Biomedical subjects

Jennifer R Calhoun

Publications and source records attributed to Jennifer R Calhoun.

4 recordsLinked to original sources

PubMed2005

Artificial di-iron proteins: solution characterization of four helix bundles containing two distinct types of inter-helical loops.

Peptide-based models have an enormous impact for the development of metalloprotein models, as they seem appropriate candidates to mimic both the structural characteristics and reactivity of the natural systems. Through the de novo design of four-helix bundles, we developed the DF (Due Ferri) family of artificial proteins, as models of di-iron and di-manganese metalloproteins. The goal of our research is to elucidate how the electrostatic environment, polarity and solvent accessibility of the metal-binding site, influence the functional properties of di-iron proteins. The first two subsets of the DF protein family, DF1 and DF2, consist of two non-covalently associated helix-loop-helix motifs, which bind the di-metal cofactor near the center of the structure. The DF2 subset was designed to improve the properties of DF1: DF2 and DF2t have several changes in their sequences to improve solubility and metal ion access, as well as a change in the loop connecting the two helices. In order to evaluate how these changes affect the overall structure of the model proteins, we solved the NMR structures of the di-Zn(II) complexes of DF2 and DF2t, and compared these structures with those recently obtained from X-ray crystallography. Further, we examined the thermodynamic consequences associated with the mutations, by measuring the stability of DF2t in the presence of different metal ions, and comparing the results with the data already obtained for DF2. Taken together, analysis of all the data showed the importance of the turn conformation in the design and stability of four-helix bundle.

Amino Acid Sequence↗

PubMed2005

Artificial diiron proteins: from structure to function.

De novo protein design provides an attractive approach for the construction of models to probe the features required for the function of complex metalloproteins. These minimal models contain the essential elements believed necessary for activity of the protein. In this article, we summarize the design, structure determination, and functional properties of a family of artificial diiron proteins.

Amino Acid Sequence↗

PubMed2003

Computational design and characterization of a monomeric helical dinuclear metalloprotein.

The de novo design of di-iron proteins is an important step towards understanding the diversity of function among this complex family of metalloenzymes. Previous designs of due ferro (DF) proteins have resulted in tetrameric and dimeric four-helix bundles having crystallographically well-defined structures and active-site geometries. Here, the design and characterization of DFsc, a 114 residue monomeric four-helix bundle, is presented. The backbone was modeled using previous oligomeric structures and appropriate inter-helical turns. The identities of 26 residues were predetermined, including the primary and secondary ligands in the active site, residues involved in active site accessibility, and the gamma beta gamma beta turn between helices 2 and 3. The remaining 88 amino acid residues were determined using statistical computer aided design, which is based upon a recent statistical theory of protein sequences. Rather than sampling sequences, the theory directly provides the site-specific amino acid probabilities, which are then used to guide sequence design. The resulting sequence (DFsc) expresses well in Escherichia coli and is highly soluble. Sedimentation studies confirm that the protein is monomeric in solution. Circular dichroism spectra are consistent with the helical content of the target structure. The protein is structured in both the apo and the holo forms, with the metal-bound form exhibiting increased stability. DFsc stoichiometrically binds a variety of divalent metal ions, including Zn(II), Co(II), Fe(II), and Mn(II), with micromolar affinities. 15N HSQC NMR spectra of both the apo and Zn(II) proteins reveal excellent dispersion with evidence of a significant structural change upon metal binding. DFsc is then a realization of complete de novo design, where backbone structure, activity, and sequence are specified in the design process.

Amino Acid Sequence↗

PubMed2003

Direct analysis of protein sedimentation equilibrium in detergent solutions without density matching.

Characterizing membrane proteins by sedimentation equilibrium is challenging because detergents and/or lipid molecules, usually required for solubilization, form a complex with the protein. The most common way to overcome this problem is Tanford and Reynolds' density matching method, which eliminates the buoyant mass contributions of detergents/lipids by adjusting the solvent density with D2O/H2O mixtures to render either detergent or lipid molecules neutrally buoyant. Unfortunately, the method is practical only for detergent densities between 1.0 (H2O) and 1.1 (D2O) g ml(-1), excluding many of the more commonly used detergents for membrane protein studies. Here, we present a modern variant of Tanford and Reynolds' method that (1) is applicable to any detergent regardless of its specific density, (2) does not compromise accuracy and precision, and (3) provides additional information about the number of detergent molecules that are bound to each protein. The new method was applied successfully to Delta(1-43)A-I, an amino-terminal deletion mutant of human apolipoprotein A-I. Interestingly, we observed a significantly lower Delta(1-43)A-I/octyl-glucoside complex partial specific volume than that expected from volume additivity rules, indicative of specific protein-detergent interactions.

Apolipoprotein A-I↗