Comparative analysis of RuBisCO evolution and intrinsic dif-2

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Introduction
The most prevalent protein on earth is undoubtedly RubisCO (Ribulose-1,5-bisphosphate carboxylase oxygenase) [1].Most autotrophic organisms, starting from prokaryotes including cyanobacteria, photosynthetic bacteria, chemoautotrophic bacteria, and archaea to eukaryotes such as algae and higher plants, possess this enzyme.According to estimates, Rubisco can make up to 50% of all soluble proteins found in plant leaves or inside microbes [2].Its presence in marine phytoplankton, which is projected to contribute more than 45% of yearly world net primary production, is perhaps less evident but nonetheless pervasive [3].The enzyme RuBisCO (EC 4.1.1.39,Ribulose-1,5-bisphosphate carboxylase oxygenase) catalyzes the primary photosynthetic CO2 reduction process, which involves the binding of CO2 to the acceptor molecule Ribulose-1,5-bisphosphate (RuBP) to produce 3-phosphoglycerate [4].Besides that, it also possesses oxygenase activity through which it binds O2 with RuBP to form 2-phosphoglycolate.The discovery of the salt bridge's microenvironment is a novel concept in structural biology.Intragenic protein sequence and structural study sheds light on species variety, functions, and evolutionary relationships [5].Although there are numerous sequence and structure of Rubisco in the database, however, there is no such report on the sequence and structure analysis of this protein in terms of salt bridge and other interactions of proteins inside.The study was conducted to check the evolutionary pattern in RuBisCO protein starting from lower group of photosynthetic organism to higher group of plants through intra-protein interactions and molecular dynamics simulation studies.

Dataset:
Sequences of RuBisCO enzyme from Nostoc sp., Chlamydomonas reinhardtii, Oryza sativa, and Nicotiana tabacum were retrieved from the UniProt database [6].Diverse organisms were taken into consideration to check evolutionary significance between them.Structures of those proteins were extracted from RCSB PDB database [7].

Analysis of protein sequences:
All protein sequences were subjected to MSA for block preparation through Clustal omega [8].Non-block format was used for calculation of amino acid abundance, pI, grand average hydropathy (GRAVY), aliphatic index through the ProtParam server [9].Block format of sequences were used in calculations of hydropathy, polarity by the ProtScale server [9].

Analysis of crystal structures:
All those protein structures were minimized through the Chimera 1.15rc with amber forcefield [10][11].Identification and calculations of intra-protein interactions were done through the PIC server [12].PDBSum were used to check their type of secondary structures [13].

Molecular dynamics simulations:
GROMACS [16] and the GROMOS96 43a1 forcefield were used to do molecular dynamic simulations.After equilibration, the energy on the solvated systems was decreased using the steepest descent approach with 5000 steps.The last manufacturing run's molecular dynamic simulations lasted 50 ns at 300 K temperatures.Molecular dynamics simulations were used to estimate the radius of gyration (Rg), solvent accessible surface area (SASA), root mean square deviation (RMSD), root mean square fluctuation (RMSF), and hydrogen bonding.

Preferable amino acid abundance
Higher abundance of charged polar residues was observed in Oryza sativa followed by Nicotiana tabacum (Figure 1).However, the uncharged polar residues showed higher presence in the enzyme of Nostoc sp.Hydrophobic amino acid showed highest abundance in Chlamydomonas reinhardtii.Oryza sativa had higher presence of amino acid Pro.Due to its capacity to reduce the structural entropy of the denatured state, Pro may be able to improve protein stability.Additionally, Pro is often conserved in proteins and frequently contributes significantly to the structure and function of proteins [17].
A common method for identifying hydrophobic areas in proteins is the Kyte-Doolittle scale.Positive values indicate hydrophobic regions.Transmembrane helices predicted using hydropathy plots.The non-polar portion of the lipid membrane contains the transmembrane helices, whereas the loops are in a more polar solution [5].Nicotiana tabacum showed lowest plot followed by Oryza sativa which means they were more hydrophilic rather than others.
The specific polarity pattern is crucial to the molecule's structure and functionality.Higher polarity has been shown by protein sequence of Nicotiana tabacum followed by Oryza sativa.However, it was observed that in some specific positions in Chlamydomonas reinhardtii there are some pick-point for high polarity.Higher polarity drastically increased thermal stability of a protein [19][20].

Intra-protein interactions
Intra-protein interactions are crucial interactions to enhance the protein stability.Salt-bridges, aromatic-aromatic interactions, aromatic-sulfur interactions, cation-pi interactions have significant contributions in this field.Generally, they act as single pair called isolated, however sometime multiple isolated are connected to each other to make network formation.Strong hydrogen bonds that are formed by the interaction of two charged residues are known as salt bridges or ion pairs.In contrast to surface salt bridges, a subsurface salt bridge destabilizes by 3-4 kcal/mol when one partner is removed.Nowadays, beside the isolated and network salt bridge, a special salt-bridge i.e., cyclic salt bridge has been discovered [22].Highest number of isolated salt bridges was found in RuBisCO of Oryza sativa (23) whereas Nicotiana tabacum showed highest number of network salt bridges (10) (Table 1).Nostoc sp.showed 15 isolated and 7 network salt bridges whereas Chlamydomonas reinhardtii had 19 isolated and 9 network salt bridges.Higher formation of salt bridges in monocot and dicot makes them more stable than other two species.Important noncovalent interactions in proteins involve aromatic-aromatic interactions between several aromatic amino acids (Phe, Tyr, and Trp).Nostoc sp.showed highest number of isolated aromatic-aromatic interactions i.e., 7 with 3 network aromatic-aromatic interactions.Chlamydomonas reinhardtii showed 5 isolated and 3 network aromatic-aromatic interactions.RuBisCO of monocot Oryza sativa had 5 isolated and 6 network formation which was highest formation of network aromatic-aromatic interactions.Nicotiana tabacum showed equal number of isolated and network aromatic-aromatic interactions.Aromatic-sulphur interactions was higher in Chlamydomonas reinhardtii in the form of isolated bonds.However, the only formation of network aromatic-sulphur interactions was observed here.Formation of isolated cation-pi interactions was almost same in every species except Chlamydomonas reinhardtii.However, the network formation was higher in Nicotiana tabacum whereas, others showed equal number i.e., 2 network formations.Formation of higher number of intra-protein interactions in RuBisCO of dicot and monocot gives advantage to them to gain more stability over the algae and cyanobacteria.

Stability through simulation Study
50 ns molecular dynamics simulations provide the details of RMSD, RMSF, Rg and SASA.From the RMSD, it was observed that Nostoc sp. had highest RMSD than others.It started to deviate from 0.2 nm and ended at o.6 nm.On other hand, Oryza sativa and Nicotiana tabacum showed lower and almost equal trajectory throughout the 50 ns RMSD analysis.From the starting at 0.2 nm become stabilized and ended at almost same range.RMSF analysis revealed all proteins showed almost similar trajectory throughout the path.The Rg plot was also showed similarity with plot to RMSD.Oryza sativa and Nicotiana tabacum showed lowest Rg than others which means they had tightest packing RuBisCO

Conclusion:
Insilico investigation on different RuBisCO revealed how the amino acid evolutions make significant changes to gain more stability and flexibility in higher group of plant.Charged amino acid residues abundance was found mainly in Nicotiana tabacum and Oryza sativa plant.Moreover, higher hydrophilicity and higher polarity enhance the stability, functionality, and flexibility.Increase of helix in secondary structure further boost the stability.Molecular dynamics simulations revealed higher stability, flexibility, and folding patterns of RuBisCO from Oryza sativa and Nicotiana tabacum.This study will be helpful to understand the protein evolution and play role for protein engineering.
[23].SASA was also high in Oryza sativa and Nicotiana tabacum.Increasing the value of SASA enhance the stability and protein folding[24].