Using our Escherichia coli expression system, we have produced five mutant recombinant (r) hemoglobins (Hbs): r Hb (alpha V96 W), r Hb Presbyterian (beta N108K), r Hb Yoshizuka (beta N108D), r Hb (alpha V96W, beta N108K), and r Hb (alpha V96W, beta N108D). These r Hbs allow us to investigate the effect on the structure-function relationship of Hb of replacing beta 108Asn by either a positively charged Lys or a negatively charged Asp as well as the effect of replacing alpha 96Val by a bulky, nonpolar Trp. We have conducted oxygen-binding studies to investigate the effect of several allosteric effectors on the oxygenation properties and the Bohr effects of these r Hbs. The oxygen affinity of these mutants is lower than that of human normal adult hemoglobin (Hb A) under various experimental conditions. The oxygen affinity of r Hb Yoshizuka is insensitive to changes in chloride concentration, whereas the oxygen affinity of r Hb Presbyterian exhibits a pronounced chloride effect. r Hb Presbyterian has the largest Bohr effect, followed by Hb A, r Hb (alpha V96W), and r Hb Yoshizuka. Thus, the amino acid substitution in the central cavity that increases the net positive charge enhances the Bohr effect. Proton nuclear magnetic resonance studies demonstrate that these r Hbs can switch from the R quaternary structure to the T quaternary structure without changing their ligation states upon the addition of an allosteric effector, inositol hexaphosphate, and/or by reducing the temperature. r Hb (alpha V96W, beta N108K), which has the lowest oxygen affinity among the hemoglobins studied, has the greatest tendency to switch to the T quaternary structure. The following conclusions can be derived from our results: First, if we can stabilize the deoxy (T) quaternary structure of a hemoglobin molecule without perturbing its oxy (R) quaternary structure, we will have a hemoglobin with low oxygen affinity and high cooperativity. Second, an alteration of the charge distribution by amino acid substitutions in the alpha 1 beta 1 subunit interface and in the central cavity of the hemoglobin molecule can influence the Bohr effect. Third, an amino acid substitution in the alpha 1 beta 1 subunit interface can affect both the oxygen affinity and cooperativity of the oxygenation process. There is communication between the alpha 1 beta 1 and alpha 1 beta 2 subunit interfaces during the oxygenation process. Fourth, there is considerable cooperativity in the oxygenation process in the T-state of the hemoglobin molecule.

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Glycerol nucleic acid (GNA) is an interesting alternative basepairing system based on an acyclic, glycerol-phosphate backbone repeat unit. The question of whether DNA polymerases can catalyze efficient template-dependent synthesis using GNA as the template is of particular interest because GNA is unable to form a stable duplex with DNA. In the present study, we screened a variety of DNA polymerases for GNA-dependent DNA synthesis. We find that Bst DNA polymerase can catalyze full-length DNA synthesis on a dodecamer GNA template. The efficiency of DNA synthesis is increased by replacing adenine with diaminopurine in both the GNA template and the DNA monomers and by the presence of manganese ions. We suggest that the BstDNA polymerase maintains a short, transient region of base-pairing between the DNA product strand and the GNA template, but that stable duplex formation between product and template strands is not required for template-dependent polymerization.information transfer ͉ polymerase N ucleic acid analogs with altered backbones or bases are of significant interest in the search for biopolymers with novel chemical and biological properties, and many such analogs have been designed and synthesized (1-3). Evaluation of the hybridization and nuclease-resistance properties of these synthetic nucleic acids has led to several nucleic acid analogues with potential biological applications (4-6). However, much less is known about the potential for information transfer between synthetic nucleic acid systems and the modern DNA/RNA system via templatedependent polymerization, a crucial aspect of the chemical etiology of nucleic acids and directed evolution of functional biopolymers based on a synthetic nucleic acid system. Recently, our group and others have studied the enzymatic synthesis of (3Ј 3 2Ј) ␣-L-threose nucleic acid (TNA; Fig. 1) (7-13). TNA was discovered by Eschenmoser and coworkers (7, 8) during a systematic evaluation of the base-pairing properties of nucleic acids containing alternative sugar-phosphate backbones. TNA was found to be capable of forming stable, antiparallel duplexes with RNA, DNA, and itself, a surprising property for a nucleic acid analog with a shorter backbone repeat unit than DNA or RNA (8,14,15). This remarkable intersystem duplex formation has been considered to be the basis of possible information transfer between TNA and DNA/RNA. Later studies by our group revealed that ␣-L-threose nucleoside 3Ј-triphosphates were substrates for efficient template-dependent enzymatic polymerization by Therminator DNA polymerase, a mutated archaeal family B DNA polymerase (10,11,16). Therminator DNA polymerase was also shown to be an efficient and accurate TNA-dependent DNA polymerase (9). The information transfer between TNA and DNA catalyzed by polymerases provides support for a possible role of TNA as a progenitor of DNA/RNA.The search for nucleic acid analogs with even simpler backbones led to studies of the glycerol nucleic acids (GNAs), which have a three-carbon, acyclic backbone (17-19) (Fi...

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