HeteroTOCSY-based experiments for measuring heteronuclear relaxation in nucleic acids and proteins.
While both 31P and 113Cd are present at locations of interest in many different macromolecular systems, heteronuclear-detected relaxation measurements on these nuclei have been restrained by limitations in either resolution or signal-to-noise ratio. We have developed heteroTOCSY-based methods to overcome both of these problems. Two-dimensional versions of these experiments were utilized to measure 31P T1 and T2 values in DNA oligonucleotides; the additional resolution offered by a second dimension allowed determination of these values for most of the 31P resonances in a DNA dodecamer. The results from the experiments indicated that there was little significant variation in T1 values for the different phosphates in the DNA dodecamer; however, the T2 values showed a clear pattern, with lower values in the interior of the sequence than at the ends of the helix. Furthermore, a significant correlation between 31P chemical shifts and T2 values was observed. One-dimensional, frequency-selective versions of these experiments were also developed for use on systems containing a smaller number of heteronuclear spins. These methods were applied to investigate the heteronuclear relaxation properties of 113Cd in 113Cd2LAC9(61), a Cys6Zn2 DNA-binding domain. Data from the experiments confirm biochemical evidence that more significant differences occur in the metal-protein interactions between the two metal-binding sites than has been previously identified for proteins containing this motif.
Schweitzer
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Department of Pediatrics, Yale University School of Medicine, New Haven, CT, USA.
Gardner
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Tucker-Kellogg
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D000964:Antimetabolites, Antineoplastic / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002104:Cadmium; D003561:Cytarabine / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry; D007554:Isotopes; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009696:Nucleic Acids / Q000737:chemistry*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D010759:Phosphorus Isotopes; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D014157:Transcription Factors
D000964:Antimetabolites, Antineoplastic; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D007554:Isotopes; C058596:LAC9 protein, Kluyveromyces lactis; D009696:Nucleic Acids; D009838:Oligodeoxyribonucleotides; D010759:Phosphorus Isotopes; D011506:Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D002104:Cadmium; D003561:Cytarabine