Margaret A. Daugherty

Single residue modification of only one dimer within the hemoglobin tetramer reveals autonomous dimer function

The mechanism of cooperativity in the human hemoglobin tetramer (a dimer of αβ dimers) has historically been modeled as a simple two-state system in which a low-affinity structural form (T) switches, on ligation, to a high-affinity form (R), yielding a net loss of hydrogen bonds and salt bridges in the dimer–dimer interface. Modifications that weaken these cross-dimer contacts destabilize the quaternary T tetramer, leading to decreased cooperativity and enhanced ligand affinity, as demonstrated in many studies on symmetric double modifications, i.e., a residue site modified in both α- or both β-subunits. In this work, hybrid tetramers have been prepared with only one modified residue, yielding molecules composed of a wild-type dimer and a modified dimer. It is observed that the cooperative free energy of ligation to the modified dimer is perturbed to the same extent whether in the hybrid tetramer or in the doubly modified tetramer. The cooperative free energy of ligation to the wild-type dimer is unperturbed, even in the hybrid tetramer, and despite the overall destabilization of the T tetramer by the modification. This asymmetric response by the two dimers within the same tetramer shows that loss of dimer–dimer contacts is not communicated across the dimer–dimer interface, but is transmitted through the dimer that bears the modified residue. These observations are interpreted in terms of a previously proposed dimer-based model of cooperativity with an additional quaternary (T/R) component.

Hydrodynamic Studies on the Quaternary Structure of Recombinant Mouse Purβ

Purβ is a gene regulatory factor belonging to a family of highly conserved nucleic acid-binding proteins related by their ability to preferentially bind single-stranded DNA or RNA sequences rich in purine nucleotides. In conjunction with Purα, Purβ has been implicated in transcriptional and translational repression of genes encoding contractile proteins found in the heart and vasculature. Although several models of sequence-specific DNA recognition, strand separation, and activator inhibition by oligomeric Purα and Purβ have been proposed, it is currently unclear whether protein-protein interaction is a prerequisite to, or a consequence of nucleic acid binding. In this study, a recombinant protein purification scheme was devised to yield homogenous mouse Purβ devoid of nucleic acid. Recombinant Purβ was then subjected to light scattering and analytical ultracentrifugation analyses to assess the size, shape, and oligomeric state of the purified protein in solution. Results of laser light scattering and sedimentation velocity experiments indicated that Purβ reversibly self-associates in the absence of nucleic acid. Both approaches independently showed that the hydrodynamic shape of the Purβ homodimer is markedly asymmetric and non-spherical. Sedimentation velocity analyses indicated that dimeric Purβ has a sedimentation coefficient of 3.96 Svedberg, a frictional coefficient ratio (f/f0) of 1.60, and a hydrodynamic radius of 4.43 nm. These values were consistent with those determined by independent dynamic light scattering studies. Sedimentation equilibrium analyses confirmed that Purβ self-associates in a reversible monomer-dimer equilibrium characterized by a Kd = 1.13 ± 0.27 μm.

Analysis of Transcription Factor Interactions at Sedimentation Equilibrium

Publisher Summary This chapter explores the large numbers of proteins participating in the assemblies that regulate and catalyze transcription. Among methods available for characterizing their interactions, sedimentation equilibrium (SE) ultracentrifugation stands out as a direct and rigorous means of determining molecular masses, interaction stoichiometries, association constants, and the influences of low molecular weight effectors, ions, and crowding on the stabilities of protein complexes. The chapter also reviews the availability of modern instrumentation and the development of improved analysis methods that have resulted in an upsurge of interest in SE during the past decade. This chapter describes the application of SE techniques to the characterization of transcription factors and their interactions. It focuses on three situations that are encountered most frequently in studies of transcription factors: self-association, heteroassociation, and the presence of inactive components. Analysis of such interactions provides crucial information on the role of protein–protein interactions in the assembly of transcription and transcription–regulatory complexes.