Types Of Amino Acid Residues Research Articles

Protein methylation

Proteins can be enzymatically modified in several ways by the addition of methyl groups from S-adenosylmethionine. Reactions forming methyl esters on carboxyl groups are potentially reversible and can modulate the activity of the target protein; in the past year, advances have been made in understanding the physiological roles of four distinct systems that modify normal and abnormal carboxyl groups on proteins. On the other hand, methylation reactions occurring on nitrogen atoms in N-terminal and side-chain positions are generally irreversible. These reactions create new types of amino acid residues and can expand the repertoire of chemistry that a protein can perform.

The kinetics of an enzyme catalyzed reaction in the presence of an unstable, irreversible modifier

1.1. The chemical modifications of enzymes is a widely used technique to gain information about the number, type and location of aminoacid residues which are essential for activity. A number of these modifiers are unstable in aqueous solutions and act irreversibly.2.2. The kinetics of such systems have previously been studied under assumptions, some of which are either unnecessary or too restrictive.3.3. We replace these assumptions by others which are more realistic and less stringent.4.4. These assumptions allow us to derive analytical expressions for the evolution of all the species involved in the reaction.5.5. From the analytical expression for the time course of the product formation an experimental design and a kinetic data analysis allowing the easy characterization of these systems are suggested.

Characterization of the inhibitor sensitivity of the coenzyme A transport system in isolated rat heart mitochondria.

The effect of protein labeling agents on coenzyme A (CoA) transport into isolated rat heart mitochondria was studied. CoA transport was substantially inhibited by sulfhydryl reagents (mersalyl, pCMB) as well as by the tyrosine-selective reagent N-acetylimidazole. The effect of pCMB was reversed by DTT. Moreover, CoA uptake was completely abolished by agents selective for lysine and amino terminal residues (pyridoxal 5-phosphate, dansyl chloride). In contrast arginine-selective reagents (2, 3-butanedione, phenylglyoxal) caused considerably less inhibition of CoA uptake. Moreover, partial inhibition of transport was observed with the stilbene disulfonic acid derivatives DIDS and SITS. Finally, measurement of the effects of the labeling agents on the mitochondrial membrane potential indicated that the inhibition of CoA transport into mitochondria is not a secondary effect that arises from an alteration in the electric potential gradient across the inner mitochondrial membrane. These results provide the first information on the types of amino acid residues that may be essential to the CoA transport mechanism and provide additional support for the existence of a CoA transport protein within the mitochondrial inner membrane. Furthermore, the identification of effective inhibitors of the CoA transport system will greatly facilitate the functional reconstitution of this transporter in a proteoliposomal system following its solubilization and purification.

Mechanism of inhibition of cholinesterases by huperzine A

Huperzine A, an alkaloid isolated from Huperzia serrata was found to reversibly inhibit acetylcholinesterases (EC 3.1.1.7) and butyrylcholinesterases (EC 3.1.1.8) with on- and off-rates that depend on both the type and the source of enzyme. Long-term incubation of high concentrations of purified cholinesterases (1–8 μM) with huperzine A did not show any chemical modification of huperzine A. A low dissociation constant K I was obtained for mammalian acetylcholinesterase-huperzine (20–40 nM) compared to mammalian butyrylcholinesterase-huperzine (20–40 μM.) This indicates that the thermodynamic stability of huperzine-cholinesterase complex may depend on the number and type of aromatic amino acid residues in the catalytic pocket region of the cholinesterase molecule.

A 1H-15N NMR study of human c-Ha-ras protein: biosynthetic incorporation of 15N-labeled amino acids.

A 1H-15N NMR study was performed on the GDP-bound form of a truncated human c-Ha-ras oncogene product (171 amino acid residues). Resonance cross peaks of the backbone amide 1H-15N nuclei of a uniformly 15N-labeled protein were observed with heteronuclear single-quantum coherence spectroscopy (HSQC). In order to resolve overlapping cross peaks, selective 15N-labeling of one or two types of amino acid residues (Ala, Arg, Asx, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Tyr and/or Val) was carried out using appropriate E. coli mutant strains. By this procedure, all the backbone 1H-15N cross peaks were classified into amino acid types.

Transfer of oxygen from an artificial protease to peptide carbon during proteolysis.

Site-specific cleavage of proteins with metal chelates is an approach for designing artificial proteolytic reagents that are directed by proximity to a peptide bond rather than by an amino acid residue type. In the presence of ascorbate and H2O2, an iron chelate attached to Cys-212 of the enzyme human carbonic anhydrase I quickly cleaved the protein between residues Leu-189 and Asp-190 to produce two discrete fragments. The transfer of an 18O atom from [18O]H2O2 (or [18O]O2) to the carboxyl group of Leu-189 was demonstrated by mass spectrometry. Quantitative experiments revealed that one molecule of H2O2 and one molecule of ascorbate afforded the hydrolysis of one peptide bond (1:1:1 stoichiometry) and that the reaction required ascorbate and H2O2. The process is catalytic, since related experiments on the protein bovine serum albumin revealed two cleavage events for each polypeptide chain cleaved. Hydroxyl radical scavengers had no significant effect. These results may be explained by generation of a highly nucleophilic oxygen species, such as peroxide coordinated to the iron chelate, that attacks a carbonyl carbon nearby.

Chemical modification of Bacillus thuringiensis subsp. thuringiensis (HD-524) trypsin-activated endotoxin: Implication of tyrosine residues in lepidopteran cell lysis

A purified protein fraction from a solubilized and trypsin-digested extract of Bacillus thuringiensis subsp. thuringiensis (HD-524) fermentation powder was lytic to cells from several lepidopteran lines. Maximum yield was obtained by alkaline carbonate-thiocyanate solubilization of washed powder followed by trypsin digestion and Sephacryl (S-300) chromatography. The alkaline carbonate-solubilized fraction consisted predominantly of two bands on sodium dodecyl sulfatepolyacrylamide gel electrophoresis with MW of 144 ± 0.9 kDa and 134 ± 1.4 kDa. After trypsin treatment and column chromatography, the cytolytic fraction consisted of a major band with a MW of 60.0 ± 1.8 kDa and a minor band of 69 ± 0.9 kDa. Cells from Trichoplusia ni (TN368) were most susceptible to lysis with 50% of cells lysed at 3 μg/ml, followed by Spodoptera frugiperda cells (SF21AE) exhibiting 50% cell lysis at 5 μg/ml and Lymantria dispar cells (Ld652Y) showing 40% lysis at 10 μg/ml. Chemical modification of the polypeptides was performed to determine the role of certain amino acid residues in the cytolytic activity. The group-specific reagent tetranitromethane was used to nitrate and oxidize tyrosine and cysteine residues, respectively. Cysteine residues alone were also modified with p-hydroxymercuribenzoic acid. Lysine residues were modified with O-methylisourea. Of the three types of amino acid residues, only the modification of tyrosine resulted in reduced cell lysis.

Modeling of agonist binding to the ligand‐gated ion channel superfamily of receptors

A generalized model is presented of agonist binding to ligand-gated ion channels (LGICs). Broad similarity in the structure of agonists suggests that the binding sites of LGICs may have evolved from a protobinding site. Aligned sequence data identified as a candidate for such a site a highly conserved 15 residue stretch of primary structure in the N-terminal extracellular region of all known LGIC subunits. We modeled this subregion, termed the cys-loop, as a rigid, amphiphilic beta-hairpin and propose that it may form a major determinant of a conserved structural binding cleft. In the model of the binding complex (1) an invariant aspartate residue at position 11 of the cys-loop is the anionic site interacting with the positively charged amine group of agonists, (2) a local dipole within the pi-electron system of agonists is favorably oriented in the electrostatic field of the invariant aspartate, (3) the epsilon ring-proton of a conserved aromatic residue at the turn of the cys-loop interacts orthogonally with the agonist pi-electron density at its electronegative center, and (4) selective recognition is partly a result of the type of amino acid residue at position 6 of the cys-loop. Additionally, formation of a hydrogen bond between the electronegative atom of the pi-electron system of agonist and a complementary group in the receptor may be important in the high-affinity binding of agonists.

Nitrogen-15 NMR spectroscopy of proteins in solution

Experimental results from 15N NMR studies of selectively and uniformly labeled proteins with molecular weights ranging between 6000 and 155,000 Da in solution are described. These results demonstrate the resolution available in one- and two-dimensional 15N NMR spectra as well as the selection of particular 15N and 1H resonances on the basis of the type of amino acid residue, the number of hydrogens bonded to a nitrogen, local dynamics, and amide hydrogen exchange kinetics.

Domain-specific bias in arginine/lysine usage by protein toxins

The content of lysine and arginine residues in a number of A-B type protein toxins has been examined. It is found that the A subunit, or its equivalent, often shows a strong bias in the type of basic amino acid residue used tending towards nearly exclusive use of either arginine or lysine rather than use of both, whereas the B subunit or its equivalent shows no such bias. Although arginine codons are GC-rich and lysine codons are AT-rich, the content of GC and AT in the genes coding for the toxins does not adequately explain this bias. Other explanations are discussed, including the possibility that the bias is linked to catalytic function or membrane interaction. Understanding this bias may yield valuable insights into toxin structure and function. Furthermore, identification of bias in sequences may be a useful tool for identifying new toxins and their domains.

Ligand-binding effects on the kringle 4 domain from human plasminogen: a study by laser photo-CIDNP 1H-NMR spectroscopy

Photo-chemically induced dynamic nuclear polarization (photo-CIDNP) one-dimensional and two-dimensional (2D) 1H-NMR techniques have been applied to the study of the kringle 4 domain of human plasminogen both ligand-free and complexed to the antifibrinolytic drugs ɛ-aminocaproic acid and p-benzylaminesulfonic acid (BASA). A number of aromatic side-chains (His 3, Trp 72, Tyr 41, Tyr 50 and Tyr 74) appear to be exposed and accessible to 3- N-car☐ymethyl-lumiflavin, the photopolarizing flavin dye, both in the presence and in the absence of ligands. A lesser exposure is observed for the Trp 25 and Trp 62 indole groups in the presence of BASA. The spin-spin (J-coupling) and dipolar (Overhauser) connectivities in the 2D experiments afford absolute assignment of aromatic resonances for the above residues, as well as of those stemming from the Trp 72 ring in the presence of BASA. Moreover, a number of H β resonances can be identified and sorted according to specific types of amino acid residues.

Sequential proton NMR assignments and secondary structure of hen egg white lysozyme in solution

Assignments for 1H NMR resonances of 121 of the 129 residues of hen egg white lysozyme have been obtained by sequence-specific methods. Spin systems were identified with phase-sensitive two-dimensional (2-D) correlated spectroscopy and single and double relayed coherence transfer spectroscopy. For key types of amino acid residues, particularly alanine, threonine, valine, and glycine, complete spin systems were identified. For other residues a less complete definition of the spin system was found to be adequate for the purpose of sequential assignment. Sequence-specific assignments were achieved by phase-sensitive 2-D nuclear Overhauser enhancement spectroscopy (NOESY). Exploitation of the wide range of hydrogen exchange rates found in lysozyme was a useful approach to overcoming the problem of spectral overlap. The sequential assignment was built up from 21 peptide segments ranging in length from 2 to 13 residues. The NOESY spectra were also used to provide information about the secondary structure of the protein in solution. Three helical regions and two regions of beta-sheet were identified from the NOESY data; these regions are identical with those found in the X-ray structure of hen lysozyme. Slowly exchanging amides are generally correlated with hydrogen bonding identified in the X-ray structure; a number of exceptions to this general trend were, however, found. The results presented in this paper indicate that highly detailed information can be obtained from 2-D NMR spectra of a protein that is significantly larger than those studied previously.

Sequence specific assignment of the proton nuclear magnetic resonance spectrum of barley serine proteinase inhibitor 2

The sequence specific assignment of the1H NMR spectrum of barley serine proteinase inhibitor 2 is described. The sequential assignment procedure has been applied to the residues 22 to 83. The 21 residues in the N-terminal part of the structure were shown to be a random coil for which sequential assignment was not immediately possible. For the C-terminal part of the protein starting at Thr-22 and ending with the C-terminal residue Gly-83, sequence specific assignment of all the HN, Hα, Hβ′, Hβ″ resonances was achieved and complete sequence specific assignment of the non labile protons of the amino acid residue types of glycine, alanine, threonine, serine, valine, isoleucine, leucine, asparagine, aspartic acid, glutamine, glutamic acid, phenylalanine, tyrosine and tryptophan is reported.

Primary structure and comparative sequence analysis of an insect apolipoprotein. Apolipophorin-III from Manduca sexta.

The amino acid sequence of an insect apolipoprotein, apolipophorin-III from Manduca sexta, was determined by a combination of cDNA and protein sequencing. The mature hemolymph protein consists of 166 amino acids. The cDNA also encodes for an amino-terminal extension of 23 amino acids which is not represented in the mature hemolymph protein. The existence of a precursor protein was confirmed by in vitro translation of fat body mRNA. Computer-assisted comparative sequence analysis revealed the following points: 1) the protein is composed of tandemly repeating tetradecapeptide units with a high potential for forming amphiphilic helical structures. Compared to mammalian apolipoproteins the repeat units in the insect apolipoprotein show considerable length variability; 2) the sequence has a striking resemblance to several human apolipoproteins including apoE, AIV, AI, and CI. However, the homology seems to be entirely functional since, although the insect and mammalian apoproteins contain very similar types of amino acid residues, the actual degree of sequence identity is quite low. Whether the mammalian and insect apoproteins are derived from a common ancestral amphiphilic helix forming, lipid-binding protein, or arose by convergent evolution can not be determined at present. This represents the first complete amino acid sequence for an insect apolipoprotein.

Kringle 4 from human plasminogen: a proton magnetic resonance study via two-dimensional photochemically induced dynamic nuclear polarization spectroscopy

Two-dimensional (2D) proton magnetic resonance techniques used in conjunction with laser photochemically induced dynamic nuclear polarization (photo-CIDNP) spectroscopy have been applied to studying the kringle 4 domain from human plasminogen at 360 MHz. Out of 11 potential CIDNP-sensitive aromatic side chains, only 5 (His3, Tyr41, Tyr50, Trp72, and Tyr74) appear to be accessible to 3-(carboxymethyl)lumiflavin, the dye used to photogenerate spin polarization. Of these, Trp72 and Tyr74 are known to be at, or near, the lysine-binding site. The spin-spin scalar (J) and phase-sensitive dipolar (Overhauser) connectivities in the 2D experiments yield absolute assignments for the aromatic signals stemming from the exposed tyrosyl and tryptophanyl rings. Moreover, a number of side-chain H beta resonances can be identified and assigned to specific types of aromatic amino acid residues.

Photochemically induced dynamic nuclear polarization NMR study of yeast and horse muscle phosphoglycerate kinase.

A photochemically induced dynamic nuclear polarization (photo-CIDNP) study of yeast and horse muscle phosphoglycerate kinase with flavin dyes was undertaken to identify the histidine, tryptophan, and tyrosine resonances in the aromatic region of the simplified 1H NMR spectra of these enzymes and to investigate the effect of substrates on the resonances observable by CIDNP. Identification of the CIDNP-enhanced resonances with respect to the type of amino acid residue has been achieved since only tyrosine yields emission peaks and the dye 8-aminoriboflavin enhances tryptophan but not histidine. By use of the known amino acid sequences and structures derived from X-ray crystallographic studies of the enzymes from the two species, assignment of the specific residues in the protein sequences giving rise to the CIDNP spectra was partially achieved. In addition, flavin dye accessibility was used to probe any changes in enzyme structure induced by substrate binding. The nine resonance peaks observed in the CIDNP spectrum of yeast phosphoglycerate kinase have been assigned tentatively to five residues: histidines-53 and -151, tryptophan-310, and tyrosines-48 and -195. The accessibility of a tyrosine to photoexcited flavin is reduced in the presence of MgATP. Since the tyrosine residues are located some distance from the MgATP binding site of the catalytic center, it is proposed either that this change is due to a distant conformational change or that a second metal-ATP site inferred from other studies lies close to one of the tyrosines. Horse muscle phosphoglycerate kinase exhibits seven resonances by CIDNP NMR.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphorus-31 nuclear magnetic resonance studies of phosphorylated proteins

This chapter focuses on the characterization of phosphorylated target protein by phosphorus-31 nuclear magnetic resonance (31P NMR). It describes the preparation of a sample of phosphorylated protein for analysis by 31P NMR. 31P NMR can be used to assign the type of amino acid residue the phosphate is covalently attached to, such as the β-hydroxyl of a serine or threonine residue, or the N-1 or N-3 nitrogen of the imidazole ring of a histidine. 31P NMR can provide a range of information about the nature and dynamics of the phosphorylation site that cannot be obtained by other methods. The greatest strength of the NMR method is that if two or more phosphorylation sites exist on a protein, each phosphoryl group can be individually monitored by 31P NMR. 31P NMR has several important practical advantages. It is a nondestructive spectroscopic method relying on the absorption of very low radiofrequency irradiation. Thus, the sample can be recovered intact after NMR analysis. The NMR experiment can be done under the conditions of temperature and pH that are favorable for the stability of the phosphorylated protein.

Correlation of sequence hydrophobicities measures similarity in three-dimensional protein structure

The degree of similarity in the three-dimensional structures of two proteins can be examined by comparing the patterns of hydrophobicity found in their amino acid sequences. Each type of amino acid residue is assigned a numerical hydrophobicity, and the correlation coefficient r H is computed between all pairs of residues in the two sequences. In tests on sequences from two properly aligned proteins of similar three-dimensional structure, r H is found in the range 0.3 to 0.7. Improperly aligned sequences or unrelated sequences give r H near zero. By considering the observed frequency of amino acid replacements among related structures, a set of optimal matching hydrophobicities (OMHs) was derived. With this set of OMHs, significant correlation coefficients are calculated for similar three-dimensional structures, even though the two sequences contain few identical residues. An example is the two similar folding domains of rhodanese ( r H = 0.5). Predictions are made of similar three-dimensional structures for the alpha and beta chains of the various phycobiliproteins, and for delta hemolysin and melittin.

Theory of α-helix-to-random coil transitions of two-chain, coiled coils. Application to a synthetic analog of tropomyosin

A theory developed previously for the stability of the native structure in two-chain, a-helical coiled coils is applied to singly (disulfide) cross-linked tropomyosin and to a singly (disulfide) cross-linked, synthetic, 43-residue peptide analogue (called here XY5) of tropomyosin. Algorithms are obtained from fits of extant data for the helix stability parameter (s) for each amino acid residue type as a function of temperature; these reproduce the data to high precision. Extant data for the helix initiation parameter (a) are used as is. Employing these s and u values to realize the theory and using extant thermal denaturation data for XY5 and for tropomyosin, we have determined the temperature dependence of the helix-helix interaction parameter w(T) of the theory for the two substances. These w(T) functions then allow smoothed theoretical fits of thermal denaturation data and calculation of helix probability profiles along the polypeptide chains for XY5 and for tropomyosin. Theoretical predictions are generated concerning the thermal denaturation and helix probability profiles of yet unsynthesized, longer chain homologues (XY,) of the synthetic material XY5, including XY39, whose 281-residue chain would more closely model the 284-residue chain of tropomyosin. The importance of degree of polymerization in dictating helix content and profile and the difficulties in choosing models to match both the u and s(T) values that govern short-range interactions and the u(T) that governs interhelix interactions are clearly demonstrated. Thermodynamic implications of the w(T) functions obtained are discussed and shown to imply algebraic signs for standard enthalpy, entropy, and heat capacity changes that are difficult to reconcile with the expected hydrophobic and salt-bridge nature of the interactions. Predictions are generated concerning the behavior of non-cross-linked XY5, for which very few data are available. Some possible future directions of theory and experiment are outlined.

Relationship between mutability, polarity and exteriority of amino acid residues in protein evolution.

A systematic study was carried out on mutability of amino acid residues in evolving proteins in relation to their polarity and location within three-dimensional structure of proteins. Exteriority of residue sites is quantitatively defined as accessibility based on their static accessible surface area to solvent water molecule. Residue sites are classified into interior and exterior depending on their accessibility. More frequent substitution on exterior sites is confirmed to be general in eight sets of homologous protein families regardless of their biological functions and of presence or absence of a prosthetic group. Virtually all types of amino acid residues are found to have higher mutabilities on the exterior than in the interior. No correlation between mutability and polarity was observed of amino acid residues in the interior and on the exterior, respectively. Amino acid residues are classified into three depending on their polarity, polar (Arg, Lys, His, Gln, Asn, Asp and Glu), weak polar (Ala, Pro, Gly, Thr and Ser) and nonpolar (Cys, Val, Met, Ile, Leu, Phe, Tyr and Trp). Amino acid replacements during protein evolution are very conservative; 88% and 76% of them in the interior and on the exterior, respectively, are within the same group of the three. Inter-group replacements are such that weak polar residues are replaced more often by nonpolar residues in the interior and more often by polar residues on the exterior.