异亮、酪、胱和支链氨基酸伤后早期降低。烧伤患者红细胞内总游离氨基酸含量不同程度降低 ,其中 1、3、7天降低显著 (P <0 .0 5～ 0 .0 1) ;红细胞内苯丙和苯丙 酪氨酸比值未见显著性升高 ;色、蛋、精、脯氨酸含量很低或基本未测出。输注复合氨基酸注射液后未能显著改善患者血及红细胞内游离氨基酸含量。结果提示烧伤患者红细胞内游离氨基酸含量的变化趋势与血浆游离氨基酸变化趋势基本一致 ;烧伤后红细胞内苯丙氨酸及苯丙 酪氨酸比值有别于血浆变化。本研究条件下补充外源性氨基酸未能显著改变烧伤患者血浆及红细胞内游离氨基酸的含量     

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.     

Studies of fluorescent components of the “heparin” drug and its complexing with phenylalanine, tyrosine, and tryptophan

Impurities of free aromatic amino acids (Phe and Tyr) and the elastin protein were found in the heparin commercial drug (Hep) by spectral luminescent and spectrophotometric methods. The fluorescence quenching of the Trp, Tyr, and Phe amino acids by the Hep drug was studied, and the Stern-Folmer constants (K) that reflected stability of the Hep complexes with amino acids were determined. The stability of AA-Hep complexes increased in the following sequence: Trp < Tyr < Phe (K = 19 ± 2 < 39 ± 3 < 710 ± 70 M^?1, respectively). These values probably determined the dominant contribution of the phenylalanine impurity in the heparin drug. The contamination of animal elastin whose structure differed from that of the human elastin is thought to be a reason for allergic reactions and even anaphylactic shock during medical treatment with this drug.     

Human liver alanine aminopeptidase. Inhibition by amino acids.

Human liver alanine aminopeptidase is inhibited by L-amino acids having hydrophobic side chains such as Phe, Tyr, Trp, Met, and Leu. Blocking of the amino group or the carboxyl group greatly reduces the inhibitory capacity of the amino acid. Kinetic studies demonstrate that inhibition of hydrolysis of the substrate L-Ala-beta-naphthylamide is of the noncompetitive type. Inhibition of the substrate L-Leu-L-Leu is of the mixed type. Inhibition of the substrate L-Ala-L-Ala-L-Ala is of the competitive type. These changes in the mechanism of inhibition are thought to be the result of the binding of the amino acid to the third residue binding site on the enzyme. This is the part of the active center to which the third residue from the amino end of a peptide substrate is normally bound. The inhibitor constants of several alanine oligopeptides are shown to decrease with increasing length through L-Ala-L-Ala-L-Ala-L-Ala, demonstrating that alanine aminopeptidase is a multisited enzyme with three and possibly four residue sites per active center. The inhibitor constant for Gly-Gly--Phe suggesting that indeed the third residue site preferentially binds large hydrophobic residues.     

Effect of pH, urea, peptide length, and neighboring amino acids on alanine alpha-proton random coil chemical shifts

Accurate random coil alpha-proton chemical shift values are essential for precise protein structure analysis using chemical shift index (CSI) calculations. The current study determines the chemical shift effects of pH, urea, peptide length and neighboring amino acids on the alpha-proton of Ala using model peptides of the general sequence GnXaaAYaaGn, where Xaa and Yaa are Leu, Val, Phe, Tyr, His, Trp or Pro, and n = 1-3. Changes in pH (2-6), urea (0-1M), and peptide length (n = 1-3) had no effect on Ala alpha-proton chemical shifts. Denaturing concentrations of urea (8M) caused significant downfield shifts (0.10 +/- 0.01 ppm) relative to an external DSS reference. Neighboring aliphatic residues (Leu, Val) had no effect, whereas aromatic amino acids (Phe, Tyr, His and Trp) and Pro caused significant shifts in the alanine alpha-proton, with the extent of the shifts dependent on the nature and position of the amino acid. Smaller aromatic residues (Phe, Tyr, His) caused larger shift effects when present in the C-terminal position (approximately 0.10 vs. 0.05 ppm N-terminal), and the larger aromatic tryptophan caused greater effects in the N-terminal position (0.15 ppm vs. 0.10 C-terminal). Proline affected both significant upfield (0.06 ppm, N-terminal) and downfield (0.25 ppm, C-terminal) chemical shifts. These new Ala correction factors detail the magnitude and range of variation in environmental chemical shift effects, in addition to providing insight into the molecular level interactions that govern protein folding.     

Exploring the platelet proteome via combinatorial, hexapeptide ligand libraries

A combinatorial ligand library, composed of millions of diverse hexapeptide baits, able to capture and concentrate the "low-abundance" proteome while drastically cutting the concentration of the most abundant species, has been applied to the exploration of the soluble platelet proteome. Mass spectrometry analysis of untreated and library-treated platelets has resulted in the identification of 435 unique gene products. Of those, 147 entries (35% of the total) have not been described among the list of >1100 proteins in proteomic platelet investigations reported before. In addition, the analysis of excised spots from two-dimensional electrophoresis analysis allowed 57 other proteins to be added that were not found in LC-MS analysis, 33 of them not described before in proteomics studies, bringing the total number of new gene products to 180. Thus, the present data add a non-negligible number of species for continuing the "cartography" of the proteomic asset of platelets, in view of completing the mapping procedure for a deeper understanding of the physiology and pathology of this blood cell. Because the capturing process is performed under physiological conditions, by exploiting, for binding to the combinatorial library, the native protein configuration, the described technique is not adapted to capture highly hydrophobic proteins, which need strong denaturing and solubilizing conditions that are incompatible with our working procedure. Thus, our list reports essentially hydrophilic proteins, with negative GRAVY indexes.