A Folded Monomeric Intermediate in the Formation of Lambda Cro Dimer±DNA Complexes
The folding, dimerization and DNA binding equilibria of the bacterio- phage lambda Cro repressor have been characterized. Comparison with four engineered variants shows that a folded monomeric species is sub- stantially populated under conditions used for the formation of dimer± DNA complexe...
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| Format: | Article |
| Idioma: | anglès |
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JMB
2025
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| Matèries: | |
| Accés en línia: | https://repository.auw.edu.bd/handle/123456789/1033 |
| _version_ | 1857448968630304768 |
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| author | . Mollah, A. K. M. M |
| author_facet | . Mollah, A. K. M. M |
| author_sort | . Mollah, A. K. M. M |
| collection | institutional Repository |
| description | The folding, dimerization and DNA binding equilibria of the bacterio-
phage lambda Cro repressor have been characterized. Comparison with
four engineered variants shows that a folded monomeric species is sub-
stantially populated under conditions used for the formation of dimer±
DNA complexes. Although Cro dimers are the only DNA-bound species
observed in electrophoretic mobility shift assays, cooperativity in Cro ±
DNA binding isotherms shows that the predominant free protein species
is monomeric at nanomolar concentrations. Micromolar dissociation con-
stants for Cro dimers have been measured in the absence of DNA by
sedimentation equilibrium and gel ®ltration chromatography. Denatura-
tion of Cro dimers in the 10 to 100 micromolar concentration range by
guanidine hydrochloride (GdnHCl) is well modeled as a two-state pro-
cess, with folded dimers and unfolded monomers as the only signi®-
cantly populated species. However, linear extrapolation of this composite
unfolding and dimer dissociation free energy predicts a nanomolar dis-
sociation constant in the absence of denaturant. This extrapolation is
clearly inconsistent with the DNA binding and hydrodynamic measure-
ments. Our interpretation of these results is that the monomeric species
detected in DNA binding and hydrodynamic experiments is predomi-
nantly folded. The stability of the folded monomeric species can be calcu-
lated as the difference between the dimerization free energy determined
from hydrodynamic measurements and the folding free energy extrapo-
lated from GdnHCl denaturation. The calculated stability of the Cro
F58W monomer is greater than that of the wild-type Cro monomer.
Thus, residue 58, which makes critical intermolecular contacts across the
dimer interface, is also involved in intramolecular stabilization of the
monomeric intermediate. |
| format | Article |
| id | 123456789-1033 |
| institution | Asian University for Women |
| language | English |
| publishDate | 2025 |
| publisher | JMB |
| record_format | dspace |
| spelling | 123456789-10332026-02-18T06:15:29Z A Folded Monomeric Intermediate in the Formation of Lambda Cro Dimer±DNA Complexes . Mollah, A. K. M. M monomer ±dimer equilibrium; dimerization; bacteriophage repressor; DNA binding protein; covalent dimer The folding, dimerization and DNA binding equilibria of the bacterio- phage lambda Cro repressor have been characterized. Comparison with four engineered variants shows that a folded monomeric species is sub- stantially populated under conditions used for the formation of dimer± DNA complexes. Although Cro dimers are the only DNA-bound species observed in electrophoretic mobility shift assays, cooperativity in Cro ± DNA binding isotherms shows that the predominant free protein species is monomeric at nanomolar concentrations. Micromolar dissociation con- stants for Cro dimers have been measured in the absence of DNA by sedimentation equilibrium and gel ®ltration chromatography. Denatura- tion of Cro dimers in the 10 to 100 micromolar concentration range by guanidine hydrochloride (GdnHCl) is well modeled as a two-state pro- cess, with folded dimers and unfolded monomers as the only signi®- cantly populated species. However, linear extrapolation of this composite unfolding and dimer dissociation free energy predicts a nanomolar dis- sociation constant in the absence of denaturant. This extrapolation is clearly inconsistent with the DNA binding and hydrodynamic measure- ments. Our interpretation of these results is that the monomeric species detected in DNA binding and hydrodynamic experiments is predomi- nantly folded. The stability of the folded monomeric species can be calcu- lated as the difference between the dimerization free energy determined from hydrodynamic measurements and the folding free energy extrapo- lated from GdnHCl denaturation. The calculated stability of the Cro F58W monomer is greater than that of the wild-type Cro monomer. Thus, residue 58, which makes critical intermolecular contacts across the dimer interface, is also involved in intramolecular stabilization of the monomeric intermediate. 2025-08-07T10:34:25Z 2025-08-07T10:34:25Z 1997 Article https://repository.auw.edu.bd/handle/123456789/1033 en application/pdf JMB |
| spellingShingle | monomer ±dimer equilibrium; dimerization; bacteriophage repressor; DNA binding protein; covalent dimer . Mollah, A. K. M. M A Folded Monomeric Intermediate in the Formation of Lambda Cro Dimer±DNA Complexes |
| title | A Folded Monomeric Intermediate in the Formation of Lambda Cro Dimer±DNA Complexes |
| title_full | A Folded Monomeric Intermediate in the Formation of Lambda Cro Dimer±DNA Complexes |
| title_fullStr | A Folded Monomeric Intermediate in the Formation of Lambda Cro Dimer±DNA Complexes |
| title_full_unstemmed | A Folded Monomeric Intermediate in the Formation of Lambda Cro Dimer±DNA Complexes |
| title_short | A Folded Monomeric Intermediate in the Formation of Lambda Cro Dimer±DNA Complexes |
| title_sort | folded monomeric intermediate in the formation of lambda cro dimer dna complexes |
| topic | monomer ±dimer equilibrium; dimerization; bacteriophage repressor; DNA binding protein; covalent dimer |
| url | https://repository.auw.edu.bd/handle/123456789/1033 |
| work_keys_str_mv | AT mollahakmm afoldedmonomericintermediateintheformationoflambdacrodimerdnacomplexes AT mollahakmm foldedmonomericintermediateintheformationoflambdacrodimerdnacomplexes |