Amino acid size, charge, hydropathy indices and matrices for protein structure analysis.

BACKGROUND: Prediction of protein folding and specific interactions from only the sequence (ab initio) is a major challenge in bioinformatics. It is believed that such prediction will prove possible if Anfinsen's thermodynamic principle is correct for all kinds of proteins, and all the information necessary to form a concrete 3D structure is indeed present in the sequence. RESULTS: We indexed the 200 possible amino acid pairs for their compatibility regarding the three major physicochemical properties--size, charge and hydrophobicity--and constructed Size, Charge and Hydropathy Compatibility Indices and Matrices (SCI & SCM, CCI & CCM, and HCI & HCM). Each index characterized the expected strength of interaction (compatibility) of two amino acids by numbers from 1 (not compatible) to 20 (highly compatible). We found statistically significant positive correlations between these indices and the propensity for amino acid co-locations in real protein structures (a sample containing total 34630 co-locations in 80 different protein structures): for HCI: p < 0.01, n = 400 in 10 subgroups; for SCI p < 1.3E-08, n = 400 in 10 subgroups; for CCI: p < 0.01, n = 175). Size compatibility between residues (well known to exist in nucleic acids) is a novel observation for proteins. Regression analyzes indicated at least 7 well distinguished clusters regarding size compatibility and 5 clusters of charge compatibility. We tried to predict or reconstruct simple 2D representations of 3D structures from the sequence using these matrices by applying a dot plot-like method. The location and pattern of the most compatible subsequences was very similar or identical when the three fundamentally different matrices were used, which indicates the consistency of physicochemical compatibility. However, it was not sufficient to choose one preferred configuration between the many possible predicted options. CONCLUSION: Indexing of amino acids for major physico-chemical properties is a powerful approach to understanding and assisting protein design. However, it is probably insufficient itself for complete ab initio structure prediction.

In search of the nature of specific nucleic acid-protein interactions.

The theory of "codon-amino acid coevolution" was first proposed by Woese in 1967. It suggests that there is a stereochemical matching - that is, affinity - between amino acids and certain of the base triplet sequences that code for those amino acids. We have constructed a Common Periodic Table of Codons and Amino Acids, where the Nucleic Acid Table showed perfect axial symmetry for codons and the corresponding Amino Acid Table also displayed periodicity regarding the biochemical properties (charge and hydrophobicity) of the 20 amino acids and the position of the stop signals. The Table indicates that the middle (2nd) amino acid in the codon has a prominent role in determining some of the structural features of the amino acids. The possibility that physical contact between codons and amino acids might exist was tested on restriction enzymes. Many recognition site-like sequences were found in the coding sequences of these enzymes and as many as 73 examples of codon-amino acid co-location were observed in the 7 known 3D structures (December 2003) of endonuclease-nucleic acid complexes. These results indicate that the smallest possible units of specific nucleic acid-protein interaction are indeed the stereochemically compatible codons and amino acids.

Seven fundamental, unsolved questions in molecular biology. Cooperative storage and bi-directional transfer of biological information by nucleic acids and proteins: an alternative to "central dogma".

The Human Genome Mapping Project provided us a large amount of sequence data. However our understanding of these data did not grow proportionally, because old dogmas still set the limits of our thinking. The gene-centric, reductionistical side of molecular biology is reviewed and seven problems are formulated, each indicating the insufficiency of the "central dogma". The following is concluded and suggested: 1. Genes are located and expressed on both DNA strands; 2. Introns are the source of important biological regulation and diversity; 3. Repeats are the frame of the chromatin structure and participate in the chromatin regulation; 4. The molecular accessibility of the canonical dsDNA structure is poor; 5. The genetic code is co-evolved with the amino acids and there is a stereochemical matching between the codes andamino acids; 6. The flow of information between nucleic acids and proteins is bi-directional and reverse translation might exist; 7. Complex genetic information is always carried and stored by nucleic acids and proteins together.

The BlastNP: a novel, sensitive sequence similarity searching method using overlappingly translated sequences.

An alternative method to TblastX has been developed, known as blastNP. Nucleic acids in database and query sequences were translated into overlapping protein-like sequences (overlappingly translated sequences or OTSs) before searching with blastP. Thus, each nucleic acid sequence is represented by a single "protein like" sequence (instead of three hypothetical proteins in different reading frames). The BlastNP method is defined as a BlastP that is performed on an overlappingly translated nucleic acid database using a similarly converted nucleic acid query. The specificity and sensitivity of blastNP and TblastX is quantitatively very similar, except that blastNP is more sensitive to detect short sequence similarities (less than 50 residues). However, a qualitative comparison of the observed similarities showed that only 56% was detected by both methods, but 22% was indicated only by blastNP and 22% only by TblastX. For example, a statistically significant similarity between prion protein (PrP) and transcriptions factors (TF) was only detected by blastNP. A signal amplification was seen when OTS sequences were used in similarity visualisation methods (like LALIGN) instead of nucleic acids.

Codes in the codons: construction of a codon/amino acid periodic table and a study of the nature of specific nucleic acid-protein interactions.

The theory of "codon-amino acid coevolution" was first proposed by Woese in 1967. It suggests that there is a stereochemical matching - that is, affinity - between amino acids and certain of the base triplet sequences that code for those amino acids. We have constructed a common periodic table of codons and amino acids, where the nucleic acid table showed perfect axial symmetry for codons and the corresponding amino acid table also displayed periodicity regarding the biochemical properties (charge and hydrophobicity) of the 20 amino acids and the position of the stop signals. The table indicates that the middle (2/sup nd/) amino acid in the codon has a prominent role in determining some of the structural features of the amino acids. The possibility that physical contact between codons and amino acids might exist was tested on restriction enzymes. Many recognition site-like sequences were found in the coding sequences of these enzymes and as many as 73 examples of codon-amino acid co-location were observed in the 7 known 3D structures (December 2003) of endonuclease-nucleic acid complexes. These results indicate that the smallest possible units of specific nucleic acid-protein interaction are indeed the stereochemically compatible codons and amino acids.

A common periodic table of codons and amino acids.

A periodic table of codons has been designed where the codons are in regular locations. The table has four fields (16 places in each) one with each of the four nucleotides (A, U, G, C) in the central codon position. Thus, AAA (lysine), UUU (phenylalanine), GGG (glycine), and CCC (proline) were placed into the corners of the fields as the main codons (and amino acids) of the fields. They were connected to each other by six axes. The resulting nucleic acid periodic table showed perfect axial symmetry for codons. The corresponding amino acid table also displaced periodicity regarding the biochemical properties (charge and hydropathy) of the 20 amino acids and the position of the stop signals. The table emphasizes the importance of the central nucleotide in the codons and predicts that purines control the charge while pyrimidines determine the polarity of the amino acids. This prediction was experimentally tested.

Inhibitory effect of the uterus on plasma and pituitary FSH in rats.

Plasma concentrations of FSH and LH were measured in ovariectomized, ovohysterectomized, hysterectomized and sham-operated adult, non-pregnant rats at 3, 14, 21 and 28 days after operation. From day 21 after the operation onwards, there were higher concentrations of FSH in plasma in ovohysterectomized than in ovariectomized animals. The concentration of LH was not influenced by hysterectomy. The inhibitory response of FSH and LH to a single dose of oestradiol was not altered by any of the operations. By 2 weeks after surgery, pituitary FSH content had increased in ovohysterectomized animals compared with ovariectomized ones, but this difference was eliminated when ovohysterectomized animals were treated with crude uterine extract. Pituitary contents of LH and prolactin were not influenced by hysterectomy or by treatment with uterine extract, thus indicating the specificity of an inhibitory effect of the uterus on FSH levels. Treatment of hysterectomized and intact animals with uterine extract resulted in a reduction in the weight of the ovaries of 23-38% (P less than 0.05), indirectly showing the presence of an FSH-inhibiting substance in the extract. Fractionated uterine extract inhibited FSH synthesis by rat pituitary cells in vitro, but had no effect on LH synthesis. Chromatographic analysis indicated that the FSH-inhibiting substance in the uterus has a molecular weight of 10,000-20,000.

Growth hormone releasing and inhibin-like activity in rat uterus: an in vitro study.

Cytosol from uteri of intact rats stimulated growth hormone (GH) release from anterior pituitary cells in vitro. Cytosol from uteri from ovariectomized (OVX) animals had the same effect, but approximately 30-times less. Estradiol treatment in vivo increased such activity in the uteri of OVX rats. To characterize and partially purify this GH releasing activity, crude extracts from uteri of intact and OVX rats, from liver, and plasma were fractionated on Sephadex G-100 superfine columns and the effects of different fractions on the synthesis and release of GH studied and compared to the effects on the synthesis of thyrotropin (TSH), follitropin (FSH), and lutotropin (LH). Fractions containing molecules with high molecular weight (MW greater than or equal to 40,000, HMWF) in these tissues and plasma stimulated GH release. Furthermore, fractions containing molecules with lower MW (10,000-20,000, LMWF) from intact uteri and plasma, but not from uteri of OVX rats and liver had similar GH-releasing activity. LMWF from uteri of both intact and OVX rats but not from plasma and liver inhibited FSH synthesis without having any effects on LH synthesis. Tissue and plasma fractions did not show any effects on TSH and LH synthesis. It was concluded that the rat uterus contained two GH-releasing activities: one which was associated with HMWF and the other one with LMWF. The GH-releasing activity in LMWF was probably induced by estradiol and secreted into the plasma. Furthermore, inhibin-like activity was present in LMWF from uteries.

Effects of the uterus and the ovaries on growth, somatotropin and somatomedin A in developing rats.

The effects of hysterectomy and ovariectomy on plasma concentrations of GH, somatomedin A, TSH and thyroxine (T4) were studied in developing rats. Four groups of 24-day-old rats were ovariectomized, ovohysterectomized, hysterectomized or sham-operated. Their weights, lengths and plasma hormone concentrations were measured at 26, 43, 64, 78 and 92 days of age to investigate pre- and postpubertal differences caused by the uterus or ovaries. Plasma concentrations of the hormones examined showed a successive rise with time, but GH and somatomedin A concentrations rose mainly after the opening of the vagina (days 50-55). Higher GH and somatomedin A concentrations were found in the plasma of ovariectomized animals than in ovohysterectomized controls before puberty (GH: 260-300%, P less than 0.01; somatomedin A: 25-30%, P less than 0.05). Ovariectomized animals weighed more than ovohysterectomized females after puberty (4.5-6%, P less than 0.01). This indicated that the uterus exerted a stimulatory effect on GH-somatomedin A regulation and body weight gain in the absence of the ovaries. Significantly lower plasma somatomedin A (but not GH) concentrations were found in hysterectomized and sham-operated animals than in their respective controls after puberty (30-39%, P less than 0.01) and their final body weight was lower (22-26%, P less than 0.001). There were no consecutive uterus- or ovary-related changes in plasma TSH and T4 levels. It was concluded that both the uterus and ovaries had significant as well as opposite effects on somatomedin A and body weight with the effects of the ovaries being greater than those of the uterus.

Effects of hysterectomy and uterine extracts on the gonadotrophic hormones and the weight of endocrine organs of female rats.

The previous observation that hysterectomy of rats results in increased pituitary nucleic acid turnover was followed up by studies on pituitary function in hysterectomized adult female rats with or without treatment with charcoal-treated crude uterine aqueous extracts. Hysterectomy had no effect on the pituitary or plasma LH and FSH concentrations. Administration of crude uterine extracts diminished the post-castrational rise of the pituitary FSH content without affecting the plasma FSH or LH concentrations. Hysterectomy decreased body weight but increased adrenal weight of ovariectomized animals. However, the body weight and pituitary weight of both the intact and ovariectomized animals increased significantly following 14 days of treatment with uterine extracts. The ovarian weight of the intact rats was reduced by this treatment. The pituitary nucleic acid content of castrated but oestradiol-substituted rats was reduced by hysterectomy. Plasma corticosterone concentrations were not affected by the above mentioned treatments. Uterine extracts inhibited the basal and GnRH induced LH release from isolated pituitary cells in culture. The effects of hysterectomy and/or administration of crude uterine aqueous extracts show that the uterus contains biologically active, non-steroidal substances which affect pituitary function of adult female rats.

Frequent occurrence of short complementary sequences in nucleic acids.

The hypothesis, that nucleic acids which code specifically interacting receptor and ligand proteins contain complementary sequences was tested. Human insulin mRNA (HSINSU) contained 16 sequences which were 23.8 +/- 1.4 nucleotides long and were complementary to the insulin receptor mRNA (HSIRPR, 74.8 +/- 1.9% complementary matches, p less than 0.001 compared to randomly occurring matches). However, when examining 10 different nucleic acids (coding proteins not interacting with the insulin receptor), 81 additional sequences were found which were also complementary to HSIRPR. Although the finding of short complementary sequences was statistically highly significant, we concluded that this is not specific for nucleic acids coding specifically interacting proteins.

Estrogen-induced proteins: a new class of regulatory substances.

The discovery of estrogen-induced growth factors (estromedins) identified a missing link in understanding endocrine regulation. Estromedins are probably not on in vitro mitogens or auto-/paracrine growth regulators but play an important endocrine regulatory role. A review of the literature and our own previous in vivo endocrine experiments with uterus and uterine extracts support this concept. We have also discussed the effects of some other newly discovered endocrine regulators on the synthesis/release and peripheral action of gonadotropins and lactogens. We conclude that our view on endocrine regulation is fundamentally changing. In addition to the classical endocrine axis (hypothalamus-pituitary-peripheral tissue) several new factors and conditions have to be taken into consideration to understand the regulation of an endocrine effect on a target organ.