TY - JOUR
T1 - Stopped-flow NMR spectroscopy
T2 - Real-time unfolding studies of 6-19F-tryptophan-labeled Escherichia coli dihydrofolate reductase
AU - Hoeltzli, Sydney D.
AU - Frieden, Carl
PY - 1995/9/26
Y1 - 1995/9/26
N2 - Escherichia coli dihydrofolate reductase (DHFR; EC 1.5.1.3) contains five tryptophan residues that have been replaced with 6-19F-tryptophan. The 19F NMR assignments are known in the native, unliganded form and the unfolded form. We have used these assignments with stopped-flow 19F NMR spectroscopy to investigate the behavior of specific regions of the protein in real time during urea-induced unfolding. The NMR data show that within 1.5 sec most of the intensities of the native 19F resonances of the protein are lost but only a fraction (≈20%) of the intensities of the unfolded resonances appears. We postulate that the early disappearance of the native resonances indicates that most of the protein rapidly forms an intermediate in which the side chains have considerable mobility. Stopped-flow far-UV circular dichroism measurements indicate that this intermediate retains native-like secondary structure. Eighty percent of the intensities of the NMR resonances assigned to the individual tryptophans in the unfolded state appear with similar rate constants (k ≈ 0.14 sec-1), consistent with the major phase of unfolding observed by stopped-f low circular dichroism (representing 80% of total amplitude). These data imply that after formation of the intermediate, which appears to represent an expanded structural form, all regions of the protein unfold at the same rate. Stopped-flow measurements of the fluorescence and circular dichroism changes associated with the urea-induced unfolding show a fast phase (half-time of about 1 sec) representing 20% of the total amplitude in addition to the slow phase mentioned above. The NMR data show that ≈20% of the total intensity for each of the unfolded tryptophan resonances is present at 1.5 sec, indicating that these two phases may represent the complete unfolding of the two different populations of the native protein.
AB - Escherichia coli dihydrofolate reductase (DHFR; EC 1.5.1.3) contains five tryptophan residues that have been replaced with 6-19F-tryptophan. The 19F NMR assignments are known in the native, unliganded form and the unfolded form. We have used these assignments with stopped-flow 19F NMR spectroscopy to investigate the behavior of specific regions of the protein in real time during urea-induced unfolding. The NMR data show that within 1.5 sec most of the intensities of the native 19F resonances of the protein are lost but only a fraction (≈20%) of the intensities of the unfolded resonances appears. We postulate that the early disappearance of the native resonances indicates that most of the protein rapidly forms an intermediate in which the side chains have considerable mobility. Stopped-flow far-UV circular dichroism measurements indicate that this intermediate retains native-like secondary structure. Eighty percent of the intensities of the NMR resonances assigned to the individual tryptophans in the unfolded state appear with similar rate constants (k ≈ 0.14 sec-1), consistent with the major phase of unfolding observed by stopped-f low circular dichroism (representing 80% of total amplitude). These data imply that after formation of the intermediate, which appears to represent an expanded structural form, all regions of the protein unfold at the same rate. Stopped-flow measurements of the fluorescence and circular dichroism changes associated with the urea-induced unfolding show a fast phase (half-time of about 1 sec) representing 20% of the total amplitude in addition to the slow phase mentioned above. The NMR data show that ≈20% of the total intensity for each of the unfolded tryptophan resonances is present at 1.5 sec, indicating that these two phases may represent the complete unfolding of the two different populations of the native protein.
KW - Circular dichroism
KW - Fluorescence
KW - Unfolding intermediate
UR - http://www.scopus.com/inward/record.url?scp=0029079665&partnerID=8YFLogxK
U2 - 10.1073/pnas.92.20.9318
DO - 10.1073/pnas.92.20.9318
M3 - Article
C2 - 7568125
AN - SCOPUS:0029079665
SN - 0027-8424
VL - 92
SP - 9318
EP - 9322
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 20
ER -