Conformation states of concanavalin A: Kinetics of transitions induced by interaction with Mn2+ and Ca2+ ions

Rodney D. Brown, Curtis F. Brewer, Seymour H. Koenig

Research output: Contribution to journalArticle

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Abstract

Using measurements of the magnetic-field dependence of the nuclear magnetic relaxation rate (1/T1) of solvent water protons over a wide range of field values (corresponding to proton Larmor frequencies from 0.01 to 50 MHz), we have investigated the interaction of Mn2+ and Ca2+ ions with concanavalin A (Con A) over the pH range 5.3 to 6.4, at 5 and 25°C. Particular attention was given to time-dependent effects that occur upon addition or removal of metals. Limited amounts of Mn2+ added to solutions of apo-Con A bind at S1 (the usual "transition-metal" site) to form a binary complex characterized by a large and pH-dependent dissociation constant, rapid exchange of Mn2+ ions with solvent, and a relatively large and pH-independent contribution to the proton relaxation rate. With S1 occupied, Ca2+ ions can bind at S2 (the usual "calcium-binding" site) to form a metastable ternary complex characterized by a relatively large and pH-dependent dissociation constant for Ca2+ ions, rapid exchange of Ca2+ ions with solvent, and a relatively low and pH-independent contribution to the proton relaxation rate. We find that this metastable ternary complex undergoes a first-order transition to a stable ternary complex, with a pH-independent time constant of 17 ± 1 min at 5°C and an activation energy of 22 kcal M-1. This stable ternary complex has the same relaxation contribution as the initial metastable complex, but differs in that the dissociation constant of Ca2+ is very low; the off-rate of both metals is of the order of days at 25°C. Saccharide binding and agglutination studies are generally done with this form of Con A. We have also found that, in the absence of Ca2+, Mn2+ can bind at S2 as well as at S1 (S2 was previously thought to bind only Ca1 and Cd2+) to form a metastable ternary complex which, like the metastable Mn2+-Ca2+-Con A complex, undergoes a transition to a stable state, but with a time constant that is much larger than for the Ca2+-containing ternary complex. In contrast to the stable Ca2+-Mn2+-Con A complex, the stable Mn2+-Con A ternary complex has a rather large dissociation constant, and the bound Mn2+ ions are in rapid equilibrium with solvent. The Mn2+ ions can be removed rapidly by the addition of ethylenediaminetetraacetic acid to produce apo-Con A in a metastable state that has different metal-binding properties than apo-Con A prepared by acid demetallization of the protein. Metals reintroduced to this metastable form of apo-Con A produce the stable ternary complexes with no observable time-dependent effects. This metastable form of apo-Con A reverts to its initial state after several days at 25°C. We have fit the kinetic and thermodynamic data for the interaction of Mn2+ and Ca2+ ions with Con A with a model that postulates two conformation states for Con A that differ only slightly in their ground-state free energies, and are separated by an energy barrier of 22 kcal M-1. The conformation with the lower free energy is determined by the presence or absence of a metal ion at S2. The height of the energy barrier suggests that a cis-trans isomerization of a proline amide bond distinguishes the two conformations, implying that the difference between the conformations is in the secondary rather than the tertiary structure of the protein.

Original languageEnglish (US)
Pages (from-to)3883-3896
Number of pages14
JournalBiochemistry
Volume16
Issue number17
StatePublished - 1977

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Concanavalin A
Conformations
Ions
Kinetics
Metals
Protons
Ion Exchange
Energy barriers
Free energy
Ion exchange
Magnetic relaxation
Agglutination
Magnetic Fields
Isomerization
Tertiary Protein Structure
Thermodynamics
Proline
Amides
Edetic Acid
Ground state

ASJC Scopus subject areas

  • Biochemistry

Cite this

Conformation states of concanavalin A : Kinetics of transitions induced by interaction with Mn2+ and Ca2+ ions. / Brown, Rodney D.; Brewer, Curtis F.; Koenig, Seymour H.

In: Biochemistry, Vol. 16, No. 17, 1977, p. 3883-3896.

Research output: Contribution to journalArticle

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title = "Conformation states of concanavalin A: Kinetics of transitions induced by interaction with Mn2+ and Ca2+ ions",
abstract = "Using measurements of the magnetic-field dependence of the nuclear magnetic relaxation rate (1/T1) of solvent water protons over a wide range of field values (corresponding to proton Larmor frequencies from 0.01 to 50 MHz), we have investigated the interaction of Mn2+ and Ca2+ ions with concanavalin A (Con A) over the pH range 5.3 to 6.4, at 5 and 25°C. Particular attention was given to time-dependent effects that occur upon addition or removal of metals. Limited amounts of Mn2+ added to solutions of apo-Con A bind at S1 (the usual {"}transition-metal{"} site) to form a binary complex characterized by a large and pH-dependent dissociation constant, rapid exchange of Mn2+ ions with solvent, and a relatively large and pH-independent contribution to the proton relaxation rate. With S1 occupied, Ca2+ ions can bind at S2 (the usual {"}calcium-binding{"} site) to form a metastable ternary complex characterized by a relatively large and pH-dependent dissociation constant for Ca2+ ions, rapid exchange of Ca2+ ions with solvent, and a relatively low and pH-independent contribution to the proton relaxation rate. We find that this metastable ternary complex undergoes a first-order transition to a stable ternary complex, with a pH-independent time constant of 17 ± 1 min at 5°C and an activation energy of 22 kcal M-1. This stable ternary complex has the same relaxation contribution as the initial metastable complex, but differs in that the dissociation constant of Ca2+ is very low; the off-rate of both metals is of the order of days at 25°C. Saccharide binding and agglutination studies are generally done with this form of Con A. We have also found that, in the absence of Ca2+, Mn2+ can bind at S2 as well as at S1 (S2 was previously thought to bind only Ca1 and Cd2+) to form a metastable ternary complex which, like the metastable Mn2+-Ca2+-Con A complex, undergoes a transition to a stable state, but with a time constant that is much larger than for the Ca2+-containing ternary complex. In contrast to the stable Ca2+-Mn2+-Con A complex, the stable Mn2+-Con A ternary complex has a rather large dissociation constant, and the bound Mn2+ ions are in rapid equilibrium with solvent. The Mn2+ ions can be removed rapidly by the addition of ethylenediaminetetraacetic acid to produce apo-Con A in a metastable state that has different metal-binding properties than apo-Con A prepared by acid demetallization of the protein. Metals reintroduced to this metastable form of apo-Con A produce the stable ternary complexes with no observable time-dependent effects. This metastable form of apo-Con A reverts to its initial state after several days at 25°C. We have fit the kinetic and thermodynamic data for the interaction of Mn2+ and Ca2+ ions with Con A with a model that postulates two conformation states for Con A that differ only slightly in their ground-state free energies, and are separated by an energy barrier of 22 kcal M-1. The conformation with the lower free energy is determined by the presence or absence of a metal ion at S2. The height of the energy barrier suggests that a cis-trans isomerization of a proline amide bond distinguishes the two conformations, implying that the difference between the conformations is in the secondary rather than the tertiary structure of the protein.",
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T2 - Kinetics of transitions induced by interaction with Mn2+ and Ca2+ ions

AU - Brown, Rodney D.

AU - Brewer, Curtis F.

AU - Koenig, Seymour H.

PY - 1977

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N2 - Using measurements of the magnetic-field dependence of the nuclear magnetic relaxation rate (1/T1) of solvent water protons over a wide range of field values (corresponding to proton Larmor frequencies from 0.01 to 50 MHz), we have investigated the interaction of Mn2+ and Ca2+ ions with concanavalin A (Con A) over the pH range 5.3 to 6.4, at 5 and 25°C. Particular attention was given to time-dependent effects that occur upon addition or removal of metals. Limited amounts of Mn2+ added to solutions of apo-Con A bind at S1 (the usual "transition-metal" site) to form a binary complex characterized by a large and pH-dependent dissociation constant, rapid exchange of Mn2+ ions with solvent, and a relatively large and pH-independent contribution to the proton relaxation rate. With S1 occupied, Ca2+ ions can bind at S2 (the usual "calcium-binding" site) to form a metastable ternary complex characterized by a relatively large and pH-dependent dissociation constant for Ca2+ ions, rapid exchange of Ca2+ ions with solvent, and a relatively low and pH-independent contribution to the proton relaxation rate. We find that this metastable ternary complex undergoes a first-order transition to a stable ternary complex, with a pH-independent time constant of 17 ± 1 min at 5°C and an activation energy of 22 kcal M-1. This stable ternary complex has the same relaxation contribution as the initial metastable complex, but differs in that the dissociation constant of Ca2+ is very low; the off-rate of both metals is of the order of days at 25°C. Saccharide binding and agglutination studies are generally done with this form of Con A. We have also found that, in the absence of Ca2+, Mn2+ can bind at S2 as well as at S1 (S2 was previously thought to bind only Ca1 and Cd2+) to form a metastable ternary complex which, like the metastable Mn2+-Ca2+-Con A complex, undergoes a transition to a stable state, but with a time constant that is much larger than for the Ca2+-containing ternary complex. In contrast to the stable Ca2+-Mn2+-Con A complex, the stable Mn2+-Con A ternary complex has a rather large dissociation constant, and the bound Mn2+ ions are in rapid equilibrium with solvent. The Mn2+ ions can be removed rapidly by the addition of ethylenediaminetetraacetic acid to produce apo-Con A in a metastable state that has different metal-binding properties than apo-Con A prepared by acid demetallization of the protein. Metals reintroduced to this metastable form of apo-Con A produce the stable ternary complexes with no observable time-dependent effects. This metastable form of apo-Con A reverts to its initial state after several days at 25°C. We have fit the kinetic and thermodynamic data for the interaction of Mn2+ and Ca2+ ions with Con A with a model that postulates two conformation states for Con A that differ only slightly in their ground-state free energies, and are separated by an energy barrier of 22 kcal M-1. The conformation with the lower free energy is determined by the presence or absence of a metal ion at S2. The height of the energy barrier suggests that a cis-trans isomerization of a proline amide bond distinguishes the two conformations, implying that the difference between the conformations is in the secondary rather than the tertiary structure of the protein.

AB - Using measurements of the magnetic-field dependence of the nuclear magnetic relaxation rate (1/T1) of solvent water protons over a wide range of field values (corresponding to proton Larmor frequencies from 0.01 to 50 MHz), we have investigated the interaction of Mn2+ and Ca2+ ions with concanavalin A (Con A) over the pH range 5.3 to 6.4, at 5 and 25°C. Particular attention was given to time-dependent effects that occur upon addition or removal of metals. Limited amounts of Mn2+ added to solutions of apo-Con A bind at S1 (the usual "transition-metal" site) to form a binary complex characterized by a large and pH-dependent dissociation constant, rapid exchange of Mn2+ ions with solvent, and a relatively large and pH-independent contribution to the proton relaxation rate. With S1 occupied, Ca2+ ions can bind at S2 (the usual "calcium-binding" site) to form a metastable ternary complex characterized by a relatively large and pH-dependent dissociation constant for Ca2+ ions, rapid exchange of Ca2+ ions with solvent, and a relatively low and pH-independent contribution to the proton relaxation rate. We find that this metastable ternary complex undergoes a first-order transition to a stable ternary complex, with a pH-independent time constant of 17 ± 1 min at 5°C and an activation energy of 22 kcal M-1. This stable ternary complex has the same relaxation contribution as the initial metastable complex, but differs in that the dissociation constant of Ca2+ is very low; the off-rate of both metals is of the order of days at 25°C. Saccharide binding and agglutination studies are generally done with this form of Con A. We have also found that, in the absence of Ca2+, Mn2+ can bind at S2 as well as at S1 (S2 was previously thought to bind only Ca1 and Cd2+) to form a metastable ternary complex which, like the metastable Mn2+-Ca2+-Con A complex, undergoes a transition to a stable state, but with a time constant that is much larger than for the Ca2+-containing ternary complex. In contrast to the stable Ca2+-Mn2+-Con A complex, the stable Mn2+-Con A ternary complex has a rather large dissociation constant, and the bound Mn2+ ions are in rapid equilibrium with solvent. The Mn2+ ions can be removed rapidly by the addition of ethylenediaminetetraacetic acid to produce apo-Con A in a metastable state that has different metal-binding properties than apo-Con A prepared by acid demetallization of the protein. Metals reintroduced to this metastable form of apo-Con A produce the stable ternary complexes with no observable time-dependent effects. This metastable form of apo-Con A reverts to its initial state after several days at 25°C. We have fit the kinetic and thermodynamic data for the interaction of Mn2+ and Ca2+ ions with Con A with a model that postulates two conformation states for Con A that differ only slightly in their ground-state free energies, and are separated by an energy barrier of 22 kcal M-1. The conformation with the lower free energy is determined by the presence or absence of a metal ion at S2. The height of the energy barrier suggests that a cis-trans isomerization of a proline amide bond distinguishes the two conformations, implying that the difference between the conformations is in the secondary rather than the tertiary structure of the protein.

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