Random Amino Acid Sequences Research Articles

Folded proteins occur frequently in libraries of random amino acid sequences.

A library of synthetic genes encoding 80- to 100-residue proteins composed mainly of random combinations of glutamine (Q), leucine (L), and arginine (R) has been expressed in Escherichia coli. These genes also encode an epitope tag and six carboxyl-terminal histidines. Screening of this library by immunoblotting showed that 5% of these QLR proteins are expressed at readily detectable levels. Three well-expressed QLR proteins were purified and characterized. Each of these proteins has significant alpha-helical content, is largely resistant to degradation by Pronase, and has a distinct oligomeric structure. In addition, one protein unfolds in a highly cooperative manner. These properties of the QLR proteins demonstrate that they possess folded structures with some native-like properties. The QLR proteins differ from most natural proteins, however, in being remarkably resistant to denaturant-induced and thermal-induced unfolding and in being relatively insoluble in the absence of denaturants.

The evolution of proteins from random amino acid sequences. I. Evidence from the lengthwise distribution of amino acids in modern protein sequences

We examine in this paper one of the expected consequences of the hypothesis that modern proteins evolved from random heteropeptide sequences. Specifically, we investigate the lengthwise distributions of amino acids in a set of 1,789 protein sequences with little sequence identify using the run test statistic (ro) of Mood (1940, Ann. Math. Stat. 11, 367-392). The probability density of ro for a collection of random sequences has mean = 0 and variance = 1 [the N(0,1) distribution] and can be used to measure the tendency of amino acids of a given type to cluster together in a sequence relative to that of a random sequence. We implement the run test using binary representations of protein sequences in which the amino acids of interest are assigned a value of 1 and all others a value of 0. We consider individual amino acids and sets of various combinations of them based upon hydrophobicity (4 sets), charge (3 sets), volume (4 sets), and secondary structure propensity (3 sets). We find that any sequence chosen randomly has a 90% or greater chance of having a lengthwise distribution of amino acids that is indistinguishable from the random expectation regardless of amino acid type. We regard this as strong support for the random-origin hypothesis. However, we do observe significant deviations from the random expectation as might be expected after billions years of evolution. Two important global trends are found: (1) Amino acids with a strong alpha-helix propensity show a strong tendency to cluster whereas those with beta-sheet or reverse-turn propensity do not. (2) Clustered rather than evenly distributed patterns tend to be preferred by the individual amino acids and this is particularly so for methionine. Finally, we consider the problem of reconciling the random nature of protein sequences with structurally meaningful periodic "patterns" that can be detected by sliding-window, autocorrelation, and Fourier analyses. Two examples, rhodopsin and bacteriorhodopsin, show that such patterns are a natural feature of random sequences.

Selection of antibody ligands from a large library of oligopeptides expressed on a multivalent exposition vector

Practically any oligopeptide can be exposed on the surface of the bacteriophage capsid by fusion to the major coat protein of filamentous bacteriophages. A phage expressing a particular peptide tag can be selected from a mixture of tens of millions of clones, exposing oligopeptides of random sequence, by affinity purification with a protein ligand. In this respect, pVIII can be used as an alternative and complement to the exposition vectors based on the product of gene III (pIII). We have constructed a phagemid vector that contains gene VIII under the control of the pLac promoter. This vector can be conveniently used to construct libraries of oligopeptides with a random amino acid sequence. An antipeptide monoclonal antibody was used to affinity-purify phagemids exposing oligopeptides which can interact with the monoclonal antibody. DNA sequencing of the amino terminus of gene VIII of the recovered clones predicts the synthesis of hybrid proteins whose amino-terminal amino acid sequence is related to that of the oligopeptide used to raise the antibody. In other words, only oligopeptides that bind a very small portion of the immunoglobulin G surface are affinity-purified by this method, implying that the antigen binding site possesses molecular properties that renders it much stickier than the remainder of the molecule.

Random sequences and protein folding

Key features of the structures of globular proteins are considered to show to what extent they demand the specific amino acid sequences strategically selected during biological evolution. It is shown that: (a) the length distributions of α- and β-regions in water-soluble globular proteins are similar to what can be expected for a random copolymer of polar and nonpolar residues; (b) the alteration of α- and β-regions in globular proteins is similar to that of the random one; (c) typical folding patterns (i.e., crude three-dimensional structures) of globular proteins coincide with the thermodynamically most stable folding patterns of random sequences (and even of chemically homogeneous polypeptides with unpolar side groups). The possibility cannot be excluded that even the tight packing inside protein molecules (making the protein molecule stable relative to thermal fluctuations) does not demand a very strict selection of amino acid sequences. The conclusion is drawn that primary structures of proteins are basically just the examples of random amino acid sequences which have only been “edited” during biological evolution in order to impart to them the additional (functional) meaning and the additional stability.

Random sequences and protein folding

Key features of the structures of globular proteins are considered to show to what extent they demand the specific amino acid sequences strategically selected during biological evolution. It is shown that: (a) the length distributions of α- and β-regions in water-soluble globular proteins are similar to what can be expected for a random copolymer of polar and nonpolar residues; (b) the alteration of α- and β-regions in globular proteins is similar to that of the random one; (c) typical folding patterns (i.e., crude three-dimensional structures) of globular proteins coincide with the thermodynamically most stable folding patterns of random sequences (and even of chemically homogeneous polypeptides with unpolar side groups). The possibility cannot be excluded that even the tight packing inside protein molecules (making the protein molecule stable relative to thermal fluctuations) does not demand a very strict selection of amino acid sequences. The conclusion is drawn that primary structures of proteins are basically just the examples of random amino acid sequences which have only been “edited” during biological evolution in order to impart to them the additional (functional) meaning and the additional stability.

Base pairing in messenger RNA's for small peptides.

Longest runs of Watson-Crick pairing in hypothetical m-RNA's for a number of natural peptides were no greater than those in the hypothetical m-RNA's for a large number of randomized amino acid sequences from these peptides. This shown that even if base-pairing in m-RNA were a biological requirement, it would little constrain the amino acid sequence.

Amino acid sequence studies on the branched, synthetic polypeptide antigens of the immune response‐1 gene system

Three immunogens with side chains of random amino acid sequence, poly (L Phe, l glu)-poly (DL Ala)--poly (L Lys) [(Phe, G)-A--L 223], poly (L Tyr, L Glu)-poly (DL Ala)--poly (L Lys) [(T, G)-A--L 509] and (T, G)-A-L 52, as well as two immunogens with side chains of defined amino acid sequences, GGT-A--L and TG-A--L, were sequenced using a Beckman automated sequenator. Despite the lack of a unique amino acid sequence for the amino terminus, reasonable results for the sequence studies were obtained using the Edman reaction. GGT-A--L and TG-A--L had 70% and 80% of their side chains respectively, with the desired sequence. The three compounds of random amino acid sequence were found to contain a large proportion of their A--L side chains unsubstituted. The side chains had a much greater probability of terminating in the aromatic amino acid than in the glutamic acid. The distribution of the length of side chains and their amino acid sequences was completely heterogeneous.

Distinguishing Homologous from Analogous Proteins

Fitch, W. M. (Dept. Physiological Chetn., U. Wisconsin, Madison 53706) 1970. Distinguishing homologous from analogous proteins. Syst. Zool., 19:99–113.—This work provides a means by which it is possible to determine whether two groups of related proteins have a common ancestor or are of independent origin. A set of 16 random amino acid sequences were shown to be unrelated by this method. A set of 16 real but presumably unrelated proteins gave a similar result. A set of 24 model proteins which was composed of two independently evolving groups, converging toward the same chemical goal, was correctly shown to be convergently related, with the probability that the result was due to chance being <10−21. A set of 24 cytochromes composed of 5 fungi and 19 metazoans was shown to be divergently related, with the probability that the result was due to chance being < 10−9. A process was described which leads to the absolute minimum of nucleotide replacements required to account for the divergent descent of a set of genes given a particular topology for the tree depicting their ancestral relations. It was also shown that the convergent processes could realistically lead to amino acid sequences which would produce positive tests for relatedness, not only by a chemical criterion, but by a genetic (nucleotide sequence) criterion as well. Finally, a realistic case is indicated where truly homologous traits, behaving in a perfectly expectable way, may nevertheless lead to a ludicrous phylogeny.