Apo-dihydrofolate reductase from Escherichia coli samples two distinct environments slowly on the NMR time scale at room temperature. Several assigned resonances belong to residues in, or proximal to, a loop (loop I) which is comprised of residues 9-24. This exchange process was altered (either removed or made fast on the NMR time scale) by deleting three hairpin turn forming residues from the loop and filling the gap with a single glycine [Li, L., Falzone, C. J., Wright, P. E., & Benkovic, S. J. (1992) Biochemistry 31, 7826-7833]. An approximate value of 35 s-1 for the exchange rate associated with loop I in apo-DHFR was obtained in two-dimensional nuclear Overhauser spectra by analyzing the time dependence of the cross-peak volume for N epsilon H of Trp-22, a residue which is located in this loop and which has resolved cross-peaks. Owing to the critical role that this loop plays in catalysis, the correspondence between this rate of conformational exchange and off-rates for tetrahydrofolate and the reduced nicotinamide cofactor from product and substrate complexes suggests that loop movement may be a limiting factor in substrate turnover.

In order to characterize the structural and dynamic factors that determine the assembly in b hemoproteins, the solution structure of the 98-residue protein apocytochrome b5 was determined by NMR methods. Over 800 experimental restraints derived from a series of two- and three-dimensional experiments were used. Holocytochrome b5, the protein with iron protoporphyrin-IX liganded to His-39 and His-63, contains in sequence the following elements of secondary structure: beta 1-alpha 1-beta 4-beta 3-alpha 2-alpha 3-beta 5-alpha 4-alpha 5-beta 2-alpha 6 [Mathews, F.S., Czerwinski, E. W., & Argos, P. (1979) The Porphyrins, Vol. 7, pp. 107-147, Academic Press, New York]. The folded holoprotein possesses two hydrophobic cores: an extensive, functional core around the heme (core 1), and a smaller, structural core remote from the heme (core 2). The apoprotein was found to contain a stable four-stranded beta-sheet encompassing beta 1, beta 2, beta 3, and beta 4 and three alpha-helices, corresponding to alpha 1, alpha 2, and alpha 6. Two short alpha-helices (alpha 3 and alpha 5) appear to form partially, and alpha 4 is not detected. These three helices and beta 5 border the heme binding pocket and are disordered in the apoprotein NMR structure. According to backbone 1H-15N NOE results, the most flexible region of the apoprotein, except for the termini, extends from Ala-50 (in beta 5) to Glu-69 (in alpha 5). The polypeptide segment bearing His-63 (located immediately prior to alpha 5) exhibits faster internal motions than that bearing His-39 (at the C-terminal end of alpha 2). The latter imidazole samples a restricted region of space, whereas the former can adopt many orientations with respect to the stable core. It was concluded that heme removal affects the structure and dynamics of most of core 1 whereas it leaves core 2 largely intact. The results provide guidelines for the rational design of b hemoproteins: a modular structure including a packed, stable core and a partially folded binding site is anticipated to present strong kinetic and thermodynamic advantages compared to approaches relying on the complete formation of secondary structure prior to heme binding.

RNA is known to fold into a variety of structural elements, many of which have sufficient sequence complexity to make the thermodynamic study of each possible variant impractical. We previously reported a method for isolating stable and unstable RNA sequences from combinatorial libraries using temperature gradient gel electrophoresis (TGGE). This method was used herein to analyze a six-nucleotide RNA hairpin loop library. Three rounds of in vitro selection were performed using TGGE, and unusually stable RNAs were identified by cloning and sequencing. Known stable tetraloops were found, including sequences belonging to the UNCG motif closed by a CG base pair, and the CUUG motif closed by a GC base pair. In addition, unknown tetraloops were found that were nearly as stable as cUNCGg, including sequences related through substitution of the U with a C (Y), the C with an A (M), or both. These substitutions allow hydrogen bonding and stacking interactions in the UNCG loop to be maintained. Thermodynamic analysis of YNMG and variant loops confirmed optimal stability with Y at position 1 and M at position 3. Similarity in structure and stability among YNMG loops was further supported by deoxyribose substitution, CD, and NMR experiments. A conserved tertiary interaction in 16S rRNA exists between a YAMG loop at position 343 and two adenines in the loop at position 159 (Escherichia coli numbering). NMR and functional group substitution experiments suggest that YNAG loops in particular have enhanced flexibility, which allows the tertiary interaction to be maintained with diverse loop sequences at position 159. Taken together, these results support the existence of an extended family of UNCG-like tetraloops with the motif cYNMGg that are thermodynamically stable and structurally similar and can engage in tertiary interactions in large RNA molecules.

The glbN gene for the hemoglobin of Synechoccocus sp. PCC 7002, a cyanobacterium incapable of nitrogen fixation, was cloned and overexpressed in Escherichia coli. The 123-residue protein was purified from inclusion bodies and reconstituted with iron protoporphyrin IX to obtain the ferric form of the holoprotein. Mass spectrometric analysis confirmed the identity of the polypeptide. NMR and optical data demonstrated that the protein so prepared contained a hexacoordinate heme group, as observed in the related globin from Synechocystis sp. PCC 6803 [Scott, N. L., and Lecomte, J. T. J. (2000) Protein Sci. 9, 587-597]. The data were consistent with a similar bis-histidine coordination scheme involving His46 (E10) on the distal side and His70 (F8) on the proximal side. Several aromatic residues were identified in the vicinity of the heme and were used to establish the orientation of the prosthetic group in the polypeptide matrix. In this protein, as in that from Synechocystis sp. PCC 6803, there was a marked preference for the heme orientation in which pyrroles C and D contact the C-E corner of the protein. Both hemoglobins were found capable of forming a product in which the heme is cross-linked to the polypeptide through modification of a vinyl group.

The recombinant product of the hemoglobin gene of the cyanobacterium Synechocystis sp. PCC 6803 forms spontaneously a covalent bond linking one of the heme vinyl groups to a histidine located in the C-terminal helix (His117, or H16). The present report describes the (1)H, (15)N, and (13)C NMR spectroscopy experiments demonstrating that the recombinant hemoglobin from the cyanobacterium Synechococcus sp. PCC 7002, a protein sharing 59% identity with Synechocystis hemoglobin, undergoes the same facile heme adduct formation. The observation that the extraordinary linkage is not unique to Synechocystis hemoglobin suggests that it constitutes a noteworthy feature of hemoglobin in non-N(2)-fixing cyanobacteria, along with the previously documented bis-histidine coordination of the heme iron. A qualitative analysis of the hyperfine chemical shifts of the ferric proteins indicated that the cross-link had modest repercussions on axial histidine ligation and heme electronic structure. In Synechocystis hemoglobin, the unreacted His117 imidazole had a normal p K(a) whereas the protonation of the modified residue took place at lower pH. Optical experiments revealed that the cross-link stabilized the protein with respect to thermal and acid denaturation. Replacement of His117 with an alanine yielded a species inert to adduct formation, but inspection of the heme chemical shifts and ligand binding properties of the variant identified position 117 as important in seating the cofactor in its site and modifying the dynamic properties of the protein. A role for bis-histidine coordination and covalent adduct formation in heme retention is proposed.