Charged Amino Acid Frequencies of Proteins over Macroevolutionary Time Scale

doi:10.4236/eng.2013.510B086

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Engineering, 2013, 5, 420-423

http://dx.doi.org/10.4236/eng.2013.510B086 Published Online October 2013 (http://www.scirp.org/journal/eng)

Charged Amino Acid Frequencies of Proteins over

Macr oevolution ary T ime Scale

Yu-Juan Zhang1*, Jian-Jun Li2, You-Jin Hao3, Bin Chen3

1College of Life Sciences, Chongqing Normal University, Chongqing, China

2Art and Sciences Division, Chengdu College University of Electronic Science and Technology of China, Chengdu, China

3College of Life Sciences, Chongqing Normal University, Chongqing, China

Email: *yuj ua [email protected], [email protected], [email protected], [email protected]

Received 2013

ABSTRACT

Charged amino acids (AAs) are targets for selective forces in protein evolution. To fully explore the trend of charged

AA frequencies evolution in macroevolutionary process from prokaryotes to eukaryotes, we extend the analysis of five

charged AAs separately and total basic and acidic AAs in protein sequences of 158 prokaryotic and 63 eukaryotic pre-

dicted proteomes and 456 clusters of orthologous groups (COGs). Also, we eliminate the biases that may caused by

extreme organisms in both predicted proteomes and COGs analyses. More basic AAs, His, Lys and Glu were found in

eukaryotic proteins compared with prokaryotic proteins by predicted proteomes analysis. By COG s analysis, we found

that basic AAs and Lys frequencies are higher in eukaryotic orthologous proteins than their prokaryotic companions,

while the trend of Arg frequency is the opposite. We discussed the agreements and disagreements of two analyses and

gained a more credible trend of charged AAs evolution in macroevolutionary time scale.

Keywords: Ch a rged Am i no Ac id; Evolution

1. Introduction

Eukaryotes as a large complex have evolved from a pro-

karyote-like predecessor [1,2]. The eukaryotic proteins

are much more diverse than prokaryotic proteins. At-

tempts have been made to study the evolution and diver-

sity of eukaryotes at protein sequence and higher struc-

tures [3], for example, homolog duplication, gaining or

losing of domains [4], and so on. However, few atten-

tions have been paid on the evolution of the amino acid

(AA) frequency, especially for charged AAs, which play

an important role in protein structure and protein-protein

interactions.

The charged AAs include acidic and basic AA. The

acidic AAs are: Aspartic acid (Asp, D), Glutamic acid

(Glu, E); basic AAs are: lysine (Lys, K), arginine (Arg,

R), and histidine (His, H). Charged AAs play a critical

role in protein-protein in teraction by creating salt bridg es

and salt bridge networks, and introducing specificity in

binding [5]. Depending on function and structure of a

protein, charged AAs apparently can be important targets

for selective forces in protein evolution [6].

We are interested of charged AA frequency evolution

under the macroevolutionary time scale from prokaryotes

to eukaryotes. How did they change through this process?

Is there a global trend in charged AA frequency? A dis-

cussion of “universal trend” of AA changes has been gi-

ven by Jordan et al, in 15 taxa [7]. Their study didn’t

exclude the bias may generated by protein sequences re-

trieved from extreme organism, since life style and

growth temperatures may affect the charged AA frequen-

cy of organisms living in extreme environment [8], the

“universal trend” they detected are mixed products made

by ecosystem, macroevolution and so on, and then cloud

our visions of true evolutionary story happened in ma-

croevolutionary time scale. On the other hand, their study

is not based on one-to-one comparison because the the

data they used are whole geomes.

In the present study we conducted the comprehensive

investigation of charged AAs frequency on all available

prokaryotic and eukaryotic predicte d proteomes. Also we

computed the charged AA in cluster of orthologous group

(COG), which including proteins in the same orthologous

group. Orthologous proteins evolved from a common

ancestral gene usually share the same structure and func-

tion [9]. Statistic analysis performed for sets of ortholo-

gous proteins can be considered to have no bias. Mean-

while, to eliminate the biases may be caused by extreme

organisms, only sequences retrieved from mesophilic or-

ganism s were used.

At the predicted proteomes level, our results showed

*Corresponding a uthor.

Y.-J. ZHANG ET AL.

421

eukaryotic proteins contain more total basic AAs, Lys

and His, per protein than prokaryotic proteins. Compari-

son at the COGs level indicated that orthologous proteins

in eukaryotes have higher basic AAs and Lys than those

of prokaryotes’ companions. Our study suggested that a

trend of carrying more basic AAs and Lys on proteins

from prokaryotes to eukaryotes. This study gives new in-

sights into how charged AA frequencies have been

changed over macroevolutionary time scale.

2. Materials and Methods

2.1. Sequence Retrieving and Analyzing

1) Predicted proteomes sequence

158 mesophilic prokaryotic and 63 eukaryotic predict-

ed proteomes were retrieved from NCBI

(ftp://ftp.ncbi.nih.gov/genomes) and Ensembl

(http://www.ensembl.org). Mesophilic organisms were

classified (organism lives between 50˚C and 15˚C) ac-

cording to PGTdb (http://pgtdb.csie.ncu.edu.tw).

2) Clusters of Orthologous Grou ps

COGs classification at the NCBI [10] w as used, which

currently contain 4873 orthologous groups that are present

in varying degrees in different species. According to

PGTdb description, hyperthermophiles, thermophiles and

psychrophiles sequences were excluded; only 456 COGs

in both prokaryotes and eukaryotes (distributed in 44

mesophilic species) were used in our ana lysis

2.2. Atom Frequency Calculation

The charged AA frequencies of a protein were calculated

as: One sequence’s [*AA frequency] = [sum of *AA]/ L ;

Where L is the sequence length, *represented 5 kinds

charged AAs (Asp, His, Lys, Asp and Glu) and total

acidic (Asp + Glu), basic AAs (Asp + His + Lys). One

group’s charged AA frequency is the mean charged AA

frequency of all protein sequences in this group. They

were all calculated by special Perl scripts.

2.3. Statistical Tests

The statistical calculations were performed by using

SPSS version 13.0. For robustness and consistency we

only considered significant differences at the probability

level of p < 0.001. See detailed results in Tables 1 and 2.

3. Results

3.1. Charged AAs Frequenci es of Proteins in

Predicted Proteomes

We calculated the frequency of five kinds of charged AA

separately, and total acidic, basic (Asp + His + Lys) in

proteins of 158 prokaryotic and 63 eukaryotic predicted

proteomes. The results were showed in Figure 1 and

Table 1.

The average frequency of basic AAs is 0.130 per AA

throughout prokaryotic proteins, and 0.141 for eukaryotic

proteins. Eukaryotic proteins have a higher frequency of

total basic AAs than those of prokaryotes’ (Mann-Whit-

ney Test, P < 0.001). Among basic AA, the frequency of

His and Lys AA is high er in euk aryotes (0 .024, 0.062 for

each) than in prokaryotes (0.021, 0.049 for each) (P <

0.001), but the frequency of Arg were not significantly

different.

The average acidic AAs frequency in prokaryotic pro-

teins is not different with that in eukaryotic proteins (p >

0.1). The frequency of Glu is higher in eukaryotes (0.065)

than in prokaryotes (0.062) with statistical support. For

Asp, no significant difference was observed at the proba-

bility level of p < 0.001.

3.2. Charged AAs Freque ncies of COGs

The differences we found in charged AA frequency be-

tween prokaryotic and eukaryotic predicted proteomes

might due to the diverse origins of the protein sequences

we used. Eukaryotic genomes have higher frequency of

basic AAs, His, Lys and Glu because the new originated

eukaryotic proteins have higher frequency of these AAs.

While the old ones inherited from prokaryotes, may have

no changes. To test this hypothesis, only proteins from

the same orthologous group were furthe r analyzed.

456 sets of COG from 41 mesophilic prokaryotes and

3 eukaryotes were retrieved, and then charged AAs fre-

Table 1. Statistical tests for Figure 1.

[R] [H] [K] [D] [E] [Acid] [Basic]

Mean*frequency of prokaryotic proteome 0.060 0.021 0.049 0.055 0.062 0.118 0.131

Mean*frequency of eukaryotic proteome 0.054 0.024 0.062 0.052 0.065 0.117 0.141

P value（Mann-Whitney Test） 0.012 2E−10 1E−6 0.006 4E−4 0.102 6E−15

Table 2. Statistical tests for Figure 2.

[R] [H] [K] [D] [E] [Acid] [Basic]

Mean*frequency of prokaryotic prot eome 0.056 0.023 0.055 0.055 0.065 0.120 0.134

Mean*frequency of eukaryotic proteome 0.048 0.023 0.070 0.054 0.064 0.118 0.141

P value (Mann-Whitney Test) 4E−21 0.642 5E−41 0.074 0.442 0.068 6E−7

Y.-J. ZHANG ET AL.

422

Figure 1. The comparisons of charged AAs frequency in

prokaryotic and eukaryotic predicted proteomes.

quencies in each full-length protein sequence were cal-

culated and compared. The results were showed in Fig-

ure 2 and Table 2. It is showed that the average fre-

quency of basic AAs is higher in eukaryotic orthologous

proteins (0.141 per AA) than in prokaryotic orthologous

proteins (0.134 per AA) with statistical support. Among

basic AAs, it is worth mentioning that only the frequency

of Lys is increased significantly in eukaryotic ortholog-

ous proteins while the frequency of Arg is decreased sig-

nificantly, and the frequency of His didn’t change. For

acidic AAs (total of Asp and Glu), Asp and Glu, no ob-

vious differences were detected.

4. Discussion

The profile of charged AA frequencies in whole pre-

dicted proteomes analysis showed that eukaryotic pro-

teins contain more basic AAs, His, Lys and one kind of

acidic AA, Glu, than that of prokaryotic proteins’. Other

charged AAs didn’t change obviously; COGs analysis

showed that total basic AAs and Lys are higher in euka-

ryotic proteins than their prokaryotic companions, while

the frequency of Arg is significantly lower in eukaryotic

proteins, other charged AAs didn’t change significantly.

Meanwhile, we exclude the possible bias that may caused

by extreme microbes in both of the two analyses.

COGs analysis ba sed on one-to-one comparison shows

Figure 2. The comparisons of charged AAs frequency in

proteins of Clusters orthologous groups (COGs). The figure

is illustrated as described in Fig 1 legend.

us more reliable trend of AAs changes under macroevo-

lutionary time scal e. Pred ict ed p ro teo mes analys is is based

on much more data. Each analysis has its advantage, re-

sults supported by both analyses streng then the reliability

and credibility. Both of the results showed that total basic

AAs and Lys were increased in eukaryotic proteins, total

acidic AAs have no significant difference. Charged AAs

are significant contributor to the protein-protein interac-

tion by creating salt bridges and salt bridge networks,

and introducing specificity in binding. We proposed that

the increase of more total basic AAs and Lys in eukaryo-

tic proteins might help maintaining binding among dif-

ferent eukaryotic proteins. More importantly, this result

is supported by comparison between eukaryotic and pro-

karyotic orthologous proteins, it means eukaryotic pro-

teins that possess similar function and structure as their

prokaryotic ancestor, have a higher basic AAs and Lys

than their prokaryotic ancestor.

In two results, some disagreements existed. The trends

of Arg, His and Glu frequency are discordant in two ana-

lyses. Firstly, for Arg, eukaryotic proteins possess sig-

nificant lower Arg than prokaryotic proteins in COGs

analysis, while no difference found in predicted prote-

omes analysis. Diverse origins of the protein sequence

we u sed could explain this disagreement; 1) newly orig i-

nated eukaryotic proteins might have higher Arg or 2)

prokaryotic proteins that have no descendant in euka-

ryote might have higher Arg frequency. Both of them

might produce no difference in COG analysis but differ-

Y.-J. ZHANG ET AL.

423

ence in COG analysis, because this part of data was used

in predicted proteomes analysis but could not be included

in the COGs analysis. Secondly, for His and Glu, the

disagreement might also due to the same reason. For

example, eukaryotic histone proteins possess especially

more His. Histone proteins were included in predicted-

proteome analysis but could not be included in COGs

analysis. Here we thought COGs analysis is relatively

more convincing when considering under macroevolutio-

nary time scale.

Our study gives a trend of charged AAs changes under

macroevolutionary time scale from prokaryotes to euka-

ryotes. More importantly, our findings provide the first

suggestion that total basic AAs and Lys increased on pro-

teins over macroevolutionary time scale to help proteins

carry more information, which lays a material basis for

the evolution of primary sequences and higher structures

complexity for proteins in eukaryotes. This study could

help to bet ter unde rs t a nd prote ins evolution.

5. Acknowledgements

This work was supported by grants from National Natu-

ral Science Foundation of China (No. 31200947), the

National Institute of Health (R01 AI095184), the Key

Scientific and Technological Project of Chongqing

(CSTC2012GG-YYJSB80002) and Par-Eu Scholars

Program.

The vertical axis is the value of AA frequency. The

black bar in box stands for the average charged AA fre-

quency in proteins throughout the different prokaryotic

(left plot, p) or eukaryotic (right plot, e) predicted prote-

omes. The boxes rep resen t the upper 25 % and lower 25 %

of the data and the bars at the top and the bottom of the

box repres ent the total ran ge of the data.

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