Characterizing protein-protein complexes and oligomers by nuclear magnetic resonance spectroscopy.
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Carbon Isotopes↗
Biomedical subjects
K J Walters
Publications and source records attributed to K J Walters.
Structure of the soluble methane monooxygenase regulatory protein B.
The soluble methane monooxygenase (sMMO; EC 1.14.13.25) from the pseudothermophile Methylococcus capsulatus (Bath) is a three-component enzyme system that catalyzes the selective oxidation of methane to methanol. We have used NMR spectroscopy to produce a highly refined structure of MMOB, the 16-kDa regulatory protein of this system. This structure has a unique and intricate fold containing seven beta-strands forming two beta-sheets oriented perpendicular to each other and bridged by three alpha-helices. The rate and efficiency of the methane hydroxylation by sMMO depend on dynamic binding interactions of the hydroxylase with the reductase and regulatory protein components during catalysis. We have monitored by NMR the binding of MMOB to the hydroxylase in the presence and absence of the reductase. The results of these studies provide structural insight into how the regulatory protein interacts with the hydroxylase.
Amino Acid Sequence↗
Structure and mobility of the PUT3 dimer.
The solution structure and backbone dynamics of the transcriptional activator PUT3 (31-100) has been characterized using NMR spectroscopy. PUT3 (31-100) contains three distinct domains: a cysteine zinc cluster, linker, and dimerization domain. The cysteine zinc cluster of PUT3 closely resembles the solution structure of GAL4, while the dimerization domain forms a long coiled-coil similar to that observed in the crystal structures of GAL4 and PPR1. However, the residues at the N-terminal end of the coiled-coil behave very differently in each of these proteins. A comparison of the structural elements within this region provides a model for the DNA binding specificity of these proteins. Furthermore, we have characterized the dynamics of PUT3 to find that the zinc cluster and dimerization domains have very diverse dynamics in solution. The dimerization domain behaves as a large protein, while the peripheral cysteine zinc clusters have dynamic properties similar to small proteins.
Amino Acid Sequence↗
Sequence dependence of purine C8H exchange kinetics in the dodecamer 5'-d(CGCGAATTCGCG)-3'.
Proton nmr spectroscopy is used to measure the deuterium exchange rates of C8 protons in individual purines of the dodecamer 5'-d(CGCGAATTCGCG)-3' and their temperature dependence. In perfect agreement with results from tritium labeling and laser Raman spectroscopy, we find that the DNA secondary structure retards the rates of purine C8H exchange. The largest effects are observed for the C8 protons of adenines whose rates of exchange at 40 degrees C are 3- to 4-fold lower than that in 5'-adenosine monophosphate. Moreover, the retardation of exchange at the central adenine is greater than that at its 5'-neighbor. For the guanines, the exchange rates are up to 2-fold lower than that in 5'-guanosine monophosphate, and the largest retardation is observed for the bases at positions 10 and 12. A dependence on base sequence is also observed for the activation energy for exchange. The activation energy is largest for the adenines and its value is 4 kcal/mol higher than that in 5'-adenosine monophosphate. The lowest activation energy is observed for the guanine in position 4 and the value is the same as in 5'-guanosine monophosphate. These results demonstrate the sensitivity of the purine C8H exchange kinetics to sequence-dependent conformational features of B-DNA in solution state.
Base Sequence↗
The significance of aortoiliac atherosclerosis as assessed by Doppler ultrasound.
A simple, noninvasive method of assessing atherosclerotic aortoiliac obstruction is described using Doppler ultrasound with a concurrent electrocardiogram. The method is significantly more accurate than clinical examination. The pulse wave velocity profile at the common femoral artery is recorded with a nondirectional Doppler probe. The time delay from the R wave of the concurrent electrocardiogram to the ultrasound waveform peak and to a point half-way up the waveform upslope is measured. By evaluating the mean of ten such measurements at each point and then by taking the ratio of the former to the latter, a Proximal Damping Quotient (PDQ) may be derived. If the PDQ is greater than 1.4, significant proximal obstruction is probable. Conversely, a PDQ of less than 1.4 suggests a functionally clear aortoiliac segment. Any patient with a PDQ of less than 1.3 in whom reconstructive surgery is being correlated may thus be spared an aortogram and the affected limb may be investigated by femoral angiography alone. A low PDQ is supporting evidence of an adequate "run-in" to the distal segment when a distal arterial reconstruction is proposed. Similarly, if a femoro-femoral crossover graft is to be used, then significant aortoiliac atherosclerosis proximal to the donor femoral artery may be excluded without recourse to aortography.
Adult↗