Regularities in the primary structure of proteins

In this paper the latest protein database consisting of more than a million amino acids is analyzed to characterize the short range regularities in the primary structure. The amino acid distributions along the polypeptide chain and among the proteins have been studied first. Their influence on the amino acid pair statistics was taken into account. We are primarily interested in the distances of the covalent structure, where the amino acid pair frequencies show non-random characters. The amino acid pairs separated by at least 20 residues in the covalent structure exhibit an exact Gaussian distribution. We found that there is a range of non-random pairing in the covalent structure. We conclude that the pair preference characters are different for each of the 20 × 20 amino acid pairs. The range of the non-random pairing varies from pair to pair, and in most cases it does not extend beyond the 9th neighbour. The preferences of a certain pair in a certain position can not be derived from the character of that pair in another position. The preference values of 400 amino acid pairs are listed for up to the pairs in 9th neighbour position. Some fields of potential application of these data have also been discussed.     

PROTEIN THERMOSTABILITY.

The amino acid compositions of more than 20 enzymes and protein from various closely related mesophilic and thermophilic micro-organisms (esp. Bacillus) have been used to calculate a variety of macroscopic parameters. These included the hydrophobic index (Hø), the ratio of polar to non-polar volumes (rho), the ratios of Arg/(Arg + Lys), and (Arg + Lys) or (Glx + Asx) to total amino acids, % H-bonding amino acids, %α-helix- or β-sheet-forming amino acids, the theoretical melting temperature (T_m^Ccalc), the total volume to total amino acid ratio (V_R), and the % non-polar residues (NPS). In contrast to previous similar comparisions with proteins from divergent sources, it was found that thermophilic vs mesophilic proteins from the same genus show correlations between thermostability and increased Hø, decreased rho, and increased Arg/(Arg + Lys), as well as increased α-index and β-index. Weaker correlations were seen for V_R, T_m^Calc, aliphatic index, and NPS, all derived from, or related to, Hø. No correlations existed for the other calculated parameters. These results are consistent with recent results of Argos et al. (1979) [Biochemistry 18, 5698–5703] on sequence analyses of glyceraldehyde-3-P dehydrogenases, where thermophilic proteins showed multiple amino acid replacements that caused increased internal hydrophobicity and increased external polarity. No trends were observed in any of the parameters calculated from amino acid compositions for crude cytoplasmic protein extracts from mesophilic vs thermophilic Bacilli.     

Conformational properties of pairs of amino acids

Crystal structure data of 31 different globular proteins were analysed. Potentials for all 400 pairs of 20 amino acid residues in helix, extended structure, chain reversals and coil state were obtained. Analysis of these potentials showed that tripeptides of amino acid residues are not linear combinations of amino acids. The association effect of these tripeptides depends on the types of amino acids associated, the position of amino acids in the pair and the secondary structure in which the pair exists. This built-in information in tripeptides should be used in polypeptide and protein folding studies.     

The usage of oligopeptides in proteins correlates negatively with molecular weight

We scanned the Swissprot databank to study the distribution of oligopeptides in sequenced proteins. We observed that in 7839000 amino acids in the bank, the frequency is strongly and negatively correlated with molecular weight. The correlation coefficient is -0.63 for single amino acids, -0.58 for dipeptides, -0.54 for tri- and -0.46 for tetrapeptides. In subsets of proteins of man, mouse, rat, drosophila, yeast and E. coli, the correlations were very similar to those for the total database. The intensity of the correlation diminishes linearly with the length of the peptide. We attributed the loss of dominance of molecular weight in determining the frequency of oligopeptides of higher order, to the emergence of function in longer oligopeptides. In the case of dipeptides, we observed that those composed of the same amino acid are in great excess in comparison with their expectation. The WW dipeptide is singular, in the sense that its observed/expected ratio is 13.5 standard deviations above the average ratio.     

Amino acid composition and zinc content of milk-clotting protease from Bacillus mesentericus strain 76

The milk-clotting protease from Bacillus mesentericus strain 76 is free of carbohydrate and lacks cysteine and cystine. The amino acid composition indicates a single peptide chain with 304 residues. Five amino acids - aspartic acid, threonine, serine, glycine and alanine - represent one-half of the total residues. The enzyme contains 35 aromatic amino acids and 103 ionic amino acids. The observed constant value of the ratio M_enzyme:A_Zinc is 1:1 for active and acid denatured enzyme preparations, indicating that 0.1 M acetic acid causes denaturation of the enzyme but it can not eliminate the zinc ions.     

山羊角提取物中氨基酸的种类研究与含量测定

目的研究山羊角提取物中氨基酸的种类和含量,为建立山羊角提取物质量控制提供依据。方法采用分子排阻色谱法检测山羊角提取物中物质的分子量范围。利用稳定同位素iTRAQ标记/液质联用法对山羊角提取物进行42种全谱氨基酸检测。结果山羊角提取物中物质的分子量范围在50³89 Da。090903批、090904批、091001批山羊角提取物中分别包含34种氨基酸(蛋白质氨基酸19种、非蛋白氨基酸15种),31种氨基酸(蛋白质氨基酸19种、非蛋白氨基酸12种)、33种氨基酸(蛋白质氨基酸19种、非蛋白氨基酸14种),山羊角提取物中未检出多肽及大分子物质。天门冬氨酸及谷氨酸加热易破坏,故在提取物制备过程中应严格控制干燥时间和温度。结论本研究建立了快速、稳定检测山羊角提取物中42种全谱氨基酸种类及含量的方法,为建立山羊角提取物质量检测标准提供参考依据。     

Analysis of γβ, βγ, γγ, ββ multiple turns in proteins

Abstract: The number of γ‐turns in a representative protein dataset selected from the current Protein Data Bank has increased almost seven times during the past decade. Eighty percent classic γ‐turns and 57% inverse γ‐turns are associated as multiple turns with either another γ‐turn or a β‐turn. We refer to these as multiple turns of the (γβ)_1,2,3 or (βγ)_1,2,3 type, depending upon whether the γ‐turn is before or after the β‐turn along the protein chain, respectively. However, for multiple turns involving only γ‐turns, we follow the nomenclature analogous to that proposed earlier for the multiple (or double) β‐turns. Fifty‐eight per cent β‐turns are associated as multiple turns with another β‐turn. We extracted multiple turns from the protein dataset and classified them on the basis of individual γ‐ or β‐turn types and the number of overlapping residues. Furthermore, we evaluated the amino acid positional potentials and determined the statistically significant amino acid preferences, hydrogen bond/side‐chain interaction preferences in the multiple turns and secondary structure preferences for residues immediately flanking these turns. The results of our analysis would be useful in the modeling, prediction or design of multiple turns in proteins. The amino acid sequence corresponding to the multiple turn, position in the protein chain, PDB Code/chain in which multiple turn is present and the individual turn types constituting the multiple turns are available from our website and this information would also be integrated in our Database of Structural Motifs in Proteins ( http://www.cdfd.org.in/dsmp.html ).     

脾氨肽口服冻干粉中游离氨基酸及肽的含量测定

目的：建立定量分析脾氨肽口服冻干粉中游离氨基酸与肽含量的方法.方法：采用氨基酸分析仪,以外标法定量测定游离氨基酸与酸水解后总氨基酸的含量,总氨基酸与游离氨基酸的差值即为肽的含量.结果：各氨基酸在各自测定浓度20％～180％的范围内线性关系均良好（r均在0.999以上）;游离氨基酸回收率在97.2％ ～ 103.9％之间,总氨基酸的回收率在93.1％～ 101.7％之间,RSD均小于2.0％（n=9）.结论：本方法能够准确地测定样品中游离氨基酸及肽的含量.     

Reabsorption of neutral amino acids mediated by amino acid transporter LAT2 and TAT1 in the basolateral membrane of proximal tubule

In order to understand the renal reabsorption mechanism of neutral amino acids via amino acid transporters, we have isolated human L-type amino acid transporter 2 (hLAT2) and human T-type amino acid transporter 1 (hTAT1) in human, then, we have examined and compared the gene structures, the functional characterizations and the localization in human kidney. Northern blot analysis showed that hLAT2 mRNA was expressed at high levels in the heart, brain, placenta, kidney, spleen, prostate, testis, ovary, lymph node and the fetal liver. The hTAT1 mRNA was detected at high levels in the heart, placenta, liver, skeletal muscle, kidney, pancreas, spleen, thymus and prostate. Immunohistochemical analysis on the human kidney revealed that the hLAT2 and hTAT1 proteins coexist in the basolateral membrane of the renal proximal tubules. The hLAT2 transports all neutral amino acids and hTAT1 transports aromatic amino acids. The basolateral location of the hLAT2 and hTAT1 proteins in the renal proximal tubule as well as the amino acid transport activity of hLAT2 and hTAT1 suggests that these transporters contribute to the renal reabsorption of neutral and aromatic amino acids in the basolateral domain of epithelial proximal tubule cells, respectively. Therefore, LAT2 and TAT1 play essential roles in the reabsorption of neutral amino acids from the epithelial cells to the blood stream in the kidney. Because LAT2 and TAT1 are essential to the efficient absorption of neutral amino acids from the kidney, their defects might be involved in the pathogenesis of disorders caused by a disruption in amino acid absorption such as blue diaper syndrome.     

Quantitative analyses of hydrophobicity of di- to pentapeptides having un-ionizable side chains with substituent and structural parameters.

With experimental conditions established recently, we measured the partition ratio (P') of 124 di- to pentapeptides composed of amino acids having un-ionizable side chains in a 1-octanol:pH 7.0 aqueous phosphate buffer system as an approximate zwitterionized "molecular" partition coefficient (P). Empirical equations of good quality, correlating the variations in log P' value of peptides with free energy-related physicochemical parameters for the side chain substituents and substructures, were formulated. The significant side chain parameters were those representing the intrinsic hydrophobicity, the steric effect on the relative solvation of functional groups on the backbone, and the conformational potential index derived from the Chou-Fasman beta-turn potential parameters. For polar side chains, specific indicator variables were required for intramolecular hydrogen-bond formations and the "polar proximity effect" for reductions of hydrophilicity observed when polar groups are crowded together. The proline residue was shown to contribute to the log P' value depending not only on its location on the backbone but also on the number of residues in peptides. On the basis of the analyses, we proposed a new "effective" hydrophobicity index for un-ionizable side chains which could predict the secondary structure of oligopeptides.     

Predicting isomorphic residue replacements for protein design

The replaceability of amino acids as reflected in their neighbourhood selectivity is analyzed in this paper. Neighbourhood selectivity was found to be more characteristic of the individual amino acids than the other commonly used parameters. The residue replacement rank obtained was compared with other proposed ranks and was tested on naturally accepted point mutations in homologous proteins.     

口服水解蛋白的成分分析

目的 分析口服水解蛋白的组成成分及分子量分布范围.方法 通过PITC柱前衍生法对口服水解蛋白的游离氨基酸含量进行测定,并通过质谱扫描及凝胶色谱方法测定分子量分布范围,分析口服水解蛋白的组成成分.结果 口服水解蛋白分子量范围在100～300 Da左右,口服水解蛋白含有18种氨基酸,其中8种为人体必需氨基酸,人体必需氨基酸含量占游离氨基酸总和的56.1％.口服水解蛋白的短肽含量与游离氨基酸含量比值为1.08.结论 本实验分析了口服水解蛋白的组成成分,口服水解蛋白是无脂,主要以短肽、混合氨基酸为主,含有少量糖类及矿物质的高蛋白营养补充剂.     

Structural Specificity of Mucosal-Cell Transport and Metabolism of Peptide Drugs: Implication for Oral Peptide Drug Delivery

The brush border membrane of intestinal mucosal cells contains a peptide carrier system with rather broad substrate specificity and various endo- and exopeptidase activities. Small peptide (di-/ tripeptide)-type drugs with or without an N-terminal -amino group, including -lactam antibiotics and angiotensin-converting enzyme (ACE) inhibitors, are transported by the peptide transporter. Poly-peptide drugs are hydrolyzed by brush border membrane proteolytic enzymes to di-/tripeptides and amino acids. Therefore, while the intestinal brush border membrane has a carrier system facilitating the absorption of di-/tripeptide drugs, it is a major barrier limiting oral availability of polypeptide drugs. In this paper, the specificity of peptide transport and metabolism in the intestinal brush border membrane is reviewed.     

High-performance liquid chromatography of epimeric N-protected peptide acids and esters for assessing racemization

The separations by reversed phase high-performance liquid chromatography on a μBondapak-C₁₈ column of 53 epimeric N-substituted di-, tri- and tetrapeptide acids and esters have been attempted, with success in three quarters of the cases. Substituents include acetyl, benzoyl, benzyloxycarbonyl, tert.-butoxycarbonyl and 9-fluorenylmethoxycarbonyl. The series N-benzyloxycarbonylglycyl-Xxx-valine ethyl ester with Xxx = alanyl, valyl, leucyl and phenylalanyl, is recommended for use in studies on racemization. Results on racemization attending the coupling of an amino acid ester as compared with a di- and tripeptide ester vary with the coupling method.     

Hydrophobic Contribution Constants of Amino Acid Residues to the Hydrophobicities of Oligopeptides

Purpose. The main purpose of this study is to explore the additive-constitutive nature of common amino acids in their contribution to the partition coefficients of small peptides. Methods. The Log P values and other physico-chemical parameters of the peptides studied are taken from the literature. The frequency of appearance (n_i) of each individual amino acid is calculated as the number of the amino acids in a given peptide. Results. The partition coefficients (Log P_(oct./buff.) at PH 7) of 87 N-acetyl-peptide-amides have been correlated with the frequency of appearance of amino acids. From the correlation obtained, the de novo hydrophobic contribution constants of 19 amino acid residues are derived for the first time. The contribution constants are extended to 59 unmodified regular peptides with the inclusion of the pk_a values of both N-terminal and C-terminal amino acids. The models thus obtained have been validated with additional 27 peptides (both N-acetyl-peptide-amides and unmodified). Conclusions. The Log P of oligopeptides is very well correlated with the de novo hydrophobic contribution constants of amino acids. The models we have derived are reasonably accurate in predicting the hydrophobicities of new oligopeptides (tetrapeptides) at a fixed pH (e.g., 7).     

酶解鱼可溶性肽分子组成结构及营养评价

本文叙述了用胃蛋白酶、胰蛋白酶水解的鱼可溶性肤类水解物,经凝胶高效液相色谱和氨基酸分析仪分析测定其肤类分子组成结构和氨基酸组成成分.测得水解产物中肽类相对分子质量在7400以下,其中相对分子质量6600～7400、由52～58个氨基酸组成的较长肽链占1.74%;相对分子质量2500～5300、由20～41个氨基酸组成的中长肽链占29.75%;相对分子质量在1000以下的由2～10个氨基酸组成的寡肽占50%.水解物的总氮与氨基酸态氮比为25.91,约有96%的氨基酸以肽类形式存在,4%为游离氨基酸.测得可溶性肽的总氨基酸含量为73.98%,必需氨基酸为32.39%,占总氮基酸的43.78%,与FAO/WHO相比,苯丙氨酸为第一限制性氨基酸,氨基酸分值为61.根据分析结果,深入探讨了鱼可溶性肽氨基酸组成的平衡性及其有关寡肽在动物机体的生理功能作用.     

Relevance of sequence statistics for the properties of extremophilic proteins

The amino acid composition of proteins from mesophilic and extremophilic organisms is commonly assumed to reflect the mechanisms of molecular adaptation to extremes of physical conditions. In this context, halo-philic behaviour has been attributed to significantly increased numbers of aspartic and glutamic acid residues. However, extending the analysis to a statistically relevant set of related proteins, dihydrofolate reductase from Halobacterium volcanii, as an example, shows that the increase in negative charge is found to be less significant than other exchanges of amino acids (e.g., Ala, Am, Arg, Lys, Phe, Ser). Thus, the high water binding capacity of negatively charged residues cannot be unambiguously correlated with the anomalous stability of halophilic proteins. A similar caveat holds for generalizations regarding the thermal stability of proteins. In this case, D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima was compared with a number of mesophilic and moderately thermophilic homologs. Again, ‘traffic rules of stabilization’, in terms of amino acid changes in going from mesophilic to thermophilic proteins, cannot be given.     

氨基酸分析仪在抗生素发酵研究中的应用

众多影响抗生素发酵过程生物合成的因素中，氨基酸的影响占有重要地位。氨基酸分析可定性、定量地测定发酵液中氨基酸的含量，为抗生素发酵研究提供了一个有力工具。     

Synthesis of enantiopure non‐natural α‐amino acids using tert‐butyl (2S)‐2‐[bis‐(tert‐butoxycarbonyl)amino]‐5‐oxopentanoate as key‐intermediate:the first synthesis of(S)‐2‐amino‐oleic acid

Abstract: A general method for the synthesis of enantiopure non‐natural α‐amino acids is described. The key intermediate tert‐butyl (2S)‐2‐[bis(tert‐butoxycarbonyl)amino]‐5‐oxopentanoate was obtained from l ‐glutamic acid after suitable protection and selective reduction of the γ‐methyl ester group by DIBALH. Wittig reaction of this chiral aldehyde with various ylides led to a variety of δ,ε‐unsaturated α‐amino acids. This methodology was applied to the synthesis of (S)‐2‐amino‐oleic acid.     

Are secondary structures secondary?

Crystallographically observed secondary structures in globular proteins are divided into two categories based on calculated potential values for these structures. Type I secondary structures are those which have maximum potential in observed structural state. These type I structures consist of those amino acids which intrinsically prefer that structural state. On the other hand type II secondary structures have a calculated potential value which is not maximum for the observed state. Type II structures are composed of those amino acid residues which do not intrinsically prefer the observed state and thus seem to have been formed due to tertiary interactions. All the observed secondary structures have been identified as type I or type II in 31 different globular proteins considered. It is suggested that type II structures are formed at a latter stage of protein folding mainly due to favourable tertiary interactions.