Insights into the Sensitivity of Arginine Concentration to Preserve the Folded Form of Insulin Monomer under Thermal Stress

Abstract

Arginine, although popularly known as aggregation suppressor additive, has been found to quench proteins' structure and function by destabilizing their conformations. Driven by such controversial evidence, in this work we performed a series of atomistic molecular dynamics simulations of insulin monomer, a biologically active hormone protein, in arginine solution of varying concentrations (0.5, 1, and 2 M) at ambient and elevated temperature (400 K) to explore the arginine concentration driven structurebased stability of the protein. Our study reveals that the flexibility of the protein's structure is dependent on the arginine concentration, and among all the used solutions, 2 M arginine, a "neutral crowder" that mimics the cellular environment, can preserve the native folded form of the protein at ambient temperature in an excellent manner. Further, while the protein unfolds at 400 K in pure water, this solution worked satisfactorily to preserve the protein's folded conformation more firmly than the other solutions. The replica-exchange MD of insulin in 2 M arginine solution further supports the fact. In this aspect an important issue in molecular pharmacology is to identify and recognize the physical origin of the stability of a protein, i.e, in this case, how arginine directs the conformational flexibility of the protein and preserves its native folded form. We identified that the exclusion of arginine from the protein surface increases the local structuration of water around the protein, thereby preserving its "biological water" layer, and makes the protein more hydrated at 2 M concentration as compared to the other arginine solutions. Additionally, our microscopic investigation on the interactions of the protein-solvation layer revealed that the structural heterogeneity of the protein surface, arising from the differential physicochemical nature of the amino acid residues, controls the favorable formation of sluggish water−arginine mixed solvation layer at higher arginine concentration that helps the protein to maintain its structural rigidity. Importantly, apart from the protein−solvent hydrogen-bonding interactions, the anion−pi interactions, established between the carboxyl group of arginine and the aromatic amino acid residues of insulin, were recognized to facilitate the protein to maintain its native folded form at the experimental temperatures.

Progress in Simulation Studies of Insulin Structure and Function

“…To probe the effect of varying pressure, temperature, pH, chemical modifications, and electric field on the structure of insulin, several MD studies were conducted ( 71 , 78 , 79 , 84 , 102 , 122 ). The effect of cations, various green solvents, and small molecules on the conformational stability of insulin was explored using several simulation studies ( 52 , 73 , 76 , 101 , 103 , 105 , 111 , 114 , 120 , 121 , 126 , 128 , 129 ).…”

“…The binding of arginine stabilized the insulin dimer and hindered the transition to a hexameric state. Santra and Jana ( 126 ) reported a detailed study on the effect of arginine solution of varying concentrations (0.5M, 1M, and 2M) on the stability of the insulin monomer at near-physiological (300 K) and elevated (400 K) temperatures using atomistic MD and replica exchange MD (REMD) simulations. They revealed that the insulin monomer unfolds completely at 400 K in an aqueous medium, while in a 2M arginine solution the hormone maintains its native fold.…”

Influence of Aqueous Arginine Solution on Regulating Conformational Stability and Hydration Properties of the Secondary Structural Segments of a Protein at Elevated Temperatures: A Molecular Dynamics Study

“…Majorly, two different classes of cosolvents, known as osmolytes (stabilizer) and denaturants, exist. Cosolvent-guided protein stability could be due to the solvation effects in which preferential exclusion of solvent from the protein surface occurs, thereby altering the hydration shell. − However, the contribution of hydration was still observed to be important in preserving protein conformation in the presence of cosolvent.…”

“…Although plenty of experimental and computational studies established arginine as the protein aggregation suppressor to stabilize the native state of a protein, − very little information on the role of arginine in stabilizing the folded native protein and inhibiting unfolding under thermal stress have been reported so far. In some recent studies, we investigated the interaction of insulin with different free amino acids in solutions. , Further, concentration-dependent osmolytic efficiency of arginine toward stabilizing the native folded form of insulin monomer was investigated . Among the tested solutions, 2 M arginine showed stabilizing behavior as compared to others.…”

TAR RNA Mediated Folding of a Single-Arginine-Mutant HIV-1 Tat Protein within HeLa Cells Experiencing Intracellular Crowding

“…To optimize the refolding of mutant R52Tat without EGFP, we compared the effect of an uncrowded environment on the TatR52A (purified) in E. coli cells with that of a crowded environment on EGFP in the absence of TAR RNA by BLI at 1.3 nm during a refolding period of 20 min ( Figures S4 and S5 ) [ 56 ]. We used this well-characterized system to assess the response of His-tagged Tat-BLI after binding to the TAR RNA substrate to design a BLI-detection study for mutant R52Tat folding without EGFP in the absence of TAR RNA ( Figure 5 and Figure S4 ) [ 3 , 57 ]. To normalize the refolding efficiency of the R52 mutant Tat, we monitored refolding buffer alone as a negative control.…”