Aichun Dong

Infrared methods for study of hemoglobin reactions and structures.

Publisher Summary This chapter presents the experimental approaches and interpretations of data for ligand spectra, microspectroscopy, thiol spectra, and amide I spectra. Methods using infrared (IR) spectroscopy provide important, often unique, means for the study of the reactions and structures of hemoglobins (Hb). The amide I bands, primarily because of the carbonyl groups in the peptide bonds that constitute the linkages between the amino acid residues of the Hb molecule, can be utilized for the qualitative and quantitative determination of α-helix, random, β-sheet, and turn secondary structures. Thus, the accurate measurement of an IR band at a given wavelength depends on the absorption of the medium as well as on protein concentration, intrinsic band intensity, and the distance through the sample traversed by the IR radiation. Improvements in IR instrumentation and in data analysis have greatly enhanced the sensitivity of the IR method applied to aqueous solutions.

Probing heart cytochrome c oxidase structure and function by infrared spectroscopy.

AbstractIR spectra directly probe specific vibrators in bovine heart cytochromec oxidase, yielding quantitative as well as qualitative information on structures and reactions at these vibrators. C-O IR spectra reveal that CO binds to

The anesthetic nitrous oxide affects dioxygen utilization by bovine heart and bean seed mitochondrial particles

Fe_{a_3 }^{2 + }

Comparison of the Secondary Structures of Human Class I and Class II MHC Antigens by Ftir and CD Spectroscopy

as two conformers each in isolated immobile environments sensitive to Fea and/or CuA oxidation state but remarkably insensitive to pH, medium, anesthetics, and other factors that affect activity. C-N IR spectra reveal that the one CN− that binds to fully and partially oxidized enzyme can be in three different structures. These structures vary in relative amounts with redox level, thereby reflecting dynamic electron exchange among Fea, CuA, and CuB with associated changes in protein conformation of likely significance in O2 reduction and H+-pumping. Azide IR spectra also reflect redox-dependent long-range effects. The amide I IR bands, due to C-O vibrators of peptide linkages and composed of multiple bands derived from different secondary structures, reveal high levels of α-helix (∼60%) and subtle changes with redox level and exposure to anesthetics. N2O IR spectra reveal that these anesthetic molecules at clinically relevant levels occupy three sites of different polarity within the enzyme as the enzyme is reversibly, but only partially, inhibited.

Protein Secondary Structures in Water from Second-Derivative Amide I Infrared Spectra+

Methods for obtaining the infrared spectrum of a single erythrocyte by infrared microscopy have been developed. The spectrum contains the amide I, II, and III bands characteristic of protein secondary structure near 1650, 1550, and 1300 cm-1, respectively. Bound carbon monoxide exhibits a readily measured band at 1951 cm-1 for 12C16O and 1907 cm-1 for 13C16O. Both amide and CO bands are similar to those found for purified hemoglobin A. Spectra can be obtained in H2O or D2O media under physiologically relevant conditions. Single cell infrared spectroscopy (SCIR) permits the qualitative and quantitative determination of differences among individual red cells. These results suggest many potential applications for SCIR for the measurements of properties of individual cells at the molecular level under physiologically relevant conditions.

Secondary Structure Analysis of the Scrapie-Associated Protein PrP 27-30 in Water by Infrared Spectroscopy+

Nitrous oxide affects dioxygen utilization by both bean seed and bovine heart submitochondrial particles when either succinate or reduced cytochrome c are used as substrates. Bovine heart particles exhibit reversible, dose-dependent partial inhibition of respiratory activity when exposed to N2O. Bean seed particle respiration is stimulated by low levels of N2O, but higher concentrations are inhibitory. These findings can be explained in terms of one locus of anesthetic action: cytochrome c oxidase, the terminal component of the mitochondrial respiratory chain. Alterations in respiration rates are expected to make important contributions to anesthesia in animals and to control of germination in plants.

Redox-dependent changes in .beta.-extended chain and turn structures of cytochrome c in water solution determined by second derivative amide I infrared spectra

Carbon monoxide binding to a myoglobin mutant with distal arginine in place of histidine has been examined. The mutant is derived from a cDNA clone for Mb mRNA from fetal bovine skeletal muscle. The mutation only slightly perturbs visible/Soret spectra whereas the infrared spectrum of liganded CO is greatly modified to become nearly identical to Hb Zurich beta-subunit spectrum. The mutant IR spectra differ substantially from spectra of wild-type MbCO and normal HbCO beta-subunit. For both the Mb and the Hb the distal His----Arg mutation increases the affinity for CO and reduces the number of observed conformers. These results demonstrate that this mutation greatly reduces the differences between Mb and Hb in the structure and properties of its ligand binding sites.

Destabilizing Effects of Replacing a Surface Lysine of Cytochrome c with Aromatic Amino Acids: Implications for the Denatured State?

Considerable evidence exists that the structures of class I and class II histocompatibility antigens are similar. Most of the evidence (summarized in Kappes and Strominger, 1988 and Brown et al., 1988) is based on sequence homologies and similarities in domain structure at both the protein and DNA levels. In addition, some T cells that are specific for either class I or class II molecules use the same receptor (Rupp et al, 1985; Marrack and Kappler, 1986). However, the secondary structures of purified class I and class II antigens have not been directly compared.