Mainly two opposing mechanisms are controversially discussed, according to which either hydrophobic, or polar interactions are the dominant driving force. To resolve this question, we have investigated the interactions between urea and all 20 amino acids by comprehensive molecular dynamics simulations of 22 tripeptides. Calculation of atomic contact frequencies between the amino acids and solvent molecules revealed a clear profile of solvation preferences by either water or urea.
Almost all amino acids showed preference for contacts with urea molecules, whereas charged and polar amino acids were found to have slight preferences for contact with water molecules. Particularly strong preference for contacts to urea were seen for aromatic and apolar side-chains, as well as for the protein backbone of all amino acids.
Our results suggest that hydrophobic interactions are the dominant driving force, while hydrogen bonds between urea and the protein backbone contribute markedly to the overall energetics by avoiding unfavorable unsatisfied hydrogen bond sites on the backbone. In summary, we suggest a combined mechanism that unifies the two current and seemingly opposing views. In papers with more than one author, the asterisk indicates the name of the author to whom inquiries about the paper should be addressed.
Figure 1 Contact coefficient C UW for each amino acid. High values above 1 indicate preferential interactions with urea; a value of 1 corresponds to equal probability to interact with urea or with water. The color characterizes the amino acids. Red, charged; yellow, polar; gray, aliphatic; blue, aromatic; green, apolar.
Crosses denote the C UW of the backbone alone; the dotted line at 1. Figure 2 Atomic interaction sites for urea and water. Statistical errors are below 0. The large numbers denote the weighted average.
Figure 4 Force field energies between urea and water. Such files may be downloaded by article for research use if there is a public use license linked to the relevant article, that license may permit other uses. We thank Ira Tremmel for carefully reading the manuscript. View Author Information.
Cite this: J. ACS AuthorChoice. Article Views Altmetric -. Citations Abstract High Resolution Image. Urea is a widely used protein denaturant. Despite its widespread use, however, the molecular mechanism underlying urea-induced denaturation is not well understood. Two classes of interaction models are distinguished in the literature. In the first, direct interactions between urea and the protein are considered the main denaturation driving force. That the peptide backbone is an important interaction site for urea is now widely accepted.
Hence, more detailed insights into the interactions of a denaturant with amino acids is imperative to understand how denaturants work. This study aims to elucidate and quantify by extended molecular dynamics simulations the interactions of urea with each of the natural 20 amino acids. To this aim, interaction frequencies between urea and the individual amino acids are investigated to decide whether urea interacts preferentially with polar or apolar residues or with the peptide backbone.
To quantify residue interaction with urea, a contact coefficient C UW is introduced as a measure for preferential interaction with urea relative to that with water.
Additionally, the role of hydrogen bonds between urea or water and the peptide residues is investigated and hydrogen bond energies are estimated. To separate sequence dependence and secondary or tertiary structure effects from the immediate interaction between urea and the respective amino acids, all 20 amino acids were investigated by simulations of glycine-capped tripeptides GXG.
The influence of sequence and structure on the immediate or direct interactions of the amino acids are discussed in the Conclusions. Simulation Setup.
Each of the 20 natural amino acids X was simulated in a glycine-capped tripeptide GXG. For histidine, all three protonation states were considered, resulting in 22 simulations in total, each of ns length.
All tripeptides were simulated individually in aqueous urea solution with water molecules and urea molecules, corresponding to a urea mole fraction of 0. Ion concentration might affect the results.
Additionally, a second set of simulations was performed with all electrostatic interactions switched off to estimate steric effects on the calculated contact coefficients, which may arise from different volumes of urea and water molecules. All simulations were performed using the Gromacs 30,31 program package, version 3.
A cutoff of 1. An integration time step of 2 fs was chosen. The initial size of the periodic cubic box was set to 3. To setup the simulation systems, non-overlapping urea molecules were placed at random positions in the simulation box containing the tripeptide. Subsequently, the box was filled up with water molecules. Each simulation was preceded by a step steepest-descent energy minimization, ps equilibration with position restraints on the tripeptide, and finally a 1 ns equilibration without position restraints.
The total simulation time for data collecting was 4. Contact Coefficient. To quantify the frequency of interactions between urea and the amino acids, we define the contact coefficient C UW for a particular amino acid X: where N X - U and N X - W are the numbers of atomic contacts of amino acid X with urea and water molecules, respectively.
Atoms were defined to be in contact if they are closer than 0. Values above 1. Since C UW relates to interaction frequencies, it can be regarded as a measure for the free energy of contact formation which gives rise to the first peak in the respective radial distribution function. The autocorrelation time of the instantaneous contact coefficient determined from single snapshots was found to be about ps for the analysis on the residue level and about 10 ps for the analysis on the atomic level.
Lifetimes of contacts were distributed exponentially with a similar time-constant. The correlation time was used to calculate the number of statistically independent frames within the ns simulation time for the statistical error estimate of the average contact coefficients. Note that the contact coefficient can easily be extended to quantify interaction preferences of solute molecules X in solvents consisting of more than two components S i : where k is the number of solvent components, N X - S i is the number of atomic contacts between the solute X and the solvent molecules S i , and M S i denotes the total number of atoms of all solvent molecules i.
Hydrogen Bonds. The estimated energies are for isolated hydrogen bonds and are certainly not identical with the free energy contribution of these hydrogen bonds for proteins in solution. Force Field Energies. Energies per atom were then defined as where N X and N U denote the number of atomic contacts of residue X with water or urea, respectively.
Contact Coefficients. We first focus on contact coefficients C UW Figure 1. The other amino acids exhibit a C UW between 1. For each amino acid the C UW of the backbone alone is higher than for the complete amino acid. The C UW of the backbone alone, averaged over all residues, is 1. High Resolution Image. In summary, urea interacts mainly with aromatic and nonpolar residues, as well as with the protein backbone.
Polar and especially charged residues interact less frequently with urea, the charged amino acids ASP and GLU show even more interactions with water than with urea. To elucidate which parts of the amino acids show contact preferences for either urea or water, we calculated C UW atomwise.
This in-depth analysis was further motivated by the difference in average C UW for the backbones and the complete residues. Figure 2 shows atomic interaction sites for urea and water for all amino acids. Again, clear differences in the C UW are seen for the different the amino acids.
For both amino acids, also the side-chain CH 2 groups and even the backbone show reduced interactions with urea due to the charge of the carboxyl group. The amino groups are the main interaction sites for water, whereas the backbones exhibit high C UW values and are not significantly affected by the charged amino groups due to the long apolar side-chains of both amino acids.
The peptide backbone shows preferential interaction with urea for all amino acids. Overall, we observe for the residue, as well as for the atomic level, that polar parts with large partial charges mainly interact with water while less polar parts with small partial charges interact mainly with urea. Hydrogen Bonding. Hydrogen bonds between water, urea, and the amino acids were analyzed, and their strength was estimated via the Espinosa formula. Macromolecules , 46 10 , Specificity in Cationic Interaction with Poly N-isopropylacrylamide.
The Journal of Physical Chemistry B , 17 , Guinn , Jeffrey J. McDevitt , Wolf E. Merker , Ryan Ritzer , Gregory W. Muth , Samuel W. Engelsgjerd , Kathryn E. Mangold , Perry J. Thompson , Michael J. Kerins , and M. Journal of the American Chemical Society , 15 , Okur , Jaibir Kherb , and Paul S. Journal of the American Chemical Society , 13 , Macromolecules , 46 5 , Macromolecules , 46 3 , Langmuir , 28 47 , Langmuir , 28 45 , Pazos and Feng Gai.
The Journal of Physical Chemistry B , 41 , The Journal of Physical Chemistry B , 30 , Biomacromolecules , 13 7 , Chua , Peter J. Roth , Hien T. Duong , Thomas P. Davis , and Andrew B. Macromolecules , 45 3 , The Journal of Physical Chemistry B , 4 , Burke , and Harald D. Macromolecules , 44 22 , The Journal of Physical Chemistry B , 45 , Macromolecules , 44 21 , The Journal of Physical Chemistry B , 28 , Elder , Nicole M. Dangelo , Stephanie C.
Kim , and Newell R. Biomacromolecules , 12 7 , Madhusudhana Reddy and P. The Journal of Physical Chemistry B , 16 , ACS Nano , 5 4 , The Journal of Physical Chemistry B , 12 , The Journal of Physical Chemistry B , 49 , Langmuir , 26 16 , Canchi , Dietmar Paschek and Angel E. Equilibrium Study of Protein Denaturation by Urea. The exact manner in which urea degrades proteins is still the subject of some mystery.
Research on the subject has shown that the probable answer is, in all likelihood, a combination of the above named factors. Experimental methods are an unlikely source of gathering insight as to how proteins are denatured by urea. Future research and improvement in atomic-level microscopy will, no doubt, shed more light on the issue and reveal the exact mechanism by which protein denaturation by urea occurs.
Vee Enne is a U. Military Veteran who has been writing professionally since She writes for Demand Studios in many categories, but prefers health and computer topics. Enne has an associate's degree in information systems, and a bachelor's degree in information technology IT from Golden Gate University. Numbers and energies of the hydrogen bonds were analyzed between water and water, urea and water, and urea and urea. The total number of hydrogen bonds per water molecule was almost independent of urea concentration.
In this respect, urea seemes to substitute well for water in the hydrogen bond network. Although, geometrically, urea incorporates well into the hydrogen bond network, we found hydrogen bonds between urea and water to be significantly weaker than those between water molecules. The hydrogen bonds between urea molecules were even weaker. This progression in bond-strength caused a slight self-aggregation of urea due to the hydrophobic effect.
Figure 2 : Urea green in water blue. Urea incorporates well into the hydrogen bond network and shows only a small degree of self-aggregation. In the light of urea-induced protein denaturation, the weak urea hydrogen bonds point towards a preferential interaction of urea with less polar parts of the protein.
0コメント