The infrared absorption of amino acid side chains

Amino acid side chains play fundamental roles in stabilising protein structures and in catalysing enzymatic reactions. These fields are increasingly investigated by infrared spectroscopy at the molecular level. To help the interpretation of the spectra, a review of the infrared absorption of amino acid side chains in H₂O and ²H₂O is given. The spectral region of 2600–900 cm⁻¹ is covered.     

DNA-catalyzed covalent modification of amino acid side chains in tethered and free peptide substrates

This study focuses on the development of DNA catalysts (deoxyribozymes) that modify side chains of peptide substrates, with the long-term goal of achieving DNA-catalyzed covalent protein modification. We recently described several deoxyribozymes that modify tyrosine (Tyr) or serine (Ser) side chains by catalyzing their reaction with 5'-triphosphorylated RNA, forming nucleopeptide linkages. In each previous case, the side chain was presented in a highly preorganized three-dimensional architecture such that the resulting deoxyribozymes inherently cannot function with free peptides or proteins, which do not maintain the preorganization. Here we describe in vitro selection of deoxyribozymes that catalyze Tyr side chain modification of tethered and free peptide substrates, where the approach can potentially be generalized for catalysis involving large proteins. Several new deoxyribozymes for Tyr modification (and several for Ser modification as well) were identified; progressively better catalytic activity was observed as the selection design was strategically changed. The best new deoxyribozyme, 15MZ36, catalyzes covalent Tyr modification of a free tripeptide substrate with a k(obs) of 0.50 h(-1) (t(1/2) of 83 min) and up to 65% yield. These findings represent an important advance by demonstrating, for the first time, DNA catalysis involving free peptide substrates. The new results suggest the feasibility of DNA-catalyzed covalent modification of side chains of large protein substrates and provide key insights into how to achieve this goal.     

An essential carboxylic acid group in human prostate acid phosphatase.

Treatment of homogenous human prostatic acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) with low concentrations of Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate) leads to a rapid loss of enzymic activity. The rate of inactivation of the enzyme is reduced in the presence of the competitive inhibitors phosphate and L-(+)-tartrate, but not in the presence of non-inhibitory D-tartrate. Measurement of the ethylamine produced upon hydrolysis of enzyme modified in the presence of D- and of L-tartrate permitted the quantitative estimation of the number of carboxylic acid residues at the active site. The data indicate that two carboxyl groups per (dimeric) enzyme molecule are essential for catalytic activity. It is proposed that one function of the active site carboxyl group may be to protonate the leaving alcohol or phenol portion of the phosphomonoester substrate during the formation of the covalent phosphoenzyme intermediate.     

Conformational constraints of amino acid side chains in alpha-helices

The conformational freedom of amino acid side chains is strongly reduced when the side chains occur on an α-helix. A quantitative evaluation of this freedom has been carried out by means of conformational energy computations for all naturally occurring amino acids and for α-aminobutyric acid when they are placed in the middle of a right-handed poly(L-alanine) α-helix. One of the three possible rotameric states for rotation around the C^α ? C^β bond (viz. g⁺) is excluded completely on the helix because of steric hindrance, and the relative populations of the other two rotamers (t and g^?) are altered because of steric interactions and the reduction of hydrogen-bonding possibilities. The computed tendencies of the changes in distributions of rotamers, on going from an ensemble of all backbone conformations to the α-helix, agree with the observed tendencies in proteins. Minimum-energy side-chain conformations in an α-helix have been tabulated for use in conformational energy computations on polypeptides.     

Conformational preferences of amino acid side chains in collagen

Conformational energy computations were carried out on collagenlike triple-stranded conformations of several poly(tripeptide)s with the general structure CH₃CO? (Gly? X? Y)₃? NHCH₃. The sequences considered had various amino acid residues in position X or Y of the central tripeptide, with either Pro or Ala as a neighbor, i.e., Gly-X-Pro, Gly-X-Ala, Gly-Pro-Y, and Gly-Ala-Y. Minimum-energy conformations were computed for the side chains, and their distributions were compared for the four sequences. The residues used were Abu (= α-aminobutyric acid), Leu, Phe, Ser, Asp, Asn, Val, Ile, and Thr. The conformational energy of a ? Ch₂? CH₃ side chain in Abu was mapped as a function of the dihedral angle χ¹. Intrastrand interactions with neighboring residues do not affect the conformations of a side chain in position Y, and they have a minor effect on it in the X-Ala sequence, but they strongly restrict the conformational freedom of the side chain in the X-Pro sequence. Conversely, interstrand interactions do not affect side chains in position X, but they strongly restrict the conformational freedom of a side chain in position Y if there is a nearby Pro residue in a neighboring strand. Hydrogen bonds with the backbone can be formed in some conformations of long polar side chains, such as Asp, Asn, or Gln. All amino acid residues can be accommodated in collagen. Because of the interactions mentioned above, steric and energetic constraints can be correlated with observed preferences of certain amino acids for positions X or Y in collagen. Hence, these preferences may be explained, in part, in terms of differences in the conformational freedom of the side chains in the triple-stranded structure.     

Easily polarizable proton transfer hydrogen bonds between the side chains of histidine and the carboxylic acid groups of glutamic and aspartic acid residues in proteins

The nature of the hydrogen bonds formed between glutamic acid and histidine residues between aspartic acid and histidine residues is studied by i.r. spectroscopy. These studies were performed with $\text{(}\text{l}\text{-Glu}\text{)}{}_{\mathrm{n}}\text{+(}\text{l}\text{-His}\text{)}{}_{\mathrm{n}}$ and with associates of monomeric Glu + His and Asp + His systems in solutions whereby these amino acids had protected α-amino and α-carboxylic groups. It is shown that the OH …N?^?O?H⁺N bonds are easily polarizable proton transfer hydrogen bonds. The residence time of the proton at the His is a little larger in the case of the Asp + His than in the case of the Glu + His systems. Polar environments shift this proton transfer equilibrium in favour of the proton limiting structure ^?O?H⁺N, and less polar ones in favour of the structure OH?N. These results demonstrate that the large proton polarizability of the hydrogen bonded system in the active centre of chymotrypsin is responsible for the charge shift caused by the substrate, and thus for the increase in reactivity of the serine residue and the catalytic activity of the enzyme.     

Theoretical study of the acidic strength of amino acid side chains

The acidic strengths in gas phase of three groups (NH₄⁺, H₂S, and HCOOH) that mimic the most common amino acid side chains of enzymes are studied by means of quantum mechanical methods. The results demonstrate that in gas phase the acidities of such groups change drastically with respect to those reported in aqueous phase. Moreover, the dependence between the energetics of the proton-transfer process and the distance separating the acid and base groups is stated. The biological implications of these results are discussed.     

Oligosaccharide side chains of wall molecules are essential for cell-wall lysis in Chlamydomonas reinhardtii

The glycoproteins of the cell walls of Chlamydomonas are lysed during the reproductive cycle by proteases (autolysins) which are specific for their substrates. The autolysin which digests the wall of sporangia to liberate the zoospore daughter cells in the vegetative life cycle is a collagenase-like enzyme which attacks only selected domains in its wall substrates containing (hydroxy)-proline clusters. Cell-wall fractions obtained by salt-extraction (NaClO₄) and oxidizing agents (NaClO₂) and the insoluble residue were tested as substrates. The most-crosslinked insoluble inner part of the wall is the best substrate for the sporangia autolysin. Oligosaccharides obtained from the insoluble cell-wall fraction of sporangia by hydrolysis with Ba(OH)₂ inhibit autolysin action. We conclude that the oligosaccharide side chains of wall substrates are essential for forming the reactive enzyme-substrate complex.Abbreviations CSW chlorite-soluble cell-wall fraction - ICW insoluble cell-wall fraction - PSW salt-soluble fraction - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Pectic arabinan side chains are essential for pollen cell wall integrity during pollen development

Pectin is a complex polysaccharide and an integral part of the primary plant cell wall and middle lamella, contributing to cell wall mechanical strength and cell adhesion. To understand the structure–function relationships of pectin in the cell wall, a set of transgenic potato lines with altered pectin composition was analysed. The expression of genes encoding enzymes involved in pectin acetylation, degradation of the rhamnogalacturonan backbone and type and length of neutral side chains, arabinan and galactan in particular, has been altered. Upon crossing of different transgenic lines, some transgenes were not transmitted to the next generation when these lines were used as a pollen donor, suggesting male sterility. Viability of mature pollen was severely decreased in potato lines with reduced pectic arabinan, but not in lines with altered galactan side chains. Anthers and pollen of different developmental stages were microscopically examined to study the phenotype in more detail. Scanning electron microscopy of flowers showed collapsed pollen grains in mature anthers and in earlier stages cytoplasmic protrusions at the site of the of kin pore, eventually leading to bursting of the pollen grain and leaking of the cytoplasm. This phenomenon is only observed after the microspores are released and the tapetum starts to degenerate. Timing of the phenotype indicates a role for pectic arabinan side chains during remodelling of the cell wall when the pollen grain is maturing and dehydrating.     

Effective concentrations of amino acid side chains in an unfolded protein.

Preferential interactions between chain segments are studied in unfolded cytochrome c. The method takes advantage of heme ligation in the unfolded protein, a feature unique to proteins with covalently attached heme. The approach allows estimation of the effective concentration of one polypeptide chain segment relative to another, and is successful in detecting differences for peptide chain segments separated by different numbers of residues in the linear sequence. The method uses proton NMR spectroscopy to monitor displacement of the histidine heme ligands by imidazole as guanidine hydrochloride unfolded cytochrome c is titrated with deuterated imidazole. When the imidazole concentration exceeds the effective (local) concentration of histidine ligands, the protein ligands are displaced by deuterated imidazole. On displacement, the histidine ring proton resonances move from the paramagnetic region of the spectrum to the diamagnetic region. Titrations have been carried out for members of the mitochondrial cytochrome c family that contain different numbers of histidine residues. These include cytochromes c from tuna (2), yeast iso-2 (3), and yeast iso-1-MS (4). At high imidazole concentration, the number of proton resonances that appear in the histidine ring C2H region of the NMR spectrum is one less than the number of histidine residues in the protein. So one histidine, probably His-18, remains as a heme ligand. The effective local concentrations of histidines-26, -33, and -39 relative to the heme (position 14-17) are estimated to be (3-16) X 10(-3) M.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydration of amino acid side chains: dependence on secondary structure.

Energy calculations have been used to study the hydration sites around the polar groups of serine, threonine and tyrosine side chains. These hydration sites depend not only on the hybridization of the polar group but also on the local secondary structure, the chi 1 side chain torsion angle and the position of the hydroxyl hydrogen atom. For tyrosine side chains, two solvent sites are found approximately in the plane of the ring. Even for serine and threonine side chains only two minimum energy sites are found in general of which one is in an expected position within hydrogen bonding of the hydroxyl hydrogen atom (unless this is blocked from interaction with solvent molecules by, for example, Oi-4 or Oi-3. The position of the second of these sites depends not only on the position of the hydroxyl oxygen but also on neighbouring main chain atoms to which it can also hydrogen bond. There is good agreement with the solvent distributions obtained from crystallographic data.     

The aminoacylation of transfer ribonucleic acid. Inhibitory effects of some amino acid analogues with altered side chains.

Several amino acid analogues that are able to replace amino acid residues in binding positions of the biologically active C-terminal tetrapeptide amide sequence, Trp-Met-Asp-PheNH2, of the gastrins were examined for their ability to inhibit the aminoacylation of tRNA in an Escherichia coli and rat liver system. Although in both systems the amino acid side chains are involved in the recognition process, the structural requirements of the side chain in the two systems are not comparable. Analogues of methionine and phenylalanine behaved similarly in the E. coli and rat liver systems, whereas analogues of tryptophan behaved differently. From the results it is possible to suggest structural features of the amino acid side chains which are required for recognition by the aminoacyl-tRNA synthetases.