Konda S. Reddy

Design and engineering of an O(2) transport protein.

The principles of natural protein engineering are obscured by overlapping functions and complexity accumulated through natural selection and evolution. Completely artificial proteins offer a clean slate on which to define and test these protein engineering principles, while recreating and extending natural functions. Here we introduce this method with the design of an oxygen transport protein, akin to human neuroglobin. Beginning with a simple and unnatural helix-forming sequence with just three different amino acids, we assembled a four-helix bundle, positioned histidines to bis-histidine ligate haems, and exploited helical rotation and glutamate burial on haem binding to introduce distal histidine strain and facilitate O2 binding. For stable oxygen binding without haem oxidation, water is excluded by simple packing of the protein interior and loops that reduce helical-interface mobility. O2 affinities and exchange timescales match natural globins with distal histidines, with the remarkable exception that O2 binds tighter than CO.

A Designed Four-α-Helix Bundle That Binds the Volatile General Anesthetic Halothane with High Affinity

The structural features of volatile anesthetic binding sites on proteins are being examined with the use of a defined model system consisting of a four-alpha-helix bundle scaffold with a hydrophobic core. Previous work has suggested that introducing a cavity into the hydrophobic core improves anesthetic binding affinity. The more polarizable methionine side chain was substituted for a leucine, in an attempt to enhance the dispersion forces between the ligand and the protein. The resulting bundle variant has an improved affinity (K(d) = 0.20 +/- 0.01 mM) for halothane binding, compared with the leucine-containing bundle (K(d) = 0.69 +/- 0.06 mM). Photoaffinity labeling with (14)C-halothane reveals preferential labeling of the W15 residue in both peptides, supporting the view that fluorescence quenching by bound anesthetic reports both the binding energetics and the location of the ligand in the hydrophobic core. The rates of amide hydrogen exchange were similar for the two bundles, suggesting that differences in binding affinity were not due to changes in protein stability. Binding of halothane to both four-alpha-helix bundle proteins stabilized the native folded conformations. Molecular dynamics simulations of the bundles illustrate the existence of the hydrophobic core, containing both W15 residues. These results suggest that in addition to packing defects, enhanced dispersion forces may be important in providing higher affinity anesthetic binding sites. Alternatively, the effect of the methionine substitution on halothane binding energetics may reflect either improved access to the binding site or allosteric optimization of the dimensions of the binding pocket. Finally, preferential stabilization of folded protein conformations may represent a fundamental mechanism of inhaled anesthetic action.

Role of β112 Cys (G14) in Homo- (β4) and Hetero- (α2β2) Tetramer Hemoglobin Formation

In order to assess the role of β112 Cys in homo- and hetero-tetrameric hemoglobin formation, we expressed four β112 variants (β112Cys→Asp, β112Cys→Ser, β112Cys→Thr, and β112Cys→Val) and studied assembly with α chainsin vitro. β112 Cys is normally present at β1β2 and α1β1interaction sites in homo- (β4) and hetero-tetramers (α2β2). β4 formation in vitro was influenced by the amino acid at β112. β112 Asp completely inhibited formation of homo-tetramers, whereas β112 Ser showed only slight inhibition. In contrast, β112 Thr or Val enhanced homo-tetramer formation compared with βA chains. Association constants for homo-tetramer formation increased in the order of β112Cys→Ser, βA, β112Cys→Thr, and β112Cys→Val, whereas the value for β112Cys→Asp was zero under the same conditions. These β112 changes also affected in vitroα2β2 hetero-tetramer formation. Order of α2β2 formation under limiting α-globin chain conditions showed Hb βC112S > Hb A > Hb S = Hb βC112T = Hb βC112V >>> Hb βC112D. Hb β112D can form tetrameric hemoglobin, but this β112 change promotes dissociation into α and β chains instead of αβ dimer formation upon dilution. These results indicate that amino acids at α1β1 interaction sites such as β112 on the G helix play a key role in stable αβ dimer formation. Our findings suggest, in addition to electrostatic interaction between α and β chains, that dissociation of β4 homo-tetramers to monomers and hydrophobic interactions of the β112 amino acid with α chains governs stable α1β1 interactions, which then results in formation of functional hemoglobin tetramers. Information gained from these studies should increase our understanding of the mechanism of assembly of multi-subunit proteins.

Spectroscopic, electrochemical, and ligand binding properties of the horse heart metmyoglobin His64-Tyr variant.

The distal histidine (E7) of horse heart myoglobin (Mb) has been replaced by tyrosine using site-specific mutagenesis. The resulting green Mb variant (His64-Tyr) was expressed in Escherichia coli JM101, isolated and purified to homogeneity. Spectrophotometric pH titrations of the variant exhibit a change in spectrum that occurs with a pKa of 4.7 (25 degrees C). The midpoint reduction potential of the variant is 20 mV (vs. SHE at pH 7, 25 degrees C). Cyanide and azide binding measurements indicate that the oxidized variant binds these anionic ligands with much greater affinity at pH 4.0 than at neutral pH. Extended X-ray absorption fine structure (EXAFS) spectroscopy establishes that the variant is six coordinate at pH 7.0 and pH 4.2. Higher shell contributions to the iron EXAFS observed at pH 7.0 are attributed to tyrosine. These contributions are absent at pH 4.2. Thus, the sixth heme iron ligand of the oxidized variant Mb at pH 7.0 is attributed to oxygen from the hydroxyl group of tyrosine and the sixth ligand present at pH 4.2 is attributed to the oxygen atom of a coordinated water. The EXAFS spectra, electronic absorption spectra, and ligand binding properties of the His64-Tyr Mb variant are consistent with the binding of Tyr-64 as the sixth heme iron ligand between pH 5 and 12 and with the replacement of Tyr-64 by a water molecule at low pH with a pKa of 4.7.

Role of Hydrophobic Amino Acids at β85 and β88 in Stabilizing F Helix Conformation of Hemoglobin S

Three Hb S variants containing Glu substitutions at Phe-β85 and/or Leu-β88 were expressed in yeast in an effort to evaluate the role of hydrophobic amino acids at these sites in stabilizing F helix conformation of Hb S. Helix stability of tetrameric Hb S βF85E,βL88E was measured by CD and compared with those of Hb S βF85E, Hb S βL88E, Hb A, and Hb S. The CD spectra of these Hb S variants were similar to those of Hb S and Hb A at 10°C. However, changes in ellipticity at 222 nm for Hb S βF85E in the CO form at 60°C were about 15-fold greater than that of Hb S, while those for Hb S βL88E and Hb S βF85E,βL88E were similar and about 30-fold greater than Hb S. Thermal stability measured by continuous scanning of spectral changes revealed the three Hb S variants were much more unstable than Hb S, and stability of Hb S βF85E,βL88E was similar to that of Hb S βL88E rather than Hb S βF85E. These results suggest that Glu insertion at both β85 and β88 makes heme insertion into the heme pocket more difficult; however, once inserted, stability of Hb S βF85E,βL88E is similar to Hb S βL88E rather than Hb S βF85E. Furthermore, these results suggest that both Phe-β85 and Leu-β88 are critical for F helix stabilization and that Glu insertion at β88 leads to more destabilization than insertion at β85.

Polymerization of Recombinant Hb S-Kempsey (Deoxy-R State) and Hb S-Kansas (Oxy-T State)

In order to investigate the role of the R (relaxed) to T (tense) structural transition in facilitating polymerization of deoxy-Hb S, we have engineered and expressed two Hb S variants which destabilize either T state (Hb S-Kempsey, αβ) or R state structures (Hb S-Kansas, αβ). Polymerization of deoxy-Hb S-Kempsey, which shows high oxygen affinity and increased dimer dissociation, required about 2- and 6-fold higher hemoglobin concentrations than deoxy-Hb S for polymerization in low and high phosphate concentrations, and its kinetic pattern of polymerization was biphasic. In contrast, oxy- or CO Hb S-Kansas, which shows low oxygen affinity and increased dimer dissociation, polymerized at a slightly higher critical concentration than that required for polymerization of deoxy-Hb S in both low and high phosphate buffers. Polymerization of oxy- and CO Hb S-Kansas was linear and showed no delay time, which is similar to oversaturated oxy- or CO Hb S. These results suggest that nuclei formation, which occurs during the delay time prior to deoxy-Hb S polymerization, does not occur in T state oxy-Hb S-Kansas, even though the critical concentration for polymerization of T state oxy-Hb S-Kansas is similar to that of T state deoxy-Hb S.

Inhibition of hemoglobin S polymerization in vitro by a novel 15-mer EF-helix β73 histidine-containing peptide

Our mutational studies on Hb S showed that the Hb S beta73His variant (beta6Val and beta73His) promoted polymerization, while Hb S beta73Leu (beta6Val and beta73Leu) inhibited polymerization. On the basis of these results, we speculated that EF-helix peptides containing beta73His interact with beta4Thr in Hb S and compete with Hb S, resulting in inhibition of Hb S polymerization. We, therefore, studied inhibitory effects of 15-, 11-, 7-, and 3-mer EF-helix peptides containing beta73His on Hb S polymerization. The delay time prior to Hb S polymerization increased only in the presence of the 15-mer His peptide; the higher the amount, the longer the delay time. DIC image analysis also showed that the fiber elongation rate for Hb S polymers decreased with increasing concentration of the 15-mer His peptide. In contrast, the same 15-mer peptide containing beta73Leu instead of His and peptides shorter than 11 amino acids containing beta73His including His alone showed little effect on the kinetics of polymerization and elongation of polymers. Analysis by protein-chip arrays showed that only the 15-mer beta73His peptide interacted with Hb S. CD spectra of the 15-mer beta73His peptide did not show a specific helical structure; however, computer docking analysis suggested a lower energy for interaction of Hb S with the 15-mer beta73His peptide compared to peptides containing other amino acids at this position. These results suggest that the 15-mer beta73His peptide interacts with Hb S via the beta4Thr in the betaS-globin chain in Hb S. This interaction may influence hydrogen bond interaction between beta73Asp and beta4Thr in Hb S polymers and interfere in hydrophobic interactions of beta6Val, leading to inhibition of Hb S polymerization.

Inhibition of hemoglobin S polymerization in vitro by a novel 15-mer EF-helix beta73 histidine-containing peptide.

Our mutational studies on Hb S showed that the Hb S beta73His variant (beta6Val and beta73His) promoted polymerization, while Hb S beta73Leu (beta6Val and beta73Leu) inhibited polymerization. On the basis of these results, we speculated that EF-helix peptides containing beta73His interact with beta4Thr in Hb S and compete with Hb S, resulting in inhibition of Hb S polymerization. We, therefore, studied inhibitory effects of 15-, 11-, 7-, and 3-mer EF-helix peptides containing beta73His on Hb S polymerization. The delay time prior to Hb S polymerization increased only in the presence of the 15-mer His peptide; the higher the amount, the longer the delay time. DIC image analysis also showed that the fiber elongation rate for Hb S polymers decreased with increasing concentration of the 15-mer His peptide. In contrast, the same 15-mer peptide containing beta73Leu instead of His and peptides shorter than 11 amino acids containing beta73His including His alone showed little effect on the kinetics of polymerization and elongation of polymers. Analysis by protein-chip arrays showed that only the 15-mer beta73His peptide interacted with Hb S. CD spectra of the 15-mer beta73His peptide did not show a specific helical structure; however, computer docking analysis suggested a lower energy for interaction of Hb S with the 15-mer beta73His peptide compared to peptides containing other amino acids at this position. These results suggest that the 15-mer beta73His peptide interacts with Hb S via the beta4Thr in the betaS-globin chain in Hb S. This interaction may influence hydrogen bond interaction between beta73Asp and beta4Thr in Hb S polymers and interfere in hydrophobic interactions of beta6Val, leading to inhibition of Hb S polymerization.

Inhibition of Hb S Polymerization In Vitro by a Novel 15-mer EF Helix β73 His-Containing Peptide

Our mutational studies on Hb S showed that the Hb S beta73His variant (beta6Val and beta73His) promoted polymerization, while Hb S beta73Leu (beta6Val and beta73Leu) inhibited polymerization. On the basis of these results, we speculated that EF-helix peptides containing beta73His interact with beta4Thr in Hb S and compete with Hb S, resulting in inhibition of Hb S polymerization. We, therefore, studied inhibitory effects of 15-, 11-, 7-, and 3-mer EF-helix peptides containing beta73His on Hb S polymerization. The delay time prior to Hb S polymerization increased only in the presence of the 15-mer His peptide; the higher the amount, the longer the delay time. DIC image analysis also showed that the fiber elongation rate for Hb S polymers decreased with increasing concentration of the 15-mer His peptide. In contrast, the same 15-mer peptide containing beta73Leu instead of His and peptides shorter than 11 amino acids containing beta73His including His alone showed little effect on the kinetics of polymerization and elongation of polymers. Analysis by protein-chip arrays showed that only the 15-mer beta73His peptide interacted with Hb S. CD spectra of the 15-mer beta73His peptide did not show a specific helical structure; however, computer docking analysis suggested a lower energy for interaction of Hb S with the 15-mer beta73His peptide compared to peptides containing other amino acids at this position. These results suggest that the 15-mer beta73His peptide interacts with Hb S via the beta4Thr in the betaS-globin chain in Hb S. This interaction may influence hydrogen bond interaction between beta73Asp and beta4Thr in Hb S polymers and interfere in hydrophobic interactions of beta6Val, leading to inhibition of Hb S polymerization.

Optical and X-Ray Techniques in the Study of Rapid Ligand Binding: A Ligand „Docking“ Site in the Reaction of Mb and Co At 40 K

Methods for the study of rapid reactions region at cryogenic temperatures are reviewed from the standpoint of range of kinetic constants, signal-to-noise ratio and compatability of optical methods with X-ray absorption spectroscopy. Alternation of optical monitoring with X-ray absorption spectroscopy and optical pumping of the sample are treated, and appropriate apparatus designs are reviewed. Typical results of structural studies at 4 and 40 K are described and the accumulation of the ligand at about 3Â from the iron atom in a protein structural crevice or “energy minimum” is described. The generality of the idea of metal atom substrate binding sites, supplemented with adjacent protein binding sites, which act to “dock” the ligand and allow transfer to the active site with appropriate orientation and collision frequency, is suggested. The possibility that the “docking site” mechanism extends to other proteins and enzymes is suggested.