Proteins Rich in Prime Mass Residues and Their Role in Cell Biology

Abstract

Background:

Prime mass amino acids residues, assigned based upon the nominal mass of their repeating structure in peptides and proteins, have been found to occur more often than by chance. These 9 such residues are predominantly hydrophobic in character and thus play an important role in protein stability and folding. This study investigates their prevalence in human proteins and the function of such proteins.

Objectives:

To identify human proteins rich in the prime mass residues and assess their functions.

Results:

Prime-rich proteins are found to proliferate among those important to transcriptional events, cell adhesion, and the formation of cell surface membranes. Eighteen prime-rich proteins are identified that are abundant in 3 or 4 prime residues, the latter notably contain alanine, cysteine, histidine or threonine, and proline. The percentage of prime residues in these prime-rich proteins exceeds one-half (>50%) of all residues in many cases, and these residues are found to be concentrated in particular regions of the protein.

Conclusion:

Proteins rich in prime residues are found to play an important role in biological events important to cellular life. Such prime residues represent those associated with the highest hydrophobicity, and those essential to protein folding and function. Many prime residues were recruited early into the genetic code according to the composition of the last universal common ancestor (LUCA) proteins. Consideration is given to whether the formation of evolution of these proteins is predicated by the primality of the residues or their physicochemical and structural properties. This remains an open question and awaits further inquiry and debate.

Introduction

Amino acids have long been defined based on their physicochemical properties ranging from charged or non-charged, hydrophobic or hydrophilic, polar or non-polar, acidic or basic, and aliphatic or aromatic. Such definitions have their limitations ¹ since many residues exhibit multiple characteristics based on the nature of their side-chains, and measures of these properties differ both for the free amino acids and within the protein polymers comprised of them. Further, the number of scales for any one property is very large; there are more than a hundred scales related to hydrophobicity alone. ² This is confounding and computationally burdensome when attempting to accurately predict protein properties and define them numerically. ³

A more rigid definition of amino acids is associated with their physical mass. This is calculated from their elemental composition, through a summation of the masses of their component atoms. The nominal mass is even for the most abundant isotopes for carbon, nitrogen, oxygen, and sulphur; namely 12 (standardised), 14, 16 and 32 respectively. Only that for hydrogen is odd (ie, 1). ⁴ It has been found that amino acid residues with an odd number of hydrogen atoms have residue masses that are prime more often than by chance. ⁴

Prime mass residues are defined as those whose repeating unit [–NH–CH(R)–C(=O)–] in a peptide or protein chain have an odd number of hydrogen atoms and where the mass of this unit is divisible only by 1 and that mass. ⁴ Further, the total number of each element, carbon, hydrogen, nitrogen, oxygen, and sulphur, across all prime residues is also prime. ⁴

It is logical therefore to classify such molecular entities as prime based on their atomic construct.

Since all natural numbers can be expressed as a multiple of primes, prime numbers are considered to be the building blocks of natural numbers, as atoms are the building blocks of all matter.^5,6 Prime numbers have been found to have a real world significance in biology.^7-9 There is also an interest in being able to express proteins and other biological information in the form of numbers in order to construct a computable language.

There are 9 prime mass residues: alanine, cysteine, histidine, isoleucine, leucine, methionine, proline, threonine, and tyrosine. The majority are considered hydrophobic and thus play an important role in folding and structure formation by expelling water from a protein’s core. Cysteine, that forms bridges that restrict protein structures, is also a prime residue, as is its sulphur-containing counterpart methionine. Histidine and tyrosine are both metal-binding residues that help bridge residue side chains are also prime. Proline is also a prime residue. Its side chain attaches itself twice to the backbone at both the alpha-carbon and amide nitrogen atom. As a consequence, the flexibility of the protein’s backbone is restricted and this residue is known to disrupt alpha-helix formation and promote beta-turns.

In a symbol of their collective biological importance, two-thirds of prime residues (namely histidine, isoleucine, leucine, methionine, threonine, and even tyrosine) represent amino acids that are essential to the diet of humans. It is also noteworthy, from an evolutionary and coding perspective, that there is a strong correlation between the occurrence of each amino acid residue within proteins of the human proteome and their codon frequency ⁴ compared with the non-prime residues.

In this study their prevalence is examined more closely through the identification of all human proteins that are rich in prime residues. Further, the biological function of the identified proteins is reported together with their evolutionary significance in the early stages of life.

Materials and Methods

Designation of Prime Residues

Prime residues have been defined as those whose repeating unit [–NH–CH(R)–C(=O)–] in a peptide or protein chain have an integer or nominal mass that is a prime number, namely a number that is only divisible by the number 1 and itself. ⁴ Nine of a total fifteen odd mass residues, all of which contain an odd number of hydrogen atoms, have prime masses. These are alanine (A), cysteine (C), histidine (H), isoleucine (I), leucine (L), methionine (M), proline (P), threonine (T), and tyrosine (Y). ⁴ In single letter code, they form the word LYMPHATIC that can be conveniently recalled to remember them. Such residues are referred to interchangeably as prime mass and prime residues in this work.

Search of Uniprot/Swiss-Prot Databases for Prime-Rich Residues

The ScanProsite ¹³ online web-based tool (https://prosite.expasy.org/scanprosite/) was used to search the Prosite database. ¹⁰ The tool adopts ProRules dependent annotation templates to identify proteins within a database with particular structural and functional characteristics. The Prosite database contains the protein domains, families and functional sites ¹¹ derived from the combined Uniprot/SWISS-PROT (UniProtKB) database (Release 2025_02 of 09/04/25 containing 573,230 entries). Human proteins (lineage = Homo sapiens) rich in prime residues were identified using increasing numbers and combinations of the Proscan descriptors for each of the residues: PS50310 for alanine (A), PS50311 for cysteine (C), PS50316 for histidine (H), PS50317 for isoleucine (I), PS50319 for leucine (L), PS50320 for methionine (M), PS50099 for proline (P), PS50325 for threonine (T), and PS50328 for tyrosine (Y). As no hits were found for isoleucine, it was excluded from further analysis. The maximum number of residues found in prime rich proteins was found to be 4 with the combinations [A, C, H, P] and [A, C, T, P].

Identification of Amino Acid Composition in Prime-Rich Proteins and Their Components

The ProtParam ¹² online web-based tool (https://web.expasy.org/protparam/) was used to compute the amino acid compositions and the level of prime residue enrichment in each of the human proteins identified that were rich in 3 or 4 prime residues. The tool also reports domains rich in each prime residue, those residues of a particular physicochemical character, and those associated with particular structural motifs, disorder, or function. The same tool was also used to interrogate the prime-rich domains alone.

Results

Identification of Prime-Rich Proteins and Their Residue Enrichments

The Uniprot knowledgebase (UniprotKB) was searched for all human proteins rich in prime mass residues using the ScanProsite tool. ¹³ Using PROSITE signatures (of the form PS50XXX), ¹¹ the algorithm can search the combined Uniprot/SWISS-PROT protein database for proteins rich in specified residues; in this case prime-rich residues.

The combined protein database was searched accordingly for increasing numbers of the prime residues: alanine, cysteine, histidine, isoleucine, leucine, methionine, proline, threonine, and tyrosine. The results of searches for single and 2 prime-rich residues are shown in Supplemental Table 1. The number of proteins identified ranged from 0 for isoleucine to 1702 for proline alone (see shaded boxes). In combination (unshaded), the majority of hits were identified for prime pairs containing alanine, cysteine, histidine and proline. As no hits were found when searches were conducted for isoleucine, this residue was subsequently excluded from all further analyses.

To narrow the number of matches, a series of additional searches were performed for 3 of the remaining 8 prime residues at a time. These results are summarised in Supplemental Table 2. At most, the searches returned 1 hit each with 9 and 4 sequences, but most hits identified either 1 or 0 sequences (Table 2). Seven combinations of 3 prime residues that identified at least 1 protein are: [A, C, H], [A, H, P], [C, L, T], [L, T, P], [M, P, T], [A, T, P], [A, C, T]. Adding a fourth prime residue to each of these combinations, only 2, namely [A, C, H, P] and [A, C, T, P], returned a single protein hit each.

Table 2.

Percentage of Prime Residues in Prime-Rich Proteins.

Protein ID	No. of amino acids	% Total prime mass residues	% Each prime mass residue
Q9BXQ6	578	50.0	A	14.2
			C	3.5
			H	2.2
			I	2.1
			L	12.3
			M	0.9
			P	8.7
			T	4.0
			Y	2.1
Q9H1U4	602	51.2	A	8.6
			C	7.0
			H	1.8
			I	3.8
			L	0.7
			M	6.5
			P	10.0
			T	10.1
			Y	2.7
Q8NDT2	890	40.8	A	9.6
			C	0.6
			H	3.6
			I	2.1
			L	9.0
			M	1.1
			P	7.6
			T	3.9
			Y	3.3
Q4VCS5	1084	46.8	A	12.8
			C	0.7
			H	3.4
			I	2.9
			L	7.9
			M	2.4
			P	9.2
			T	5.4
			Y	2.1
P58012	376	56.2	A	17.3
			C	1.6
			H	3.7
			I	1.9
			L	6.1
			M	2.7
			P	16.2
			T	2.4
			Y	4.3
Q5VYV0	432	55.8	A	14.8
			C	0.9
			H	9.3
			I	2.3
			L	6.2
			M	2.3
			P	15.0
			T	2.5
			Y	2.5
P20264	500	47.8	A	14.0
			C	0.8
			H	6.6
			I	1.6
			L	6.6
			M	2.2
			P	10.8
			T	4.2
			Y	1.0
Q99626	313	49.6	A	12.8
			C	0.6
			H	4.5
			I	1.3
			L	7.7
			M	1.3
			P	13.7
			T	3.2
			Y	4.5
Q2VWA4	1015	45.8	A	11.9
			C	1.9
			H	4.6
			I	2.1
			L	7.9
			M	0.9
			P	12.8
			T	2.2
			Y	1.5
Q8NFD5	2319	44.0	A	9.7
			C	0.8
			H	2.9
			I	2.5
			L	6.0
			M	4.4
			P	11.6
			T	3.1
			Y	3.0
O14776	1098	43.2	A	9.9
			C	0.4
			H	1.4
			I	3.9
			L	5.1
			M	3.5
			P	10.8
			T	6.6
			Y	1.6
O60641	907	48.3	A	16.0
			C	0.4
			H	0.9
			I	2.3
			L	7.7
			M	2.9
			P	11.4
			T	8.6
			Y	1.0
P51610	2035	51.3	A	11.5
			C	1.7
			H	1.7
			I	3.8
			L	6.8
			M	1.9
			P	9.5
			T	13.2
			Y	1.2
Q14114	963	44.7	A	7.6
			C	6.5
			H	2.7
			I	4.0
			L	8.9
			M	1.1
			P	6.5
			T	5.3
			Y	2.1
P07359	652	52.1	A	4.3
			C	1.4
			H	2.3
			I	1.8
			L	16.7
			M	0.9
			P	11.8
			T	11.7
			Y	2.1
O43670	478	54.6	A	7.1
			C	1.0
			H	2.1
			I	4.2
			L	4.6
			M	6.5
			P	19.9
			T	6.7
			Y	2.5

Table 1 lists the identified proteins rich in 3 and 4 prime residues. Together with their Uniprot identifier and reported function, the regions rich in each of the prime mass residue are shown. It is immediately apparent that the prime-rich proteins are those important to cellular events. Transcriptional regulation proteins, cell surface receptors and those associated with the adhesion of ligands, proteins involved in the formation of cell surface membranes, and cytoskeletal protein polymerisation, all feature. These biological events are all important to early life cycle events.

Table 1.

Identity of Proteins Containing 3 and 4 Prime-Rich Residue Segments and Their Biological Function.

Prime residue combination (3 and 4)	No. of seq. hits	Uniprot identifier	Protein name	Prime rich-residue regions from ScanProsite outputs	Function a
[A, C, H]	1	Q9BXQ6	Transmembrane protein 121B	A: 256-317, C: 168-177, H: 261-275	Transmembrane protein
[A, C, T]	1	Q9H1U4	Multiple epidermal growth factor-like domains protein 9	A: 24-38, C: 204-437, T: 102-174	Transmembrane protein with role in cell adhesion
[A, H, P]	9	Q8NDT2	Putative RNA-binding protein 15B	A: 270-295, H: 658-677, P: 110-129	Regulator of RNA methylation
		Q4VCS5	Angiomotin	A: 876-1032, H: 385-391, P: 973-1037	Regulates endothelial cell migration
		P58012	Forkhead box protein L2	A: 221-348, H: 289-312, P: 280-341	Transcriptional regulator
		Q5VYV0	Forkhead box protein B2	A: 220-261, H: 116-181, P: 162-214	Transcriptional regulator
		P20264	POU domain, class 3, transcription factor 3	A: 81-204, H: 133-184, P: 270-294	Transcription factor
		Q99626	Homeobox protein CDX-2	A: 46-153, H: 114-125, P: 257-284	Transcription factor
		Q9BXQ6	Transmembrane protein 121B	A: 46-153, H: 114-125, P: 257-284	Transmembrane protein
		Q2VWA4	SKI family transcriptional co-repressor 2	A: 261-448, H: 603-668, P: 285-294 + 465-711	Transcriptional repressor
		Q8NFD5	AT-rich interactive domain-containing protein 1B	A: 2-130 + 412-576, H: 164-187, P: 582-590	Regulates transcriptional activation and repression
[A, T, P]	4	O14776	Transcription elongation regulator 1	A: 178-258, T: 260-331, P: 32-132	Transcriptional regulator
		O60641	Clathrin coat assembly protein AP180	A: 361-583, T: 536-556, P: 809-897	Link clathrin to receptors in coated vesicles
		Q9H1U4	Multiple epidermal growth factor-like domains protein 9	A: 24-38, T: 102-174, P: 55-200	Transmembrane protein with role in cell adhesion
		P51610	Host cell factor 1	A: 1613-1645, T: 574-1491, P: 412-456	Transcriptional co-regulator
[C, L, T]	1	Q14114	Low-density lipoprotein receptor-related protein 8	C: 47-401, L: 9-38, T: 741-781	Cell surface receptor
[L, T, P]	1	P07359	Platelet glycoprotein Ib alpha chain	L: 3-195, T: 345-482, P: 382-456	Surface membrane protein participates plug formation
[M, P, T]	1	O43670	BUB3-interacting and GLEBS motif-containing zinc finger protein	M: 164-209, T: 310-360, P: 129-470	Promotes tubulin polymerisation bundling
[A, C, H, P]	1	Q9BXQ6	Transmembrane protein 121B	A: 256-317, C: 168-177, H: 261-275, P: 537-548	Transmembrane protein
[A, C, T, P]	1	Q9H1U4	Multiple epidermal growth factor-like domains protein 9	A: 24-38, C: 204-437, T: 102-174, P: 55-200	Transmembrane protein with role in cell adhesion

^aFunction based on cited reports in Uniprot database.

The level of prime-residue enrichment, calculated for each protein entry using the ProtParam Tool, is shown in Table 2. For the most common 5 prime residues in the combinations above, namely A, C, H, T, and P, the enrichment levels range from 7.1% to 17.3% for alanine, 0.6% to 6.5% for cysteine, 1.4% to 9.3% for histidine, 2.2% to 13.2% for threonine, and 6.5% to 19.9% for proline. The levels of proline across all 16 proteins identified in Table 2 are particularly high and exceed 10% in 11 of the proteins. In a random distribution of 20 amino acids, without codon bias or structural considerations, one would expect some 5% (100%/20) of all residues to be proline.

Total prime residue enrichment ranges from 40.8% to 54.6% across the proteins, in other words, up to an exceeding one-half of all residues. In the 34.8 kDa zinc-finger protein (Uniprot entry O43670), proline levels approach 20% or one-fifth of all 478 residues. Over one-half (or 54.6%) of all residues are also prime in this protein. The proline-rich region is defined as residues 129 to 470 with a ScanProsite high score of 35.18 (Figure 1). The score indicates how well a specific profile, comprised of a weighted matrix, matches that for the domain. The ProtParam tool ¹² narrows the proline-rich domain to residues 127 to 136, 142 to 157, and 447 to 467. The microtubule binding region is located towards the N-terminus across residues 1 to 92, featuring a predominantly alpha-helical structure, while much of the outward structure of the protein is highly disordered. Clearly, the highly disordered nature of the protein offers it flexibility in terms of its function and adaption.

Figure 1.

ScanProsite output for proline-rich zinc-finger protein (Uniprot entry O43670).

In the extended proline-rich 129 to 470 region (Figure 1), proline makes up 90 or over one quarter (26.3%) of the 342 residues. Given the backbone constraints imposed by the proline residue, where its side chain is attached back to the nitrogen atom of the backbone, the next most common residue is the structural-alleviating glycine comprising 39 or 11.4% of residues. These residues are well-known to co-locate, particularly in beta-turns.

The next 2 most prime-rich proteins, where the total percentage of primes equals 56.2% and 55.8% are 2 transcriptional regulator forkhead proteins (Uniprot entries P58012 and Q5VYV0). Proline and alanine levels in these proteins are 16.2% and 17.3%, and 15.0% and 14.8%, respectively. Thus every 6th to 7th residue in each protein is either proline or alanine. The proline and alanine rich regions occupy residues 280 to 341 and 221 to 348 with ScanProsite scores of 16.16 and 14.40 respectively (Figure 2). The latter alanine-rich region, in which 41 of 128 or 32.0% of residues are alanine, is also rich in proline (24.2%). Similarly, the proline-rich segment comprising proline at 25 (or 40.3%) of 62 residues is very alanine-rich (29.0%).

Figure 2.

ScanProsite output for proline-rich forkhead proteins (Uniprot entries P58012 and Q5VYV0).

The same 2 prime residues predominate in regions 162 to 214 (ScanProsite score 18.76) and 220 to 261 (ScanProsite score 14.58) for proline and alanine respectively. The former is rich in proline (49.1%) and glutamine (22.6%) while the latter site contains 57.1% alanine.

Both forkhead proteins also are rich in histidine (Figure 2). The former protein (Uniprot entry P58012) contains a short histidine-rich segment comprising residues 116 to 181 (ScanProsite score 9.45) while the latter features a longer histidine-rich segment across residues 116 to 181 with a high ScanProsite of 27.99. This 66 residue peptide segment contains 40.9% histidine, 24.2% proline and 9.1% alanine, totalling almost three quarters (or 74.2%) of all residues.

Discussion

Prime-Rich Protein Functions, Hydrophobic Effects, and Their Role in Cellular Life

Biological events important early in the life cycle feature proteins that play fundamental roles in basic cellular processes such as translation and protein translocation. The prime-rich residues feature heavily among these. A high proportion perform roles in transcription activation, regulation, including the regulation of RNA methylation, and serve as transcription factors (Table 1). Others, such as angiomotin, regulate endothelial cell migration. Several, including the multiple epidermal growth factor-like domain protein, are transmembrane proteins with diverse roles that aid cell adhesion, act as a cell surface receptor, or serve as a surface membrane protein that participates in cytoskeletal protein binding and polymerisation. The resulting filaments and microtubules play a crucial role in cell support and movement, and are vital to normal cell function. In the early stages of life, the creation of transmembrane proteins, which span the cell membrane, and the development of systems that allow for protein translocation across membranes, are all crucial processes.

Hydrophobic effects are important to this organisation. They are responsible for the assembly of cell membranes and intracellular compartments, and the absence of strong attractive hydrophilic and charged forces makes membranes more fluid and deformable. Structures that are less rigid are more suited to the organisation of living matter, where deformability is not only a virtue, but also a necessity. ¹⁴ Hydrophobic amino acid residues have been proposed to have predominated in the prebiotic world, before today’s oxygen-rich environment. ¹⁵ Ancient protein sequences have been found to be over-represented with hydrophobic residues ¹⁶ that tend to cluster together along the protein chain. ¹⁷ These help to prevent undesirable aggregation. ¹⁸

During protein evolution, amino acids are selectively sequestered based upon their specific physicochemical properties. This leads to an abundance of specific amino acid positions in proteins, especially at sites that are critical to protein function or stability. Natural selection drives this accumulation of particular amino acids. ¹⁹

The selection of the 20 standard amino acids into the genetic code was likely driven by a combination of factors, including the ability to form stable proteins with the right folding characteristics^20,21 along with the efficiency of biosynthesis and folding. A plot of the codon frequency of each amino acid versus their frequency across all known proteins of the human proteome ⁴ revealed that the greatest correlation existed for the prime residues. All prime residue values were located along the intersecting (x = y) line, while the polar, hydrophilic acidic and basic non-prime residues were the most diverged from this line. ⁴ Thus the prime residues appear to maintain their importance during protein expression, irrespective of other evolutionary events.

Hydrophobic residues drive protein folding by expelling water from the core of their structure. ²² Prime residues (alanine, cysteine, histidine, isoleucine, leucine, methionine, proline, threonine, and tyrosine) are predominantly hydrophobic in nature, and the least hydrophobic residues among them, histidine ²³ and proline, are both known to be essential to protein biosynthesis and protein folding. Proline is known to promote beta-turns ²⁴ while disrupting alpha-helices ²⁵ thus driving particular folds at the secondary structural level. It has also been reported that metal-binding (cysteine and histidine) and sulphur-containing (cysteine and methionine) amino acids, all prime residues, were introduced in the genetic code much earlier than previously thought, also to aid protein folding. ²⁶ It has even been proposed that such hydrophobic residues were involved in the development of foldamers ²⁷ in a protein-first model of biology. ²⁸ We have also recently shown in a global study of nearly 16 000 single point substitutions across several hundred disparate proteins, that mutations which retain a prime mass residue are favoured over those that lose them. ²⁹ This suggests they may afford some evolutionary advantage.

Has the Evolution of Proteins, and Their Functions, Been Driven in Part by Their Residues’ Primality?

It is interesting to pose the question as to whether the evolutionary emergence of prime-rich proteins and their functionality can be truly attributable to the primality of residues, or is merely a consequence of the hydrophobicity and/or structural properties of such residues. It is tempting to suggest that prime residues were recruited early into the genetic code, based on studies of the last universal common ancestor (LUCA) proteins, ²⁶ because of their primality alone.

Although there is no definitive consensus, ³⁰ it is generally believed that the evolution of the current genetic code proceeded with a stepwise incorporation of individual amino acids, driven in part by the chemical and biological environment encountered by early life forms. Smaller, simple aliphatic residues, including those containing sulphur, are widely thought to have been produced first. ²⁶ Similar findings were made in a revisited spark-discharge chemistry study conducted under abiotic conditions that attempted to simulate conditions on primordial Earth. ³¹ Indeed, 5 prime residues (alanine, isoleucine, leucine, methionine, and threonine) were among the highest concentration detected in this study. ³¹ It remains difficult, however, to prove that the primality of residues somehow influenced their recruitment for protein synthesis and impacted protein expression. ³²

To be prime, residues must contain an odd number of hydrogen atoms ⁴ since the major isotopes of all other elements (carbon, nitrogen, oxygen and sulphur) are even. Most are aliphatic and have fewer nitrogen and oxygen atoms in their side chains than their non-prime, odd mass, counterparts. ⁴ The aliphatic side chains of the 4 prime residues alanine, leucine, isoleucine, and proline all contain only carbon and hydrogen atoms. Two others, cysteine and methionine, contain only 1 atom of sulphur each within their straight aliphatic side chains while threonine contains 1 oxygen atom.

All such carbon-based, aliphatic straight chain or branched side chains contain an odd number of hydrogen atoms, bar proline, and the total number of hydrogen atoms within the repeating amino acid residue structure remains odd when the 2 hydrogen atoms attached to the backbone (at the alpha-carbon and amide nitrogen) are added. In the case of proline, the even number of hydrogen atoms on its cyclic side chain is compensated for in the total by the lack of 1 at the amide nitrogen.

Yet if the proposal that such simple aliphatic residues emerged first were universally true, and was associated with their simple side-chain structures and primality, valine would be expected to be prime. This residue, with a mass of 99, is in fact non-prime. Serine, with a shorter side chain similar in structure and composition to threonine, is also non-prime with a mass of 87.

Various amino acid biochemical pathway scenarios have been proposed ³³ to explain the order in which residues have emerged, where most prime residues have been suggested to have predominated in early life forms. ²⁶ However, these pathways vary considerably among species in response to their individual metabolisms, each evolving over time in response to dietary changes. Over evolutionary time, some pathways available to certain organisms are absent in others. ³³ It has been noted previously that as many as 6 of the 9 prime mass residues (histidine, isoleucine, leucine, methionine, threonine, and even tyrosine) are not synthesised by humans and are therefore essential to our diet today. ⁴ Histidine is the only prime residue with a side chain containing nitrogen (two) atoms, while tyrosine, like threonine, has 1 oxygen atom on its side chain. While both are aromatic, each preserves an odd number of hydrogen atoms within their side chains by virtue of their chemical construct. Both are important in the binding of metal ions. This likely favoured their proposed early emergence in proteins^26,29 over their primality.

Finally, one can consider how the replacement of prime residues with their non-prime counterparts would impact upon a protein’s function. While substituting prime residues alanine or leucine, for instance, with valine is unlikely to have any dramatic effect on a protein’s structure, the substitution of any of the mostly hydrophobic prime residues with a non-prime hydrophilic residue with acidic or basic side chains would have a more dramatic effect.

Our past results suggest that retaining or introducing the more hydrophobic prime residues, and particularly alanine, ²⁹ is favoured in order to maintain or promote protein structural motifs at a local and global level. It is conceivable that the early prevalence of simple hydrophobic aliphatic prime residues, and aromatic primes which bind metal ions such as histidine, all aid in the formation and stability of proteins. The later inclusion of hydrophilic acidic and basic residues promoted additional function but not to a level that would induce significant unfolding and loss of critical structure.

Conclusions

It has been demonstrated that proteins rich in prime residues play an important role in biological events important to cellular life. These prime residues are not exclusively those associated with the highest hydrophobicity, but also those known to be essential to protein biosynthesis and folding including cysteine, histidine and proline. Human proteins have been identified that contain prime residue enrichments of between 40% and 56%; namely up to and exceeding one-half of all residues. In the 3 most prime rich proteins, with enrichments ranging from 54.0% to 56.2%, these residues are found clustered within segments of the protein with high PROSITE scores.

Further, our studies ⁴ have revealed that within the human proteome there is a strong correlation between the percentage of the prime residues they contain and the codon frequency of such residues, suggesting that prime mass residues are associated with protein encoding genes that maintain their importance during protein translation irrespective of other evolutionary events.

We have considered whether the evolutionary emergence of prime-rich proteins and their functionality can be tied to the primality of the residues, and/or their physicochemical and structural properties in light of the proposed evolutionary history of the amino acids,^31-33 where prime residues were recruited early into the genetic code according to the composition the last universal common ancestor (LUCA) proteins. ²⁶ This question remains open and unresolved.

Collectively, this study and our previous work,^4,29 promotes the importance of such residues, and the significance of prime numbers in general, in the natural world.