Effect of deuteration on the accuracy of HN-HN distance constraints.

The effect of deuteration on the measurement of HN-HN distances in moderately sized (15 kDa) proteins is discussed. Data are presented for a 15 kDa protein which is 95% deuterated on the Halpha position, and partially (70%) deuterated at other aliphatic sites. Deuteration of the protein increases the signal intensity of HN-HN cross peaks in NOESY spectra such that dipolar couplings between protons 4-5 A apart are readily detected. Experimental data and computer simulations show that either perdeuteration or partial deuteration of the protein increases the accuracy of amide-amide distance constraints. Thus, partial deuteration can be used to obtain more accurate long-range distance constraints for structure determination by NMR.

Crystal structure of the RNA-binding domain from transcription termination factor rho.

Transcription termination factor rho is an ATP-dependent hexameric helicase found in most eubacterial species. The Escherichia coli rho monomer consists of two domains, an RNA-binding domain (residues 1-130) and an ATPase domain (residues 131-419). The ATPase domain is homologous to the beta subunit of F1-ATPase. Here, we report that the crystal structure of the RNA-binding domain of rho (rho130) at 1.55 A confirms that rho130 contains the oligosaccharide/oligonucleotide-binding (OB) fold, a five stranded beta-barrel. The beta-barrel of rho130 is also surprisingly similar to the N-terminal beta-barrel of F1 ATPase, extending the applicability of F1 ATPase as a structural model for hexameric rho.

The NMR structure of the RNA binding domain of E. coli rho factor suggests possible RNA-protein interactions.

Rho protein is an essential hexameric RNA-DNA helicase that binds nascent mRNA transcripts and terminates transcription in a wide variety of eubacterial species. The NMR solution structure of the RNA binding domain of rho, rho130, is presented. This structure consists of two sub-domains, an N-terminal three-helix bundle and a C-terminal beta-barrel that is structurally similar to the oligosaccharide/oligonucleotide binding (OB) fold. Chemical shift changes of rho130 upon RNA binding and previous mutagenetic analyses of intact rho suggest that residues Asp 60, Phe 62, Phe 64, and Arg 66 are critical for binding and support the hypothesis that ssRNA/ssDNA binding is localized in the beta-barrel sub-domain. On the basis of these studies and the tertiary structure of rho130, we propose that residues Asp 60, Phe 62, Phe 64, Arg 66, Tyr 80, Lys 105, and Arg 109 participate in RNA-protein interactions.

1H, 15N and 13C resonance assignments and secondary structure determination of the RNA-binding domain of E.coli rho protein.

Protein fragments containing the RNA-binding domain of Escherichia coli rho protein have been over-expressed in E. coli. NMR spectra of the fragment containing residues 1-116 of rho protein (Rho116) show that a region of this protein is unfolded in solution. Addition of (dC)10 to this fragment stabilizes the folded form of the protein. The fragment comprising residues 1-130 of rho protein (Rho130) is found to be stably folded, both in absence and presence of nucleic acid. NMR studies of the complex of Rho130 with RNA and DNA oligonucleotides indicate that the binding-site size, affinity, and specificity of Rho130 are similar to those of intact rho protein; therefore, Rho130 is a suitable model of the RNA-binding domain of Rho protein. NMR line widths as well as titration experiments of Rho130 complexed with oligonucleotides of various lengths suggests that Rho130 forms oligomers in the presence of longer oligonucleotides. 1H, 15N and 13C resonance assignments were facilitated by the utilization of two pulse sequences, CN-NOESY and CCH-TOCSY. The secondary structure of unliganded Rho130 has been determined by NMR techniques, and it is clear that the RNA-binding domain of rho is more structurally similar to the cold shock domain than to the RNA recognition motif.

Expression and characterization of the fourth repeat of Xenopus interphotoreceptor retinoid-binding protein in E. coli.

Interphotoreceptor retinoid-binding protein (IRBP) is an extracellular glycolipoprotein which in higher vertebrates has a 4-repeat structure and carries endogenous vitamin A and fatty acids. The location of IRBP's 1-2 binding sites for retinol is unknown. To begin to understand which repeat(s) are responsible for ligand-binding, we expressed the fourth repeat of Xenopus IRBP in E. coli to determine if it could by itself bind all-trans retinol. Our expression studies used a polyhistidine fusion domain to purify the recombinant protein directly from inclusion bodies. The fusion protein could be renatured without aggregation if refolded at a sufficiently dilute concentration (< 3 microM). The recombinant fourth repeat of Xenopus IRBP binds [3H]all-trans retinol and the fluorescence of this ligand increases 8-fold upon binding. The binding is saturable with a Kd = 0.4 microM. The expression of recombinant IRBP fragments as fusion proteins in prokaryotes will be useful for defining the structural requirements for ligand binding by this interesting protein.

Infrared spectroscopic detection of light-induced change in chloride-arginine interaction in halorhodopsin.

A light-induced transient change in the ionic interaction between chloride and arginine in the transmembrane anion pump halorhodopsin (hR) is detected with infrared absorption spectroscopy. In the IR difference spectrum of hR and one of its photoproducts (hL), only a few bands have frequencies that depend on the particular halide ion (Cl-, Br-, or I-) present. Three of the halide-sensitive negative difference bands (at 1695, 1610, and 1170 cm-1) correspond in frequency to arginine C-N vibrations and undergo anion-dependent shifts that match those seen in ethylguanidinium halide model compounds. These shifts reflect the different strengths of the ionic interactions formed with the various halides. We conclude that a halide-arginine ion pair is present in the hR state; this interaction appears to be disrupted by photoconversion to hL.