A conformational rationale for the origin of the mechanism of nucleicacid-directed protein synthesis of ‘living’ organisms

The physical basis for the natural evolution of a primitive decoding system is presented using the concepts of molecular interactions. Oligoribonucleotides of five residues havingU at the 5-end, a purine at the 3-end and any combination of three bases in the middle is taken as a primitive tRNA (PIT). From conformational considerations PIT is expected to haveU-turn conformation wherein, N₃–H₃ of baseU hydrogen-bonds with phosphate, three residues ahead leaving triplet bases called primitive anticodons (PAC) into a helical conformation, and this creates a cleft betweenU and PAC. An amino acid can be comfortably nestled into the cleft with the amide hydrogens and carboxyl oxygen hydrogen-bonded to the last purine and the first uridine, while the side-chain can interact with the cleft side of PAC. The other side of PAC is free to base-pair with triplet codons on a longer RNA. Also two PACs can recognize consecutive triplet codons, and this leads to a dynamic interaction in which the amino and carboxyl ends are brought into proximity, making the formation of peptide bond feasible.The cleft formed by different anticodon triplets, broadly speaking, shows preferences for the corresponding amino acids of the presently known codon assignment.Thus the nucleicacid-directed protein synthesis, which is a unique feature of all living organisms is shown to be a natural consequence of a particular way of favourable interaction between nucleic acids and amino acids, and our model provides the missing link between the chemical evolution of small organic molecules and biological evolution through the process of mutations in nucleicacids and nucleicacid-directed protein synthesis.Contribution No. 507 from this department.     

Origins of life: conformational energy calculations on primitive tRNA nestling an amino acid

We report conformational energy calculations on our proposal of a molecular interaction theory for the origin of the nucleic acid-directed, adaptor-mediated synthesis of proteins that links the phenomena of chemical and biological evolution. A particular conformation of a pentanucleotide turns out to be a double-sided template for a primitive decoding system. It is able to neatly nestle an amino acid via hydrogen bonds, and this complex is found to be an energetically favourable conformation. The total potential energy of the complex is calculated using semi-empirical potential energy functions. A local-minimum conformation is obtained and its features are reported. The template conformation of the pentanucleotide is found to have an energy value far lower than a regular helical conformation. When the amino acid is nestled in the cleft of the template-conformation through specific hydrogen bonds, the energy is further lowered. A D-amino acid nestled into the PIT (Primitive tRNA) is found to be less stable than its L counterpart, as revealed by energy calculations.     

D-configuration of serine is crucial in maintaining the phalloidin-like conformation of viroisin.

NMR studies have revealed that the conformation of the monocyclic viroisin is dissimilar to that of the corresponding monocyclic derivative of phalloidin, dethiophalloidin, but has much similarity with the conformation of the bicyclic phalloidin. Obviously, one of three structural features found exclusively in the virotoxins is able to compensate for the conformational strain that in the bicyclic phallotoxins maintains the toxic conformation. Synthetic work on virotoxin analogues has shown that both the additional hydroxy group in allo-hydroxyproline and the methylsulfonyl moiety in the 2'-position of tryptophan are unlikely to represent the structural element in question, leaving the D-serine moiety as the supposed key element. In this study we asked whether it is the hydroxy group of this amino acid or its D-configuration that is responsible for the effect. We synthesized four viroisin analogues and submitted them to conformational analysis by NMR as well as to an actin binding assay. While the rotating-frame nuclear Overhauser effect (ROESY) spectra of the analogues with L-configured amino acids showed several sets of signals, indicating the existence of conformers interconverting more slowly than the NMR time scale, the spectra of the analogues with D-configured amino acids showed only one set of signals. Remarkably, the two viroisin analogues with D-serine and D-alanine also had distinctly higher affinities for filamentous actin than their L-configured counterparts, suggesting that the high biological activity may be correlated with the absence of multiple and slowly interconverting conformers. Anyhow, D-configuration of serine is the structural element that maintains the phalloidin-like structure, while the hydroxy group does not contribute to conformational stability but is likely to be in contact with the actin surface.     

Guest-dependent conformation of 18-crown-6 tetracarboxylic acid: relation to chiral separation of racemic amino acids

(+)-18-crown-6 tetracarboxylic acid (18C6H(4)) has been used as a chiral selector for various amines and amino acids. To further clarify the structural scaffold of 18C6H(4) for chiral separation, single crystal X-ray analysis of its glycine(+) (1), H3O+ (2), H5O2+ (3), NH4+ (4), and 2CH3NH3+ (5) complexes was performed and the guest-dependent conformation of 18C6H(4) was investigated. The crown ether ring of 18C6H4 in 3, 4, and 5 took a symmetrical C2 or C2-like conformation, whereas that in 1 and 2 took an asymmetric C1 conformation, which is commonly observed in complexes with various optically active amino acids. The overall survey of the present and related complexes suggests that the molecular conformation of 18C6H4 is freely changeable within an allowable range, depending on the molecular shape and interaction mode with the cationic guest. On the basis of the present results, we propose the allowable conformational variation of 18C6H4 and a possible transition pathway from its primary conformation to the conformation suitable for chiral separation of racemic amines and amino acids.     

Amino acid conformational preferences and solvation of polar backbone atoms in peptides and proteins

Amino acids in peptides and proteins display distinct preferences for alpha-helical, beta-strand, and other conformational states. Various physicochemical reasons for these preferences have been suggested: conformational entropy, steric factors, hydrophobic effect, and backbone electrostatics; however, the issue remains controversial. It has been proposed recently that the side-chain-dependent solvent screening of the local and non-local backbone electrostatic interactions primarily determines the preferences not only for the alpha-helical but also for all other main-chain conformational states. Side-chains modulate the electrostatic screening of backbone interactions by excluding the solvent from the vicinity of main-chain polar atoms. The deficiency of this electrostatic screening model of amino acid preferences is that the relationships between the main-chain electrostatics and the amino acid preferences have been demonstrated for a limited set of six non-polar amino acid types in proteins only. Here, these relationships are determined for all amino acid types in tripeptides, dekapeptides, and proteins. The solvation free energies of polar backbone atoms are approximated by the electrostatic contributions calculated by the finite difference Poisson-Boltzmann and the Langevin dipoles methods. The results show that the average solvation free energy of main-chain polar atoms depends strongly on backbone conformation, shape of side-chains, and exposure to solvent. The equilibrium between the low-energy beta-strand conformation of an amino acid (anti-parallel alignment of backbone dipole moments) and the high-energy alpha conformation (parallel alignment of backbone dipole moments) is strongly influenced by the solvation of backbone polar atoms. The free energy cost of reaching the alpha conformation is by approximately 1.5 kcal/mol smaller for residues with short side-chains than it is for the large beta-branched amino acid residues. This free energy difference is comparable to those obtained experimentally by mutation studies and is thus large enough to account for the distinct preferences of amino acid residues. The screening coefficients gamma(local)(r) and gamma(non-local)(r) correlate with the solvation effects for 19 amino acid types with the coefficients between 0.698 to 0.851, depending on the type of calculation and on the set of point atomic charges used. The screening coefficients gamma(local)(r) increase with the level of burial of amino acids in proteins, converging to 1.0 for the completely buried amino acid residues. The backbone solvation free energies of amino acid residues involved in strong hydrogen bonding (for example: in the middle of an alpha-helix) are small. The hydrogen bonded backbone is thus more hydrophobic than the peptide groups in random coil. The alpha-helix forming preference of alanine is attributed to the relatively small free energy cost of reaching the high-energy alpha-helix conformation. These results confirm that the side-chain-dependent solvent screening of the backbone electrostatic interactions is the dominant factor in determining amino acid conformational preferences.     

Binding modes of inhibitors to ribonuclease T1 as elucidated by the analysis of two-dimensional NMR

Aromatic proton and high field shifted methyl proton resonances of RNase T1 complexed with Guo, 2'GMP, 3'GMP or 5'GMP were assigned to specific amino acid residues by 2D-NMR spectra in comparison with the crystal structure of RNase T1-2'GMP complex. The spatial proximities of amino acid residues as elucidated by NOESY spectra were found to be quite similar among free RNase T1 and the inhibitor complexes, showing that large conformational changes did not occur upon complex formation. However, small but appreciable conformational changes were induced which were reflected by the systematic chemical shift changes of some amino acid residues in the active site. Furthermore, we confirmed that RNase T1 contains two specific binding sites, one for the guanine base and the other for the phosphate moiety. The inhibitors are forced to adapt their conformations to fit the guanine base and the phosphate moiety to each binding site on the enzyme. This is consistent with our previous studies that 2'GMP and 3'GMP take syn form as a bound conformation, while 5'GMP takes anti conformation around glycosidic bonds.     

Nuclear Overhauser effect studies on the conformations of Mg(alpha,beta-methylene)ATP bound to Escherichia coli methionyl-tRNA synthetase

Internuclear distances obtained from nuclear Overhauser effects were used in combination with a distance geometry algorithm to determine the conformation of Mg(alpha,beta-methylene)ATP bound to the Escherichia coli truncated methionyl-tRNA synthetase (delta MTS) both in the absence and presence of cognate and noncognate amino acids. Mg(alpha,beta-methylene)ATP, a nonhydrolyzable analog of ATP, was used to prevent hydrolysis of the nucleotide in the presence of either cognate or noncognate amino acids. Kinetic analysis showed that Mg(alpha,beta-methylene)ATP was a linear competitive inhibitor with respect to ATP in the ATP-pyrophosphate exchange reaction with a Ki = 1.2 mM. The pattern of internuclear Overhauser effects on Mg(alpha,beta-methylene)ATP bound to delta MTS was qualitatively consistent only with an anti glycosidic torsional angle, suggesting that the adenosine portion of the nucleotide is uniquely oriented in the binary enzyme-nucleotide complex. Nearly identical patterns of nuclear Overhauser effects were also observed in ternary complexes containing either cognate L-methionine or noncognate L-homocysteine amino acids. Distance geometry calculations permitted the range and conformational space of the allowed adenine-ribose glycosidic torsional angles in each of the complexes to be better defined and compared. Average adenine-ribose glycosidic torsional angles for enzyme-bound Mg(alpha,beta-methylene)ATP of -106 +/- 9 degrees, -99 +/- 11 degrees, and -97 +/- 11 degrees were determined for the delta MTS.Mg(alpha,beta-methylene)ATP, delta MTS.Mg(alpha,beta-methylene)ATP.L-methionine, and delta MTS.Mg(alpha,beta-methylene)ATP.L-homocysteine complexes, respectively. Comparison of the three enzyme-bound conformations showed that a single nucleotide structure having an adenine-ribose glycosidic torsional angle of -98 degrees with a 3'-endo to O4'-exo ribose sugar pucker was, within error, consistent with the experimental internuclear distances obtained in all three complexes. The nearly identical anti glycosidic torsional angles observed in all three complexes demonstrates that the conformation of the adenosine moiety of the enzyme-bound nucleotide is not sensitive to the presence or the nature of the amino acid bound at the aminoacyladenylate site. Therefore, conformational changes known to occur in the methionyl-tRNA synthetase upon ligand binding appear not to alter the bound conformation of the nucleotide. Information on the conformation and arrangement of substrates bound at the aminoacyladenylate site of delta MTS is necessary for understanding the molecular mechanisms involved in amino acid activation and discrimination.     

Origin of life: A hypothesis for the origin of adaptor-mediated ordered synthesis of proteins and an explanation for the choice of terminating codons in the genetic code

Life can be defined as a system of self-sustained chemical processes springing from the ordered synthesis of proteins directed by nucleic acids. To the notoriously difficult problem of the origin of this basic process of nucleic acid-directed protein synthesis, we give a solution of molecular interactions between pentanucleotides and amino acids. A particular conformation of a pentanucleotide forms a double sided template, with its ‘inside’ capable of nestling an amino acid while the ‘outside’ acts as an adaptor to a ‘codon’ triplet on long-chain nucleic acids. This serves as a primitive decoding system. An important aspect of our postulate is that a dynamic interaction is triggered, by this decoding system, through which amino acids are brought to juxtaposition facilitating peptide bond formation. Almost all the important and unique features of contemporary protein-synthesizing machinery are seen to be a direct and natural consequence of our postulate. The emergence of the termination codons also fits in, as a natural consequence of this molecular mechanism.     

Modeling, design, chiral aspects and role of para-substituents in aryloxypropranolamine based beta-blockers.

The conformation of the beta-blockers viz. metoprolol, atenolol, bisoprolol, betaxolol and celiprolol has been investigated using Perturbative Configuration Interaction of Localized Orbitals (PCILO) method. The conformational energy maps have been constructed for both the enantiomers (R and S) by rotating the molecule from the para-substituent end. The aryloxypropranolamine moiety adopts the same conformation for all antagonists. The graphical view of R- and S- form of these antagonists in the lowest energy conformation reveals that it is only in the S- form of beta-blockers, all the three functionalities--aromatic moiety, amino and beta-hydroxyl groups are available for interaction with beta-adrenoceptors. The para-substituents of the beta-blockers adopt a conformation which is perpendicular to the aryloxy moiety resulting in an L-shaped structure. The beta-antagonists possibly partition into the lipid bilayer through the para-substituents and the aryloxypropranolamine moiety containing the functionalities, thus, lies parallel to the plane of lipid bilayer for interaction with beta-adrenoceptors. Superimposition of S-bisoprolol in lowest energy conformation with the 3rd putative transmembranous segment of the beta-adrenoceptors reveals that the aromatic moiety, amino and beta-hydroxyl groups of antagonists are involved in interaction with the side chains of Trp-109, Asp-113 and Thr-110 respectively. This has been further substantiated by the interaction studies on the model systems.     

New tubular single-stranded helix of poly-L-amino acids suggested by molecular mechanics calculations: I. Homopolypeptides in isolated environments.

A search was made in terms of molecular mechanics calculation for tubular, or pore-forming, single-stranded helices of poly-L-amino acids. Such a helix was found in the vicinity of (phi, psi, omega) = 81 degrees, 98 degrees, 170 degrees) in the conformational space. It was the 6.2(20) helix of right-handedness. The 6.2(20) helix, here named the "mu helix," had a cylindrical pore along its helical axis. The diameter of the pore was 6.6 A on the basis of the atom centers of carbonyl carbons and amino nitrogens. The left-handed mu helix was less stable than the right-handed counterpart. The conformation energy of the mu helix, expressed relative to that of the alpha helix of the same polypeptide, depended to a great extent on amino acid composition. It varied over a range of a few kilocalories per mol per residue above and below the conformation energy of the alpha helix of the same polypeptide. This marked diversity in the relative conformation energy of the mu helix can be ascribed primarily to the difference in the relative position of alpha-carbons between the mu and the alpha helices. The conformational entropy made only a small contribution, if any, to the relative stability of the mu helix. There was a hydrogen-bonded network of side chains in the mu helices of poly-L-glutamine and poly-L-asparagine.     

Interplay between structural rigidity and electrostatic interactions in the ligand binding domain of GluR2

Using molecular dynamics (MD) simulations, computational protein modifications, and a novel theoretical methodology that determines structural rigidity/flexibility (the FIRST algorithm), we investigate how molecular structure and dynamics of the glutamate receptor ligand binding domain (GluR2 S1S2) facilitate its conformational transition. S1S2 is a two-lobe protein, which undergoes a cleft closure conformational transition upon binding an agonist in the cleft between the two lobes; hence it is expected that the mechanism of this conformational transition can be characterized as a hinge-type. However, in the rigidity analysis one lobe of the protein is identified as a single rigid cluster while the other one is structurally flexible, inconsistent with a presumed mechanical hinge mechanism. Instead, we characterize the cleft-closing transition as a load and lock mechanism. We find that when two cross-cleft hydrogen bonds are disrupted the protein undergoes a rapid cleft opening transition. At the same time, the dynamical behavior of the cleft in the presence of the glutamate ligand is only weakly affected by the S652 peptide bond in its flipped conformation observed in the crystal structure. The residue E705 plays significant role in stabilization of the closed conformation via electrostatic interactions. The presence of the E705-K730 salt bridge seems to correlate strongly withthe cleft opening transition in the MD simulations.     

Conformational features of the Dippu-AST 8 neuropeptide from the cockroach Diploptera punctata

Spatial structure and conformational properties of the Dippu-AST 8 (allatostatin III) neuropeptide have been investigated by the molecular mechanics method. The conformational energy and geometrical parameters corresponding to the low-energy states of the molecule are obtained. A single backbone conformation with a very restricted set of Ser 3, Phe 4 and Leu 9 amino acids positions is observed for Dippu-AST 8 neuropeptide.     

Insights into the effects of glycosylation and the monosaccharide-binding activity of the plant lectin CrataBL

CrataBL is a glycoprotein isolated from Crataeva tapia bark, containing two N-glycosylation sites. It has been identified to present lectin activity with some specificity for binding glucose over galactose. However, to date, no information on the effects of glycosylation or CrataBL monosaccharide-binding sites and monosaccharide specificity has been obtained. Thus, molecular docking and molecular dynamics simulations were employed to characterize the glycosylated CrataBL conformation and dynamics in aqueous solutions, as well as the molecular basis for its binding specificity. The obtained results indicate both local and distant conformational stabilization effects of N-linked glycans over CrataBL protein moiety. Regarding its lectin activity, molecular docking calculations were performed in two possible binding sites, identified through sequence-based, structure-based and evolutionary information, using α- and β-anomeric states of the monosaccharides. The obtained poses were further refined through molecular dynamics simulations, suggesting that positively-charged amino acids dictate the binding preference for glucose over galactose in both sites. In addition, a possible preference for β-monosaccharides was proposed. Such data are expected to contribute to a better comprehension of the lectins monosaccharide-binding activities and carbohydrate-binding site structures.     

Aminoacyl thiol esters and the origins of genetic specificity

Reasons for believing that primitive mechanisms of translation may have employed thiol esters of the amino acids rather than oxygen esters are summarized. It is suggested that coenzyme A (HSCoA), which fulfills the role of aminoacyl transfer in the synthesis of peptide antibiotics, is a primitive analogue of tRNA which performs a similar role in protein synthesis. HSCoA—an adenylic acid moiety containing phosphates esterified at the 3′ and 5′ positions and linked to a peptide-like structure terminating in a reactive thiol—possesses chemical features suggestive of both peptides and polynucleotides. Examination of the chemistry of HSCoA-like molecules shows that a rather similar compound can carry out a repeating intramolecular peptide synthesis in the absence of enzymes. Condensation of further nucleotides onto the adenylic acid moiety gives rise to parallel modes of peptide and oligonucleotide synthesis. A “self-improving” ability to select available amino acids is inherent in the proposed mechanism of peptide synthesis. The hypothesis plausibly explains the universal occurrence of a sulphur-containing amino acid at the N terminus of nascent proteins.     

Investigation of penetratin peptides. Part 1. The environment dependent conformational properties of penetratin and two of its derivatives.

The homeodomain, the DNA-binding domain of Antennapedia homeoprotein, is composed of three alpha-helices and one beta-turn between helices II and III. Its third helix from the N-terminal (helix III) can translocate through the cell membrane into the nucleus and can be used as an intracellular vehicle for the delivery of oligopeptides and oligonucleotides. To the best of our knowledge, this helix III, called penetratin, which consists of 16 amino acids, is internalized by cells in a specific, non-receptor-mediated manner. For a better understanding of the mechanism of the transfer, the structure of penetratin was examined in both extracellular matrix-mimetic and membrane-mimetic environments: 1H-NMR and CD spectroscopic measurements were performed in mixtures of TFE/water with different ratios. The molecular conformations of two analogue peptides [(6,14-Phe)-penetratin and a 12 amino acid penetratin derivative (peptide 3)] were also studied. An atomic level comprehensive analysis of penetratin and its two analogues was performed. In a membrane-mimetic solvent system (TFEd2/water = 9: 1), on the basis of 553 distance restraints, the 4-12 region of penetratin exhibits a bent, irregular helical structure on NMR examination. Interactions between hydrophobic amino acid residues in conjunction with H-bonds stabilize the secondary structure of the molecule. Thus, both derivatives adopt a helix-like conformation. However, while (6,14-Phe)-penetratin displays both alpha-helical and 310-helical features, the structure of peptide 3 is predominantly a 310-helix. Of the three peptides, surprisingly (6,14-Phe)-penetratin has the largest helical content. An increase in the polarity of the molecular environment gradually disintegrates these helix-like secondary structures. In a highly aqueous molecular system (TFEd2/water = 1 : 9), the fast exchange of multiple conformers leads to too few distance restraints being extracted, therefore the NMR structures can no longer be determined. The NMR data show that only short-range order can be traced in these peptides. Under these conditions, the molecules adopt nascent helix-like structures. On the other hand, CD spectra could be recorded at any TFE/water ratio and the conformational interconversion could therefore be monitored as a function of the polarity of the molecular environment. The CD data were analysed comprehensively by the quantitative deconvolution method (CCA+). All three penetratin peptides display helical conformational features in a low dielectric medium, with significant differences as a function of their amino acid composition. However, these conformational features are gradually lost during the shift from an apolar to a polar molecular environment.     

Molecular dynamics simulations give insight into the conformational change,complex formation,and electron transfer pathway for cytochrome P450 reductase

Cytochrome P450 reductase (CYPOR) undergoes a large conformational change to allow for an electron transfer to a redox partner to take place. After an internal electron transfer over its cofactors, it opens up to facilitate the interaction and electron transfer with a cytochrome P450. The open conformation appears difficult to crystallize. Therefore, a model of a human CYPOR in the open conformation was constructed to be able to investigate the stability and conformational change of this protein by means of molecular dynamics simulations. Since the role of the protein is to provide electrons to a redox partner, the interactions with cytochrome P450 2D6 (2D6) were investigated and a possible complex structure is suggested. Additionally, electron pathway calculations with a newly written program were performed to investigate which amino acids relay the electrons from the FMN cofactor of CYPOR to the HEME of 2D6. Several possible interacting amino acids in the complex, as well as a possible electron transfer pathway were identified and open the way for further investigation by site directed mutagenesis studies.     

Correlation between symmetry breaker position and the preferences of conformationally constrained homopeptides: a molecular dynamics investigation

The conformational tendencies of C(alpha,alpha)-diethylglycine (Deg)-based peptides have been studied in solution using all atom molecular dynamics simulations. Specifically, the conformational effects of breaking the symmetry of the host Tfa-(Deg)(5)-OtBu (Tfa, trifluoroacetyl; OtBu, tert-butoxy) pentapeptide with punctual replacements at different sequence positions of one Deg residue by its corresponding guest chiral analogue, L-alpha-aminobutyric acid (L-Abu), have been examined by simulating the following peptides: Tfa-(Deg)(5)-OtBu, Tfa-(Deg)(2)-L-Abu-(Deg)(2)-OtBu, Tfa-(Deg)(3)-L-Abu-Deg-OtBu, and Tfa-(Deg)(4)-L-Abu-OtBu. Simulations show that only the Deg homopeptide is able to stabilize a 2.0(5) helix, even though a kinked arrangement with all the Deg residues adopting a fully-extended conformation was found to be stable when the L-Abu residue is introduced in the middle of the sequence. On the other hand, when the L-Abu residue is closer to the C-end of the sequence, the peptide chain prefers a partially folded 3(10)-helix. Additional simulations on Tfa-(Deg)(3)-L-Abu-(Deg)(3)-OtBu highlighted that, when the size of the Deg segments increases, their tendency to adopt a 2.0(5) helix predominates over the preferred folded conformation of L-Abu. The overall picture extracted after more than 300 ns of molecular dynamics simulation is that breaking the alpha-carbon symmetry of achiral C(alpha)-tetrasubstituted amino acids is a promising strategy to build up polypeptides with modulated conformational tendencies.     

Use of Chou-Fasman amino acid conformational parameters to analyze the organization of the genetic code and to construct protein genealogies

Summary Chou-Fasman parameters, measuring preferences of each amino acid for different conformational regions in proteins, were used to obtain an amino acid difference index of conformational parameter distance (CPD) values. CPD values were found to be significantly lower for amino acid exchanges representing in the genetic code transitions of purines, GA than for exchanges representing either transitions of pyrimidines, CU, or transversions of purines and pyrimidines. Inasmuch as the distribution of CPD values in these non GA exchanges resembles that obtained for amino acid pairs with double or triple base differences in their underlying codons, we conclude that the genetic code was not particularly designed to minimize effects of mutation on protein conformation. That natural selection minimizes these changes, however, was shown by tabulating results obtained by the maximum parsimony method for eight protein genealogies with a total occurrence of 4574 base substitutions. At the beginning position of the codons GA transitions were in very great excess over other base substitutions, and, conversely, CU transitions were deficient. At the middle position of the codons only fast evolving proteins showed an excess of GA transitions, as though selection mainly preserved conformation in these proteins while weeding out mutations affecting chemical properties of functional sites in slow evolving proteins. In both fast and slow evolving proteins the net direction of transitions and transversions was found to be from G beginning codons to non-G beginning codons resulting in more commonly occurring amino acids, especially alanine with its generalized conformational properties, being replaced at suitable sites by amino acids with more specialized conformational and chemical properties. Historical circumstances pertaining to the origin of the genetic code and the nature of primordial proteins could account for such directional changes leading to increases in the functional density of proteins.In order to further explore the course of protein evolution, a modified parsimony algorithm was developed for constructing protein genealogies on the basis of minimum CPD length. The algorithm's ability to judge with finer discrimination that in protein evolution certain pathways of amino acid substitution should occur more readily than others was considered a potential advantage over strict maximum parsimony. In developing this CPD algorithm, the path of minimum CPD length through intermediate amino acids allowed by the genetic code for each pair of amino acids was determined. It was found that amino acid exchanges representing two base changes have a considerably lower average CPD value per base substitution than the amino acid exchanges representing single base changes. Amino acid exchanges representing three base changes have yet a further marked reduction in CPD per base change. This shows how extreme constraining effects of stabilizing selection can be circumvented, for by way of intermediate amino acids almost any amino acid can ultimately be substituted for another without damage to an evolving protein's conformation during the process.