Surface topography of histidine residues in lysozymes.

Several avian and mammalian c-type lysozymes were chromatographed on chelated (to iminodiacetate) and immobilized transition metal ions (Co2+, Ni2+, Cu2+ and Zn2+) under a variety of experimental conditions. The varied affinity of evolutionary variants of the lysozyme family for chelated metal ions, IDA-M(II), can be rationalized primarily in terms of the presence, multiplicity and microenvironments of histidine residues. The chromatographic resolution of some of these closely related proteins attests to the analytical power of immobilized metal-ion affinity chromatography.     

Identification of kinetically hot residues in proteins.

A number of recent studies called attention to the presence of kinetically important residues underlying the formation and stabilization of folding nuclei in proteins, and to the possible existence of a correlation between conserved residues and those participating in the folding nuclei. Here, we use the Gaussian network model (GNM), which recently proved useful in describing the dynamic characteristics of proteins for identifying the kinetically hot residues in folded structures. These are the residues involved in the highest frequency fluctuations near the native state coordinates. Their high frequency is a manifestation of the steepness of the energy landscape near their native state positions. The theory is applied to a series of proteins whose kinetically important residues have been extensively explored: chymotrypsin inhibitor 2, cytochrome c, and related C2 proteins. Most of the residues previously pointed out to underlie the folding process of these proteins, and to be critically important for the stabilization of the tertiary fold, are correctly identified, indicating a correlation between the kinetic hot spots and the early forming structural elements in proteins. Additionally, a strong correlation between kinetically hot residues and loci of conserved residues is observed. Finally, residues that may be important for the stability of the tertiary structure of CheY are proposed.     

Chemical modification of methionine residues in azurin.

Azurin from $\text{Pseudomonas}\text{aeruginosa}$ has been treated with bromoacetate at low pH to alkylate methionine residues. Two classes of methionine side chains are observed as a result of these reactions — four of the six methionines are reactive at pH 4, whereas all six are reactive at pH 3.2. The product containing four alkylated methionines maintains a significant portion of the blue color and spectroscopic characteristics of the native protein. The product which has been fully modified at the methionine residues, on the other hand, has lost all blue color and appears to be largely in a random coil form.     

Context-dependent conformation of diethylglycine residues in peptides.

Diethylglycine (Deg) residues incorporated into peptides can stabilize fully extended (C5) or helical conformations. The conformations of three tetrapeptides Boc-Xxx-Deg-Xxx-Deg-OMe (Xxx=Gly, GD4; Leu, LD4 and Pro, PD4) have been investigated by NMR. In the Gly and Leu peptides, NOE data suggest that the local conformations at the Deg residues are fully extended. Low temperature coefficients for the Deg(2) and Deg(4) NH groups are consistent with their inaccessibility to solvent, in a C5 conformation. NMR evidence supports a folded beta-turn conformation involving Deg(2)-Gly(3), stabilized by a 4-->1 intramolecular hydrogen bond between Pro(1) CO and Deg(4) NH in the proline containing peptide (PD4). The crystal structure of GD4 reveals a hydrated multiple turn conformation with Gly(1)-Deg(2) adopting a distorted type II/II' conformation, while the Deg(2)-Pro(3) segment adopts a type III/III' structure. A lone water molecule is inserted into the potential 4-->1 hydrogen bond of the Gly(1)-Deg(2) beta-turn.     

Autointoxication in residues ofAsparagus officinalis L.

Summary In a greenhouse experiment the growth of asparagus seedlings was retarded by the residue treatments in both vermiculite and sand cultures. In general, the retardation of asparagus root by residues was slightly greater than the retardation of shoot in both cultures. The retardation of the growth of asparagus seedlings by root and stem treatments was usually higher than that by old root litter. Root and stem extracts strongly inhibited the development of asparagus seedlings in the seed bioassay. The inhibition of extracts to the growth of shoot was greater than that to the growth of root. The quantities in the total phenolics and catachol type phenolics from root, stem and old root litter extracts corresponded to the autotoxicity in the seed bioassay. The soil extracts obtained from using acetone, methanol, and XAD-4 extractions strongly inhibited the shoot and root development of asparagus seedlings in the bioassay. The efficiency of phenolics extraction by the XAD-4 method was significantly higher than that by acetone and methanol extractions. The results obtained in the greenhouse experiment and bioassay revealed that phytotoxic substances present in the residues and the soil of asparagus and may be partially responsible for the asparagus replanting problems.     

Essential arginyl residues in thymidylate synthetase.

Thymidylate synthetase from amethopterin-resistant $\text{Lactobacillus}$$\text{casei}$ is rapidly and completely inactivated by 2,3-butanedione in borate buffer, a reagent that is highly selective for the modification of arginyl residues. The reversible inactivation follows pseudo-first order kinetics and is enhanced by borate buffer. dUMP and dTMP afford significant protection against inactivation while (±)-5,10-methylenetetrahydrofolate and 7,8-dihydrofolate provide little protection. Unlike native enzyme, butanedione-modified thymidylate synthetase is incapable of interacting with 5-fluoro-2′-deoxyuridylate and 5,10-(+)-methylenetetrahydrofolate to form stable ternary complex. The results suggest that arginyl residues participate in the functional binding of dUMP.     

Selective oxidation of methionine residues in proteins.

Methionine residues in peptides and proteins were oxidized to methionine sulfoxides by mild oxidizing reagents such as chloramine-T and N-chlorosuccinimide at neutral and slightly alkaline pH. With chloramine-T cysteine was also oxidized to cystine but no other amino acid was modified; with N-chlorosuccinimide tryptophans were oxidized as well. In peptides and denaturated proteins all methionine residues were quantitatively oxidized, while in native proteins only exposed methionine residues could be modified. Extent of oxidation of methionine residues was determined by quantitative modification of the unoxidized methionine residues with cyanogen bromide (while methionine sulfoxide residues remained intact), followed by acid hydrolysis and amino acid analysis. Methionine was determined as homoserine and methionine sulfoxide was reduced back to methionine. Sites of oxidation were identified in a similar way by cleaving the unoxidized methionyl peptide bonds with cyanogen bromide, followed by quantitative end-group analysis of the new amino-terminal amino acids (by an automatic sequencer).     

Chemical modification of arginine residues in alpha-bungarotoxin.

The reaction of alpha-bungarotoxin (alpha-BuTX) with 1,2-cyclohexanedione resulted in the modification of only Arg-72 but arginine at position 36 or 72, as well as both were modified by reaction of the toxin with p-hydroxyphenylglyoxal. No derivative modified at Arg-25 was obtained, indicating that this residue may be located in the interior region of alpha-BuTX molecule. Monoderivative at Arg-72 showed about 50% of the lethal toxicity and binding activity of alpha-BuTX to nicotinic acetylcholine receptor (AChR), while the activity was decreased to one-third when the invariant Arg-36 was modified, indicating that the latter residue is more closely related to the interaction of the toxin with AChR. Approx. 13% of the residual activity was observed when both arginine residues at 36 and 72 were modified. The antigenicity of alpha-BuTX was still retained essentially intact after Arg-36 or -72 was modified, whereas it decreased to 50% when both these arginine residues were modified. The present study indicates that Arg-36 and -72 in alpha-BuTX may be involved in the multipoint contact between the toxin and AChR, but neither is absolutely essential for the binding.     

Essential arginyl residues in yeast enolase.

Yeast enolase is rapidly inactivated by butanedione in borate buffer, complete inactivation correlating with the modification of 1. 8 arginyl residues per subunit. Protection against inactivation is provided by either an equilibrium mixture of substrates or inorganic phosphate, a competitive inhibitor of the enzyme. Complete protection by substrates correlates with the shielding of 1. 3 arginyl residues per subunit, while phosphate protects 1. 0 arginyl residue per subunit from modification.     

Essential arginyl residues in yeast phosphoglyceromutase.

Inactivation of yeast phosphoglyceromutase (tetramer) with 1,2-cyclohexanedione correlates with the modification of six arginyl residues per mole of the enzyme. Protection experiments using 3-phosphoglycerate suggest that four arginyl residues (one residue per subunit) are involved in the binding of the substrate to the enzyme. The modified enzyme reversibly regained its activity upon incubation with hydroxylamine. The reactivity of lysyl residues which have been shown to be involved in the active site is markedly reduced in the enzyme inactivated with 1,2-cyclohexanedione, indicating that the lysyl and arginyl residues are in close proximity in the active site.     

Essential carboxyl residues in yeast enolase.

Yeast enolase (EC 4.2.1.11) is rapidly inactivated at pH 6.1 by three different water-soluble carbodiimides — 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride, 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate, and 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)-carbodiimide iodide. Inactivation is most likely due to the modification of essential carboxyl residues at the enzyme active site.     

Cooligopeptides containing aromatic residues spaced by glycyl residues. IX. Fluorescence properties of tryptophan-containing peptides

The fluorescence properties of several cooligopeptides of glycine, phenylalanine, and tryptophan, containing one or two aromatic residues, are investigated. In particular, a detailed analysis is made of the influence of pH upon the quantum yield and the position of the emission maximum (λ_max) in H-Trp-Trp-OH, H-Trp-Gly-OH, H-Gly-Trp-OH, H-Gly-Trp-Gly-OH, H-Trp-Trp-OH, H-Trp-Trp-Gly-OH, H-Gly-Trp-Trp-OH, H-Phe-Trp-OH, H-Phe-Trp-Gly-OH, H-Gly-Phe-Trp-OH, and H-Gly-X-(Gly)_n-Trp-Gly-OH, with X = Phe or Trp, and n = 0,1,2. It is shown that raising the pH from ca. 2 to 11 results in a red shift of λ_max, and an increase in the quantum yield. These changes, mostly structure dependent, are in most cases attributable to electronic perturbations acting directly upon the λ_max of the fluorophore(s) and upon the quenching efficiency of the free amino and carbonyl groups. For the compounds having two adjacent tryptophyl residues, it is shown that the two fluorophores do not appear to have the same emission properties and the quantum yield is lower than expected. The causes of this behavior are discussed in terms of conformational effects, stacking interactions, and radiationless energy transfer. Finally, an attempt is made to correlate fluorescence data with previous circular dichroism data which had indicated the occurrence of a conformationally rigid structure for some of the compounds having two adjacent aromatic residues.