Salt bridge (protein)
Encyclopedia
Salt bridges fall into the broader category of noncovalent interactions. A salt bridge is actually a combination of two noncovalent interactions: hydrogen bonding and electrostatic interactions (Figure 1). This is most commonly observed to contribute stability to the entropically unfavorable folded conformation of proteins. Although noncovalent interactions are known to be relatively weak interactions, small stabilizing interactions can add up to make an important contribution to the overall stability of a conformer. Not only are salt bridges found in proteins, but they can also be found in supramolecular chemistry
. The thermodynamics of each are explored through experimental procedures to assess the free energy contribution of the salt bridge to the overall free energy of the state.
or glutamic acid
and the cationic ammonium (RNH3+) from lysine
or the guanidinium (RNHC(NH2)2+) of arginine
. Although these are the most common, other residues with ionizable side chains such as histidine
, tyrosine
, and serine
can also participate, depending on outside factors perturbing their pKa's. The distance between the residues participating in the salt bridge is also cited as being important. The distance required is less than 4 Å (400 pm). Amino acids greater than this distance do not qualify as forming a salt bridge. Due to the numerous ionizable side chains of amino acids found throughout a protein, the pH at which a protein is placed is crucial to its stability. This is largely due to alterations in half of what makes a salt bridge, electrostatic interactions. At pH extremes, two amino acids participating in a salt bridge could lose their ability to do so as one will lose its charge.
with asparagine (Asn) (Figure 3) was done obtaining three new mutants: Asp70Asn His31 (Mutant 1), Asp70 His31Asn (Mutant 2), and Asp70Asn His31Asn (Double Mutant).
Once the mutants have been established, two methods can be employed to calculate the free energy associated with a salt bridge. One method involves the observation of the melting temperature of the wild-type protein versus that of the three mutants. The denaturation can be monitored through a change in circular dichroism
. A reduction in melting temperature indicates a reduction in stability. This is quantified through a method described by Becktel and Schellman where the free energy difference between the two is calculated through ΔTΔS. There are some issues with this calculation and can only be used with very accurate data. In the T4 lysozyme example, ΔS of the pseudo-wild-type had previously been reported at pH 5.5 so the midpoint temperature difference of 11 °C at this pH multiplied by the reported ΔS of 360 cal/(mol·K) (1.5 kJ/(mol·K)) yields a free energy change of about −4 kcal/mol (−17 kJ/mol). This value corresponds to the amount of free energy contributed to the stability of the protein by the salt bridge.
The second method utilizes nuclear magnetic resonance spectroscopy to calculate the free energy of the salt bridge. A titration is performed, while recording the chemical shift corresponding to the protons of the carbon adjacent to the carboxylate or ammonium group. The midpoint of the titration curve corresponds to the pKa, or the pH where the ratio of protonated:deprotonated molecules is 1:1. Continuing with the T4 lysozyme example, a titration curve is obtained through observation of a shift in the C2 proton of histidine 31 (Figure 4). Figure 4 shows the shift in the titration curve between the wild-type and the mutant in which Asp70 is Asn. The salt bridge formed is between the deprotonated Asp70 and protonated His31. This interaction causes the shift seen in His31’s pKa. In the unfolded wild-type protein, where the salt bridge is absent, His31 is reported to have a pKa of 6.8 in H20 buffers of moderate ionic strength. Figure 4 shows a pKa of the wild-type of 9.05. This difference in pKa is supported by the His31’s interaction with Asp70. To maintain the salt bridge, His31 will attempt to keep its proton as long as possible. When the salt bridge is disrupted, like in the mutant D70N, the pKa shifts back to a value of 6.9, much closer to that of His31 in the unfolded state.
The difference in pKa can be quantified to reflect the salt bridge’s contribution to free energy. Using Gibbs free energy
:
ΔG = −RT ln(Keq), where R is the universal gas constant, T is the temperature in kelvins, and Keq is the equilibrium constant of a reaction in equilibrium. The deprotonation of His31 is an acid equilibrium reaction with a special Keq known as the acid dissociation constant
, Ka: His31-H+ His31 + H+. The pKa is then related to Ka by the following: pKa = −log(Ka). Calculation of the free energy difference of the mutant and wild-type can now be done using the free energy equation, the definition of pKa, the observed pKa values, and the relationship between natural logarithms and logarithms. In the T4 lysozyme example, this approach yielded a calculated contribution of about 3 kcal/mol to the overall free energy. A similar approach can be taken with the other participant in the salt bridge, such as Asp70 in the T4 lysozyme example, by monitoring its shift in pKa after mutation of His31.
A word of caution when choosing the appropriate experiment involves the location of the salt bridge within the protein. The environment plays a large role in the interaction. At high ionic strengths, the salt bridge can be completely masked since an electrostatic interaction is involved The His31-Asp70 salt bridge in T4 lysozyme was buried within the protein. Entropy plays a larger role in surface salt bridges where residues that normally have the ability to move are constricted by their electrostatic interaction and hydrogen bonding. This has been shown to decrease entropy enough to nearly erase the contribution of the interaction. Surface salt bridges can be studied similarly to that of buried salt bridges, employing double mutant cycles and NMR titrations. Although cases exist where buried salt bridges contribute to stability, like anything else, exceptions do exist and buried salt bridges can display a destabilizing effect. Also, surface salt bridges, under certain conditions, can display a stabilizing effect. The stabilizing or destabilizing effect must be assessed on a case by case basis and few blanket statements are able to be made.
is a field concerned with non-covalent interactions between macromolecules. Salt bridges have been used by chemists within this field in both diverse and creative ways, including the synthesis of molecular capsules and double helical polymers.
). Szumna and coworkers developed a novel molecular capsule with a chiral
interior. This capsule is made of two halves, like a plastic easter egg
(Figure 5). Salt bridge interactions between the two halves cause them to self-assemble in solution (Figure 6). They are stable even when heated to 60 °C.
. In one example, they incorporated platinum
to create a double helical mettalopolymer. Starting from their monomer and platinum(II) biphenyl (Figure 7), their metallopolymer self assembles through a series of ligand exchange reactions. The two halves of the monomer are anchored together through the salt bridge between the deprotonated carboxylate and the protonated nitrogens.
Supramolecular chemistry
Supramolecular chemistry refers to the area of chemistry beyond the molecules and focuses on the chemical systems made up of a discrete number of assembled molecular subunits or components...
. The thermodynamics of each are explored through experimental procedures to assess the free energy contribution of the salt bridge to the overall free energy of the state.
Salt bridges found in proteins
The salt bridge most often arises from the anionic carboxylate (RCOO-) of either aspartic acidAspartic acid
Aspartic acid is an α-amino acid with the chemical formula HOOCCHCH2COOH. The carboxylate anion, salt, or ester of aspartic acid is known as aspartate. The L-isomer of aspartate is one of the 20 proteinogenic amino acids, i.e., the building blocks of proteins...
or glutamic acid
Glutamic acid
Glutamic acid is one of the 20 proteinogenic amino acids, and its codons are GAA and GAG. It is a non-essential amino acid. The carboxylate anions and salts of glutamic acid are known as glutamates...
and the cationic ammonium (RNH3+) from lysine
Lysine
Lysine is an α-amino acid with the chemical formula HO2CCH4NH2. It is an essential amino acid, which means that the human body cannot synthesize it. Its codons are AAA and AAG....
or the guanidinium (RNHC(NH2)2+) of arginine
Arginine
Arginine is an α-amino acid. The L-form is one of the 20 most common natural amino acids. At the level of molecular genetics, in the structure of the messenger ribonucleic acid mRNA, CGU, CGC, CGA, CGG, AGA, and AGG, are the triplets of nucleotide bases or codons that codify for arginine during...
. Although these are the most common, other residues with ionizable side chains such as histidine
Histidine
Histidine Histidine, an essential amino acid, has a positively charged imidazole functional group. It is one of the 22 proteinogenic amino acids. Its codons are CAU and CAC. Histidine was first isolated by German physician Albrecht Kossel in 1896. Histidine is an essential amino acid in humans...
, tyrosine
Tyrosine
Tyrosine or 4-hydroxyphenylalanine, is one of the 22 amino acids that are used by cells to synthesize proteins. Its codons are UAC and UAU. It is a non-essential amino acid with a polar side group...
, and serine
Serine
Serine is an amino acid with the formula HO2CCHCH2OH. It is one of the proteinogenic amino acids. By virtue of the hydroxyl group, serine is classified as a polar amino acid.-Occurrence and biosynthesis:...
can also participate, depending on outside factors perturbing their pKa's. The distance between the residues participating in the salt bridge is also cited as being important. The distance required is less than 4 Å (400 pm). Amino acids greater than this distance do not qualify as forming a salt bridge. Due to the numerous ionizable side chains of amino acids found throughout a protein, the pH at which a protein is placed is crucial to its stability. This is largely due to alterations in half of what makes a salt bridge, electrostatic interactions. At pH extremes, two amino acids participating in a salt bridge could lose their ability to do so as one will lose its charge.
Methods for quantifying salt bridge stability
The contribution of a salt bridge to the overall stability to the folded state of a protein can be assessed through thermodynamic data gathered from mutagenesis studies and nuclear magnetic resonance techniques. Using a mutated pseudo-wild-type protein specifically mutated to prevent precipitation at high pH, the salt bridge’s contribution to the overall free energy of the folded protein state can be determined by performing a point-mutation, altering and, consequently, breaking the salt bridge. For example, a salt bridge was identified to exist in the T4 lysozyme between aspartic acid (Asp) at residue 70 and a histidine (His) at residue 31 (Figure 2). Site-directed mutagenesisSite-directed mutagenesis
Site-directed mutagenesis, also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, is a molecular biology technique in which a mutation is created at a defined site in a DNA molecule. In general, this form of mutagenesis requires that the wild type gene sequence be known...
with asparagine (Asn) (Figure 3) was done obtaining three new mutants: Asp70Asn His31 (Mutant 1), Asp70 His31Asn (Mutant 2), and Asp70Asn His31Asn (Double Mutant).
Once the mutants have been established, two methods can be employed to calculate the free energy associated with a salt bridge. One method involves the observation of the melting temperature of the wild-type protein versus that of the three mutants. The denaturation can be monitored through a change in circular dichroism
Circular dichroism
Circular dichroism refers to the differential absorption of left and right circularly polarized light. This phenomenon was discovered by Jean-Baptiste Biot, Augustin Fresnel, and Aimé Cotton in the first half of the 19th century. It is exhibited in the absorption bands of optically active chiral...
. A reduction in melting temperature indicates a reduction in stability. This is quantified through a method described by Becktel and Schellman where the free energy difference between the two is calculated through ΔTΔS. There are some issues with this calculation and can only be used with very accurate data. In the T4 lysozyme example, ΔS of the pseudo-wild-type had previously been reported at pH 5.5 so the midpoint temperature difference of 11 °C at this pH multiplied by the reported ΔS of 360 cal/(mol·K) (1.5 kJ/(mol·K)) yields a free energy change of about −4 kcal/mol (−17 kJ/mol). This value corresponds to the amount of free energy contributed to the stability of the protein by the salt bridge.
The second method utilizes nuclear magnetic resonance spectroscopy to calculate the free energy of the salt bridge. A titration is performed, while recording the chemical shift corresponding to the protons of the carbon adjacent to the carboxylate or ammonium group. The midpoint of the titration curve corresponds to the pKa, or the pH where the ratio of protonated:deprotonated molecules is 1:1. Continuing with the T4 lysozyme example, a titration curve is obtained through observation of a shift in the C2 proton of histidine 31 (Figure 4). Figure 4 shows the shift in the titration curve between the wild-type and the mutant in which Asp70 is Asn. The salt bridge formed is between the deprotonated Asp70 and protonated His31. This interaction causes the shift seen in His31’s pKa. In the unfolded wild-type protein, where the salt bridge is absent, His31 is reported to have a pKa of 6.8 in H20 buffers of moderate ionic strength. Figure 4 shows a pKa of the wild-type of 9.05. This difference in pKa is supported by the His31’s interaction with Asp70. To maintain the salt bridge, His31 will attempt to keep its proton as long as possible. When the salt bridge is disrupted, like in the mutant D70N, the pKa shifts back to a value of 6.9, much closer to that of His31 in the unfolded state.
The difference in pKa can be quantified to reflect the salt bridge’s contribution to free energy. Using Gibbs free energy
Gibbs free energy
In thermodynamics, the Gibbs free energy is a thermodynamic potential that measures the "useful" or process-initiating work obtainable from a thermodynamic system at a constant temperature and pressure...
:
ΔG = −RT ln(Keq), where R is the universal gas constant, T is the temperature in kelvins, and Keq is the equilibrium constant of a reaction in equilibrium. The deprotonation of His31 is an acid equilibrium reaction with a special Keq known as the acid dissociation constant
Acid dissociation constant
An acid dissociation constant, Ka, is a quantitative measure of the strength of an acid in solution. It is the equilibrium constant for a chemical reaction known as dissociation in the context of acid-base reactions...
, Ka: His31-H+ His31 + H+. The pKa is then related to Ka by the following: pKa = −log(Ka). Calculation of the free energy difference of the mutant and wild-type can now be done using the free energy equation, the definition of pKa, the observed pKa values, and the relationship between natural logarithms and logarithms. In the T4 lysozyme example, this approach yielded a calculated contribution of about 3 kcal/mol to the overall free energy. A similar approach can be taken with the other participant in the salt bridge, such as Asp70 in the T4 lysozyme example, by monitoring its shift in pKa after mutation of His31.
A word of caution when choosing the appropriate experiment involves the location of the salt bridge within the protein. The environment plays a large role in the interaction. At high ionic strengths, the salt bridge can be completely masked since an electrostatic interaction is involved The His31-Asp70 salt bridge in T4 lysozyme was buried within the protein. Entropy plays a larger role in surface salt bridges where residues that normally have the ability to move are constricted by their electrostatic interaction and hydrogen bonding. This has been shown to decrease entropy enough to nearly erase the contribution of the interaction. Surface salt bridges can be studied similarly to that of buried salt bridges, employing double mutant cycles and NMR titrations. Although cases exist where buried salt bridges contribute to stability, like anything else, exceptions do exist and buried salt bridges can display a destabilizing effect. Also, surface salt bridges, under certain conditions, can display a stabilizing effect. The stabilizing or destabilizing effect must be assessed on a case by case basis and few blanket statements are able to be made.
Supramolecular chemistry
Supramolecular chemistrySupramolecular chemistry
Supramolecular chemistry refers to the area of chemistry beyond the molecules and focuses on the chemical systems made up of a discrete number of assembled molecular subunits or components...
is a field concerned with non-covalent interactions between macromolecules. Salt bridges have been used by chemists within this field in both diverse and creative ways, including the synthesis of molecular capsules and double helical polymers.
Molecular capsules
Molecular capsules are chemical scaffolds designed to capture and hold a guest molecule (see Molecular encapsulationMolecular encapsulation
Molecular encapsulation in supramolecular chemistry is the confinement of a guest molecule inside the cavity of a supramolecular host molecule...
). Szumna and coworkers developed a novel molecular capsule with a chiral
Chirality (chemistry)
A chiral molecule is a type of molecule that lacks an internal plane of symmetry and thus has a non-superimposable mirror image. The feature that is most often the cause of chirality in molecules is the presence of an asymmetric carbon atom....
interior. This capsule is made of two halves, like a plastic easter egg
Easter egg
Easter eggs are special eggs that are often given to celebrate Easter or springtime.The oldest tradition is to use dyed or painted chicken eggs, but a modern custom is to substitute chocolate eggs, or plastic eggs filled with confectionery such as jelly beans...
(Figure 5). Salt bridge interactions between the two halves cause them to self-assemble in solution (Figure 6). They are stable even when heated to 60 °C.
Double helical polymers
Yashima and coworkers have used salt bridges to construct several polymers that adopt a double helix conformation much like DNADNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
. In one example, they incorporated platinum
Platinum
Platinum is a chemical element with the chemical symbol Pt and an atomic number of 78. Its name is derived from the Spanish term platina del Pinto, which is literally translated into "little silver of the Pinto River." It is a dense, malleable, ductile, precious, gray-white transition metal...
to create a double helical mettalopolymer. Starting from their monomer and platinum(II) biphenyl (Figure 7), their metallopolymer self assembles through a series of ligand exchange reactions. The two halves of the monomer are anchored together through the salt bridge between the deprotonated carboxylate and the protonated nitrogens.