TY - JOUR
T1 - Analysis of the conformational stability of the active domain of recombinant mouse TIMP-1 by intrinsic fluorescence
AU - Rajan, Shyamala S.
AU - Denhardt, David T.
AU - Lackland, Henry
AU - Ludescher, Richard D.
PY - 1998/1/14
Y1 - 1998/1/14
N2 - Intrinsic fluorescence was used to examine the stability of an active, N-terminal domain of mouse tissue inhibitor of metalloproteinase (TIMP-1) fused with an N-terminal polyhistidine tag. Emission and quenching studies suggested that the single tryptophan is on the protein surface partially exposed to solvent. The TIMP-1 recombinant unfolded reversibly in the presence of guanidinium chloride with the transition midpoint at 2.35 M; extrapolation gave a stabilization free energy of 5.1 kcal mol-1 at 25°C. Analysis of the temperature dependence of the fluorescence intensity gave a melting transition with midpoint at 51°C and an enthalpy and heat capacity change on unfolding of 32 kcal mol-1 and 0.45 kcal K-1 mol-1, respectively, values comparable to other single domain proteins. Comparison with literature data indicated that the stability of mouse recombinant TIMP-1 more closely resembled that of human metalloproteinase inhibitor TIMP-2 than TIMP-1 despite closer homology to the human TIMP-1 protein.
AB - Intrinsic fluorescence was used to examine the stability of an active, N-terminal domain of mouse tissue inhibitor of metalloproteinase (TIMP-1) fused with an N-terminal polyhistidine tag. Emission and quenching studies suggested that the single tryptophan is on the protein surface partially exposed to solvent. The TIMP-1 recombinant unfolded reversibly in the presence of guanidinium chloride with the transition midpoint at 2.35 M; extrapolation gave a stabilization free energy of 5.1 kcal mol-1 at 25°C. Analysis of the temperature dependence of the fluorescence intensity gave a melting transition with midpoint at 51°C and an enthalpy and heat capacity change on unfolding of 32 kcal mol-1 and 0.45 kcal K-1 mol-1, respectively, values comparable to other single domain proteins. Comparison with literature data indicated that the stability of mouse recombinant TIMP-1 more closely resembled that of human metalloproteinase inhibitor TIMP-2 than TIMP-1 despite closer homology to the human TIMP-1 protein.
UR - http://www.scopus.com/inward/record.url?scp=0032515342&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0032515342&partnerID=8YFLogxK
U2 - 10.1006/bbrc.1997.7960
DO - 10.1006/bbrc.1997.7960
M3 - Article
C2 - 9446789
AN - SCOPUS:0032515342
SN - 0006-291X
VL - 242
SP - 303
EP - 309
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 2
ER -