Constituent Amino Acid Residues Research Articles

Effects of alkali or acid treatment on the isomerization of amino acids

The effect of alkali treatment on the isomerization of amino acids was investigated. The 100×D/(D+L) values of amino acids from peptide increased with increase in the number of constituent amino acid residues. Furthermore, the N-terminal amino acid of a dipeptide was isomerized to a greater extent than the C-terminal residue.

Inactivation and reactivation of ribonuclease A studied by computer simulation

The year 2011 marked the half-centenary of the publication of what came to be known as the Anfinsen postulate, that the tertiary structure of a folded protein is prescribed fully by the sequence of its constituent amino acid residues. This postulate has become established as a credo, and, indeed, no contradictions seem to have been found to date. However, the experiments that led to this postulate were conducted on only a single protein, bovine ribonuclease A (RNAse). We conduct molecular dynamics (MD) simulations on this protein with the aim of mimicking this experiment as well as making the methodology available for use with basically any protein. There have been many attempts to model denaturation and refolding processes of globular proteins in silico using MD, but only a few examples where disulphide-bond containing proteins were studied. We took the view that if the reductive deactivation and oxidative reactivation processes of RNAse could be modelled in silico, this would provide valuable insights into the workings of the classical Anfinsen experiment.

Polycationic Gramicidin S Analogues with Both High Antibiotic Activity and Very Low Hemolytic Activity

The substitution of each constituent amino acid residue of gramicidin S (GS), cyclo(-Val(1,1')-Orn(2,2')-Leu(3,3')-D-Phe(4,4')-Pro(5,5')-)(2) with Lys residue indicated that each side chain structure of the constituent amino acid residues affect largely the antibiotic activity and hemolytic activity of GS. Further, the substitution of D-Phe(4,4') and Pro(5,5') residues with basic amino acid residues as a Lys residue results the high antibiotic activity and the very low hemolytic activity. Thus, we have found novel positions on the scaffold of GS at D-Phe(4,4') and Pro(5,5') residues whose modification will significantly increase the therapeutic index.

Wenxiang: a web-server for drawing wenxiang diagrams

The wenxiang diagram was proposed to represent α-helices in a 2D (two dimensional) space (Chou, K.C., Zhang, C.T., Maggiora, G.M. Proteins: Struct., Funct., Genet., 1997, 28, 99-108). It has the capacity to provide more information in a 2D plane about each of the constituent amino acid residues in an α-helix, and is particularly useful for studying and analyzing amphiphilic helices. To meet the increasing requests for getting the program of generating wenxiang diagrams, a user-friendly web-server called “Wenxiang” has been established. It is accessible to the public at the web-site http://www.jci-bioinfo.cn/wenxiang2 or http://icpr.jci.edu.cn/bioinfo/wenxiang2. Further- more, for the convenience of users, here we provide a step-to-step guide for how to use the Wenxiang web-server to generate the desired wenxiang diagrams.

Novel gratisin derivatives with high antimicrobial activity and low hemolytic activity

The substitution of each constituent amino acid residue of gratisin (GR) with Ala residue indicated that each side chain structure of the constituent amino acid residues affect largely the antibiotic and hemolytic activities of GR. Among them, the substitution of Pro residues at positions 5 and 5′ with a cationic amino acid residues (Lys and Arg) results the high antibiotic activity and the low toxicity against human blood cells. Thus, we have found a novel position on the scaffold of GR at Pro5,5′ residues whose modification will significantly lower the unwanted hemolytic activity and enhance the desired antibiotic activity.

Stability of the Heme Fe−N-Terminal Amino Group Coordination Bond in Denatured Cytochrome c

In the denatured states of Hydrogenobacter thermophilus cytochrome c(552) (HT) and Pseudomonas aeruginosa cytochrome c(551) (PA), and their mutants, the N-terminal amino group of the polypeptide chain is coordinated to heme Fe in place of the axial Met, the His-N(term) form being formed. The coordination of the N-terminal amino group to heme Fe leads to loop formation by the N-terminal stretch preceding the first Cys residue bound to the heme, and the N-terminal stretches of HT and PA are different from each other in terms of both the sequence and the number of constituent amino acid residues. The His-N(term) form was shown to be rather stable, and hence it can influence the stability of the denatured state. We have investigated the heme Fe coordination structures and stabilities of the His-N(term) forms emerging upon guanidine hydrochloric acid-induced unfolding of the oxidized forms of the proteins. The Fe-N(term) coordination bond in the His-N(term) form with a 9-residue N-terminal stretch of HT proteins was found to be tilted to some extent away from the heme normal, as reflected by the great heme methyl proton shift spread. On the other hand, the small heme methyl proton shift spread of the His-N(term) form with an 11-residue stretch of PA proteins indicated that its Fe-N(term) bond is nearly parallel with the heme normal. The stability of the His-N(term) form was found to be affected by the structural properties of the N-terminal stretch, such as its length and the N-terminal residue. With a given N-terminal residue, the stability of the His-N(term) form is higher for a 9-residue N-terminal stretch than an 11-residue one. In addition, with a given length of the N-terminal stretch, the His-N(term) form with an N-terminal Glu is stabilized by a few kJ mol(-1) relative to that with an N-terminal Asn. These results provide a novel insight into the stabilizing interactions in the denatured cyts c that will facilitate elucidation of the folding/unfolding mechanisms of the proteins.

Computational analysis of the binding affinities of the natural-product cyclopentapeptides argifin and argadin to chitinase B from Serratia marcescens

Molecular dynamics (MD) simulations and the molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) method were applied to study the interaction of the natural-product cyclopentapeptide chitinase inhibitors argifin and argadin with chitinase B (ChiB) from Serratia marcescens. Argadin inhibited ChiB with an inhibition constant ( K i) value of 20 nM, which was three orders of magnitude greater than that of argifin ( K i = 33,000 nM). The MM-PBSA free-energy analysis provided absolute binding free energies of −6.98 and −11.16 kcal/mol for the argifin and argadin complexes, respectively. These estimates were in good agreement with the free energies derived from the experimental K i values (−6.36 and −10.92 kcal/mol for the argifin and argadin complexes, respectively). The energetic analysis revealed that the van der Waals and nonpolar solvation energies drove the binding of both argifin and argadin. We found that the binding of argadin gained ∼12 kcal/mol more van der Waals energy than that of argifin, which was mainly responsible for the difference in binding free energy between argifin and argadin. In particular, W220 and W403 of ChiB were found to contribute to the more favorable van der Waals interaction with argadin. We also designed argifin derivatives with better binding affinity, in which a constituent amino-acid residue of argifin was mutated to one with a bulky side chain. The derivative in which d-Ala of argifin was replaced with d-Trp appeared to possess a binding affinity that was equally potent to that of argadin.

Theoretical exploration of the cooperative effect in NMF–NMF–amino acid residue hydrogen bonding system

This paper presents a theoretical study of the cooperative effect in sixteen linearly-arranged trimer systems consisting of N-methylformamide dimer and an extra amino acid residue. These trimer systems, NMF-NMF-AAR, in short, have been systematically investigated by full optimization at B3LYP/cc-pVTZ level and subsequent electronic energy calculations at PBE1PBE/cc-pVTZ, HF/cc-pVTZ and MP2/cc-pVTZ, respectively. Obvious spatial transformation due to energetic factors has been found in almost all the trimers. Systematic analysis in weak interaction energy components has shown that: (1) in these trimer systems, the bonding structure and the cooperative effect combine to determine the stability of both HB1 and HB2. For HB2, the structure of the constituent amino acid residue also plays a crucial role by interfering with the neighboring moieties; (2) the large contribution of the cooperative effect to the overall hydrogen bonding energy has claimed the importance of cooperativity in our systems; (3) the non-hydrogen bonding weak interaction components are found to be non-negligible in these trimer systems; (4) moreover, the cooperative effect between these non-hydrogen bonding components is always found to be positive. The good performances of PBE1PBE and PM6 have been established by comparisons between these methods.

Functional preference of the constituent amino acid residues in a phage-library-based nonphosphorylated inhibitor of the Grb2-SH2 domain.

A nonphosphorylated disulfide-bridged peptide, cyclo(Cys-Glu1-Leu-Tyr-Glu-Asn-Val-Gly-Met-Tyr9-Cys)-amide (termed G1) has been identified, by phage library, that binds to the Grb2-SH2 domain but not the src SH2 domain. Synthetic G1 blocks the Grb2-SH2 domain association (IC50 of 15.5 microM) with natural phosphopeptide ligands. As a new structural motif that binds to the Grb2-SH2 domain in a pTyr-independent manner, the binding affinity of G1 is contributed by the highly favored interactions of its structural elements interacting with the binding pocket of the protein. These interactions involve side-chains of amino acids Glu1, Tyr3, Glu4, Asn5, and Met8. Also a specific conformation is required for the cyclic peptide when bound to the protein. Ala scanning within G1 and molecular modeling analysis suggest a promising model in which G1 peptide binds in the phosphotyrosine binding site of the Grb2-SH2 domain in a beta-turn-like conformation. Replacement of Tyr3 or Asn5 with Ala abrogates the inhibitory activity of the peptide, indicating that G1 requires a Y-X-N consensus sequence similar to that found in natural pTyr-containing ligands, but without Tyr phosphorylation. Significantly, the Ala mutant of Glu1, i.e. the amino acid N-terminal to Y3, remarkably reduces the binding affinity. The position of the Glu1 side-chain is confirmed to provide a complementary role for pTyr3, as demonstrated by the low micromolar inhibitory activity (IC50 = 1.02 microM) of the nonphosphorylated peptide 11, G1(Gla1), in which Glu1 was replaced by gamma-carboxy-glutamic acid (Gla).

Functional and Structural Roles of Constituent Amino Acid Residues of Bovine Pancreatic Ribonuclease A

In protein engineering, wherein the goal is desirable function and high conformational stability, the characteristics of each constituent amino acid residue are important, in terms of the overall characteristics of the target protein. Bovine pancreatic riobonuclease A (RNase A) is, historically, one of the most intensively analyzed proteins, and a considerable amount of information is available on amino acid-level information. Such data would serve to aid the understanding of relationships between the distribution of various amino acid residues in the protein molecule and the unique structure and/or functions of RNase A. This review summarizes the thus-far clarified roles of 38 of the total 124 amino acid residues which comprise RNase A, with respect to protein function, stability, and folding.

Does in-source decay occur independent of the ionization process in matrix-assisted laser desorption?

The influence of the acidic and basic characters of constituent amino acid residues on the peptide fragment ions produced by in-source decay under matrix assisted laser desorption/ionization (MALDI) conditions has been studied using positive- and negative-ion experiments. Whereas the in-source decay spectra of peptides containing basic Arg and/or Lys residues near the N-terminus showed so-called c n - and a n -series ions in positive-ion mode, a peptide that has an acidic amino acid cluster near the N-terminus and a basic residue near the C-terminus characteristically formed y n - and z n -series ions in the positive-ion in-source decay spectrum. These results indicated that fragment ion series produced by in-source decay depend strongly upon the acidic and basic characters of the constituent amino acid residues and the near N- and C-termini. It was suggested that in-source decay processes occur intrinsically at NH–C α and CO–NH bonds independent of the formation of molecular-related ions, and that the cleavages at the NH–C α and CO–NH bonds occurred independently and were dependent on the matrix used.

An intriguing controversy over protein structural class prediction.

A recent report by Bahar et al. [(1997), Proteins 29, 172-185] indicates that the coupling effects among different amino acid components as originally formulated by K. C. Chou [(1995), Proteins 21, 319-344] are important for improving the prediction of protein structural classes. These authors have further proposed a compact lattice model to illuminate the physical insight contained in the component-coupled algorithm. However, a completely opposite result was concluded by Eisenhaber et al. [(1996), Proteins 25, 169 179], using a different dataset constructed according to their definition. To address such an intriguing controversy, tests were conducted by various approaches for the datasets from an objective database, the SCOP database [Murzin et al. (1995), J. Mol. Biol. 247, 536-540]. The results obtained by both self-consistency and jackknife tests indicate that the overall rates of correct prediction by the algorithm incorporating the coupling effect among different amino acid components are significantly higher than those by the algorithms without counting such an effect. This is fully consistent with the physical reality that the folding of a protein is the result of a collective interaction among its constituent amino acid residues, and hence the coupling effects of different amino acid components must be incorporated in order to improve the prediction quality. It was found by a revisiting the calculation procedures by Eisenhaber et al. that there was a conceptual mistake in constructing the structural class datasets and a systematic mistake in applying the component-coupled algorithm. These findings are informative for understanding and utilizing the component-coupled algorithm to study the structural classes of proteins.

Dicarboxylic Oligopeptide Bolaamphiphiles: Proton-Triggered Self-Assembly of Microtubes with Loose Solid Surfaces

A new family of oligopeptide-based bolaamphiphiles, glycylglycine- (1a−h), glycylglycylglycine- (2a−b), sarcosylsarcosine- (3), l-prolyl-l-proline- (4), glycylsarcosylsarcosine- (5), and glycyl-l-prolyl-l-proline (6)-based bolaamphiphiles with a dicarboxylic headgroup at each end, has been synthesized. The oligopeptide fragments were linked via amide bond to a long-chain α,ω-dicarboxylic acid as a hydrocarbon spacer. Self-assembling properties of these bolaamphiphiles in water have been studied by light and cryogenic temperature transmission electron microscopy, infrared spectroscopy, and pH titration. Only sodium or potassium salts (acid soap) of the bolaamphiphiles 1a, 1c, 1e, 2a, and 2b produced well-defined microtubes of 1−3-μm diameter with closed ends. All the tubes encapsulated a number of vesicular assemblies inside the aqueous compartment. The tube formation strongly depends on the connecting alkylene chain length, the alkylene even−odd carbon numbers, and constituent amino acid residues. Vectorial formation of acid−anion dimers and loose interpeptide hydrogen-bond networks are responsible for the microtube self-assembly. The atomic force microscopic observation of the microtube made of 1e revealed a distorted hexagonal arrangement of the headgroups on the surface. A self-assembling model and the tube formation mechanism are also discussed from the viewpoint of proton-triggered self-assembly.

A priori and a posteriori mixture distributions for using databases in protein structure determination

The twists and turns of protein molecules correspond to the rotational angles of the main and side chains in their constituent amino acid residues. Owing to stereochemical restrictions the joint distribution of these angles falls into several well-separated clusters. Consequently, we fit a statistical finite mixture model to data from the Brookhaven Protein Data Bank (PDB), obtaining a new classification of conformational states of amino acids. The results of this database modeling are important for knowledge-based approaches to protein structure determination and analysis and can be used in conjunction with experimental data in the determination of protein spatial structure. As part of this process, the mixture distributions we have fit can be used as a priori distributions. A posteriori distributions corresponding to these a priori mixture distributions are illustrated.

Reactivity of singlet oxygen toward proteins: the effect of structure in basic pancreatic trypsin inhibitor and in ribonuclease A.

The reactions of singlet oxygen, 1O2, with large peptides have been described previously. It was found that even in these systems, which in their native form are generally not supposed to possess a stable structure in solution, the polypeptide does impede the access of 1O2 to the amino acids that react readily with 1O2. Here we describe the 1O2 reaction with two proteins of well-defined structure. The quenching of 1O2 by bovine pancreatic trypsin inhibitor (BPTI) and by ribonuclease A (RNase A) was compared to that of a solution at the same concentration as those of its constituent amino acids that react readily with 1O2. The proteins were studied in their native form, when partly denatured by splitting their S-S bonds and when fully denatured. It was found that while in the native form the quenching rate constant was seven times lower in BPTI (2.2 vs 15.2 x 10(7) M-1 s-1) and three times lower in RNase A (11.0 vs 32 x 10(7) M-1 s-1) than in a mixture of its constituent amino acid residues, it increased upon denaturation reaching in the fully denatured state the value of the corresponding amino acid mixture. More striking is the effect of the protein structure when comparing the fraction of the encounters between 1O2 and protein, which cause damage to the protein, as reflected in the decrease of its biological activity. This decrease is assumed to be due to the chemical (oxidative) reactions of 1O2 in the protein. In the exceptionally stable BPTI the fraction of such encounters was 0.05 and in RNase A it was 0.2, whereas for the amino acid tryptophan in solution, 0.7 of the collisions with 1O2 led to a chemical reaction.

Removal of N-terminal formyl groups and deblocking of pyrrolidone carboxylic acid of proteins with anhydrous hydrazine vapor.

Many proteins have a blocked alpha-amino group which renders them inaccessible to sequence analysis by the classical Edman degradation procedure. Blockage typically occurs when the alpha-amino groups are acylated with acetyl or formyl groups or when the N-terminal residue is pyrrolidone carboxylic acid formed by cyclization of glutamine. We have found that N-formyl groups of proteins and peptides can be removed by exposure to hydrazine vapor at -5 degrees C for 8 h. Under these conditions, peptide-bond cleavage or modification of the constituent amino-acid residues does not occur. Deblocking of N-terminal pyrrolidone carboxylate residues by conversion to gamma-hydrazidyl glutamic acid can be achieved by exposure to hydrazine vapor at 20 degrees C for 4 h. These conditions cause partial modification of asparagine and glutamine residues to their corresponding hydrazides, and conversion of arginine residues to ornithine.

An investigation of dipeptides containing polar and nonpolar side groups by curie-point pyrolysis tandem mass spectrometry

Methionyl-leucine, leucyl-methionine, phenylalanyl-leucine, and leucyl-phenylalanine have been analyzed to determine dipeptide fragmentation mechanisms in Curie-point pyrolysis tandem mass spectrometry. Results show that fragmentations of dipeptides follow two general pathways, one involving direct cleavage of the dipeptide and the other involving cyclization of the dipeptide. Unique products and strong changes in relative mass spectral peak intensities arise, depending on constituent amino acid residues and their sequence. Also, the length and nature of the side groups strongly direct fragmentation. From these results, the major peaks in the spectra of eight other dipeptides could be readily explained; this suggests that a significant number of dipeptides follow the same general fragmentation mechanisms.

Factors affecting peptide catabolism by oral streptococci

The binding of a number of unsubstituted peptides to Streptococcus sanguis and Streptococcus mutans and their subsequent degradation by such cells were examined. Peptides were added to cell suspensions prepared from glucose-limited growth in a chemostat and, at appropriate time intervals, cell-free filtrates were analyzed for peptides and their constituent amino acid residues by high-pressure liquid chromatography techniques. The results indicated that peptide hydrophobicity plays a limited role in peptide binding, but that charge and chain-length are probably important. In S. sanguis, carboxypeptidase activity rapidly released C-terminal arginine (Arg); this amino acid was less rapidly released from the N-terminus but a number of other residues were also released by aminopeptidase activity. When Arg is buried in the peptide, the rate of its release depends upon the number and type of residues between it and the N-terminus. In contrast, S. mutans possessed only weak peptidase activities. The nature of its peptidase activities indicates that S. sanguis can obtain the metabolically important Arg from a variety of peptides.

Revised structure of the peptide lactone antibiotic, TL-119 and/or A-3302-B

The peptide lactone antibiotic TL-119 and/or A-3302-B was chemically synthesized in order to confirm the proposed structure. The synthetic compound was different from both natural TL-119 and A-3302-B in their physicochemical properties and in biological activity. Re-examination of the configuration of the constituent amino acid residues in natural TL-119 and/or A-3302-B indicated that natural TL-119 and A-3302-B contains D-aThr instead of the original L-Thr. We tentatively propose a revised structure for TL-119 and/or A-3302-B.

Heat capacity of proteins: II. Partial molar heat capacity of the unfolded polypeptide chain of proteins: Protein unfolding effects

Using the heat capacity values for amino acid side-chains and the peptide unit determined in the accompanying paper, we calculated the partial heat capacities of the unfolded state for four proteins (apomyoglobin, apocytochrome c, ribonuclease A, lysozyme) in aqueous solution in the temperature range from 5 to 125 degrees C, with an assumption that the constituent amino acid residues contribute additively to the integral heat capacity of a polypeptide chain. These ideal heat capacity functions of the extended polypeptide chains were compared with the calorimetrically determined heat capacity functions of the heat and acid-denatured proteins. The average deviation of the experimental functions from the calculated ideal ones in the whole studied temperature range does not exceed the experimental error (5%). Therefore, the heat-denatured state of a protein, in solutions with acidic pH preventing aggregation, approximates well the completely unfolded state of this macromolecule. The heat capacity change caused by hydration of amino acid residues upon protein unfolding was also determined and it was shown that this is the major contributor to the observed heat capacity effect of unfolding. Its value is different for different proteins and correlates well with the surface area of non-polar groups exposed upon unfolding. The heat capacity effect due to the configurational freedom gain by the polypeptide chain was found to contribute only a small part of the overall heat capacity change on unfolding.