bill brazil - Academia.edu (original) (raw)
Papers by bill brazil
Journal of Biological Chemistry, Feb 1, 1997
Cell Stress & Chaperones, 1999
No Abstract Available
Methods in Enzymology, 1998
We have elucidated conditions for the covalent incorporation of a non-specific hydrophobic probe,... more We have elucidated conditions for the covalent incorporation of a non-specific hydrophobic probe, bisANS, into various proteins. Using this method, we are able to map hydrophobic surfaces in proteins. In addition, we have shown that for GroEL, we are able to use the fluorescence of the incorporated bisANS to monitor conformational changes in a defined region of the protein in
Cell Stress & Chaperones, 1999
Journal of Biological Chemistry, Feb 1, 1998
Fluorescent and non-fluorescent probes have been used to show that divalent cations (Ca 2؉ , Mg 2... more Fluorescent and non-fluorescent probes have been used to show that divalent cations (Ca 2؉ , Mg 2؉ , Mn 2؉ , and Zn 2؉) significantly increase hydrophobic exposure on GroEL, whereas monovalent cations (K ؉ and Na ؉) have little effect. Zn 2؉ always induced the largest amount of hydrophobic exposure on GroEL. By using a new method based on interactions of GroEL with octyl-Sepharose, it was demonstrated that Zn 2؉ binding strengthens GroEL hydrophobic binding interactions and increases the efficiency of substrate release upon the addition of MgATP and GroES. The binding of 4,4bis(1-anilino-8-naphthalenesulfonic acid) to GroEL in the presence of Zn 2؉ has a K d Х 1 M, which is similar to that observed previously for the GroEL 4,4-bis(1-anilino-8-naphthalenesulfonic acid) complex. Urea denaturation, sedimentation velocity ultracentrifugation, and electron microscopy revealed that the quaternary structure of GroEL in the presence of Zn 2؉ had a stability and morphology equivalent to unliganded GroEL. In contrast, circular dichroism suggested some loss in both ␣-helical and -sheet secondary structure in the presence of Zn 2؉. These data suggest that divalent cations can modulate the amount of hydrophobic surface presented by GroEL. Furthermore, the influence of Zn 2؉ on GroEL hydrophobic surface exposure as well as substrate binding and release appears to be distinct from the stabilizing effects of Mg 2؉ on GroEL quaternary structure.
Methods in Enzymology, 1998
We have elucidated conditions for the covalent incorporation of a non-specific hydrophobic probe,... more We have elucidated conditions for the covalent incorporation of a non-specific hydrophobic probe, bisANS, into various proteins. Using this method, we are able to map hydrophobic surfaces in proteins. In addition, we have shown that for GroEL, we are able to use the fluorescence of the incorporated bisANS to monitor conformational changes in a defined region of the protein in
Journal of Biological Chemistry, 1997
Journal of Biological Chemistry, 1998
Fluorescent and non-fluorescent probes have been used to show that divalent cations (Ca 2؉ , Mg 2... more Fluorescent and non-fluorescent probes have been used to show that divalent cations (Ca 2؉ , Mg 2؉ , Mn 2؉ , and Zn 2؉) significantly increase hydrophobic exposure on GroEL, whereas monovalent cations (K ؉ and Na ؉) have little effect. Zn 2؉ always induced the largest amount of hydrophobic exposure on GroEL. By using a new method based on interactions of GroEL with octyl-Sepharose, it was demonstrated that Zn 2؉ binding strengthens GroEL hydrophobic binding interactions and increases the efficiency of substrate release upon the addition of MgATP and GroES. The binding of 4,4bis(1-anilino-8-naphthalenesulfonic acid) to GroEL in the presence of Zn 2؉ has a K d Х 1 M, which is similar to that observed previously for the GroEL 4,4-bis(1-anilino-8-naphthalenesulfonic acid) complex. Urea denaturation, sedimentation velocity ultracentrifugation, and electron microscopy revealed that the quaternary structure of GroEL in the presence of Zn 2؉ had a stability and morphology equivalent to unliganded GroEL. In contrast, circular dichroism suggested some loss in both ␣-helical and -sheet secondary structure in the presence of Zn 2؉. These data suggest that divalent cations can modulate the amount of hydrophobic surface presented by GroEL. Furthermore, the influence of Zn 2؉ on GroEL hydrophobic surface exposure as well as substrate binding and release appears to be distinct from the stabilizing effects of Mg 2؉ on GroEL quaternary structure.
Biochemistry, 2001
The extent of hydrophobic exposure upon bis-ANS binding to the functional apical domain fragment ... more The extent of hydrophobic exposure upon bis-ANS binding to the functional apical domain fragment of GroEL, or minichaperone (residues 191-345), was investigated and compared with that of the GroEL tetradecamer. Although a total of seven molecules of bis-ANS bind cooperatively to this minichaperone, most of the hydrophobic sites were induced following initial binding of one to two molecules of probe. From the equilibrium and kinetics studies at low bis-ANS concentrations, it is evident that the native apical domain is converted to an intermediate conformation with increased hydrophobic surfaces. This intermediate binds additional bis-ANS molecules. Tyrosine fluorescence detected denaturation demonstrated that bis-ANS can destabilize the apical domain. The results from (i) bis-ANS titrations, (ii) urea denaturation studies in the presence and absence of bis-ANS, and (iii) intrinsic tyrosine fluorescence studies of the apical domain are consistent with a model in which bis-ANS binds tightly to the intermediate state, relatively weakly to the native state, and little to the denatured state. The results suggest that the conformational changes seen in apical domain fragments are not seen in the intact GroEL oligomer due to restrictions imposed by connections of the apical domain to the intermediate domain and suppression of movement due to quaternary structure.
Journal of Biological Chemistry, Feb 1, 1997
Cell Stress & Chaperones, 1999
No Abstract Available
Methods in Enzymology, 1998
We have elucidated conditions for the covalent incorporation of a non-specific hydrophobic probe,... more We have elucidated conditions for the covalent incorporation of a non-specific hydrophobic probe, bisANS, into various proteins. Using this method, we are able to map hydrophobic surfaces in proteins. In addition, we have shown that for GroEL, we are able to use the fluorescence of the incorporated bisANS to monitor conformational changes in a defined region of the protein in
Cell Stress & Chaperones, 1999
Journal of Biological Chemistry, Feb 1, 1998
Fluorescent and non-fluorescent probes have been used to show that divalent cations (Ca 2؉ , Mg 2... more Fluorescent and non-fluorescent probes have been used to show that divalent cations (Ca 2؉ , Mg 2؉ , Mn 2؉ , and Zn 2؉) significantly increase hydrophobic exposure on GroEL, whereas monovalent cations (K ؉ and Na ؉) have little effect. Zn 2؉ always induced the largest amount of hydrophobic exposure on GroEL. By using a new method based on interactions of GroEL with octyl-Sepharose, it was demonstrated that Zn 2؉ binding strengthens GroEL hydrophobic binding interactions and increases the efficiency of substrate release upon the addition of MgATP and GroES. The binding of 4,4bis(1-anilino-8-naphthalenesulfonic acid) to GroEL in the presence of Zn 2؉ has a K d Х 1 M, which is similar to that observed previously for the GroEL 4,4-bis(1-anilino-8-naphthalenesulfonic acid) complex. Urea denaturation, sedimentation velocity ultracentrifugation, and electron microscopy revealed that the quaternary structure of GroEL in the presence of Zn 2؉ had a stability and morphology equivalent to unliganded GroEL. In contrast, circular dichroism suggested some loss in both ␣-helical and -sheet secondary structure in the presence of Zn 2؉. These data suggest that divalent cations can modulate the amount of hydrophobic surface presented by GroEL. Furthermore, the influence of Zn 2؉ on GroEL hydrophobic surface exposure as well as substrate binding and release appears to be distinct from the stabilizing effects of Mg 2؉ on GroEL quaternary structure.
Methods in Enzymology, 1998
We have elucidated conditions for the covalent incorporation of a non-specific hydrophobic probe,... more We have elucidated conditions for the covalent incorporation of a non-specific hydrophobic probe, bisANS, into various proteins. Using this method, we are able to map hydrophobic surfaces in proteins. In addition, we have shown that for GroEL, we are able to use the fluorescence of the incorporated bisANS to monitor conformational changes in a defined region of the protein in
Journal of Biological Chemistry, 1997
Journal of Biological Chemistry, 1998
Fluorescent and non-fluorescent probes have been used to show that divalent cations (Ca 2؉ , Mg 2... more Fluorescent and non-fluorescent probes have been used to show that divalent cations (Ca 2؉ , Mg 2؉ , Mn 2؉ , and Zn 2؉) significantly increase hydrophobic exposure on GroEL, whereas monovalent cations (K ؉ and Na ؉) have little effect. Zn 2؉ always induced the largest amount of hydrophobic exposure on GroEL. By using a new method based on interactions of GroEL with octyl-Sepharose, it was demonstrated that Zn 2؉ binding strengthens GroEL hydrophobic binding interactions and increases the efficiency of substrate release upon the addition of MgATP and GroES. The binding of 4,4bis(1-anilino-8-naphthalenesulfonic acid) to GroEL in the presence of Zn 2؉ has a K d Х 1 M, which is similar to that observed previously for the GroEL 4,4-bis(1-anilino-8-naphthalenesulfonic acid) complex. Urea denaturation, sedimentation velocity ultracentrifugation, and electron microscopy revealed that the quaternary structure of GroEL in the presence of Zn 2؉ had a stability and morphology equivalent to unliganded GroEL. In contrast, circular dichroism suggested some loss in both ␣-helical and -sheet secondary structure in the presence of Zn 2؉. These data suggest that divalent cations can modulate the amount of hydrophobic surface presented by GroEL. Furthermore, the influence of Zn 2؉ on GroEL hydrophobic surface exposure as well as substrate binding and release appears to be distinct from the stabilizing effects of Mg 2؉ on GroEL quaternary structure.
Biochemistry, 2001
The extent of hydrophobic exposure upon bis-ANS binding to the functional apical domain fragment ... more The extent of hydrophobic exposure upon bis-ANS binding to the functional apical domain fragment of GroEL, or minichaperone (residues 191-345), was investigated and compared with that of the GroEL tetradecamer. Although a total of seven molecules of bis-ANS bind cooperatively to this minichaperone, most of the hydrophobic sites were induced following initial binding of one to two molecules of probe. From the equilibrium and kinetics studies at low bis-ANS concentrations, it is evident that the native apical domain is converted to an intermediate conformation with increased hydrophobic surfaces. This intermediate binds additional bis-ANS molecules. Tyrosine fluorescence detected denaturation demonstrated that bis-ANS can destabilize the apical domain. The results from (i) bis-ANS titrations, (ii) urea denaturation studies in the presence and absence of bis-ANS, and (iii) intrinsic tyrosine fluorescence studies of the apical domain are consistent with a model in which bis-ANS binds tightly to the intermediate state, relatively weakly to the native state, and little to the denatured state. The results suggest that the conformational changes seen in apical domain fragments are not seen in the intact GroEL oligomer due to restrictions imposed by connections of the apical domain to the intermediate domain and suppression of movement due to quaternary structure.