Probing minimal independent folding units in dihydrofolate reductase by molecular dissection.

Molecular dissection was employed to identify minimal independent folding units in dihydrofolate reductase (DHFR) from Escherichia coli. Eight overlapping fragments of DHFR, spanning the entire sequence and ranging in size from 36 to 123 amino acids, were constructed by chemical cleavage. These fragments were designed to examine the effect of tethering multiple elements of secondary structure on folding and to test if the secondary structural domains represent autonomous folding units. CD and fluorescence spectroscopy demonstrated that six fragments containing up to a total of seven alpha-helices or beta-strands and, in three cases, the adenine binding domain (residues 37-86), are largely disordered. A stoichiometric mixture of the two fragments comprising the large discontinuous domain, 1-36 and 87-159, also showed no evidence for folding beyond that observed for the isolated fragments. A fragment containing residues 1-107 appears to have secondary and tertiary structure; however, spontaneous self-association made it impossible to determine if this structure solely reflects the behavior of the monomeric form. In contrast, a monomeric fragment spanning residues 37-159 possesses significant secondary and tertiary structure. The urea-induced unfolding of fragment 37-159 in the presence of 0.5 M ammonium sulfate was found to be a well-defined, two-state process. The observation that fragment 37-159 can adopt a stable native fold with unique, aromatic side-chain packing is quite striking because residues 1-36 form an integral part of the structural core of the full-length protein.     

Down-Regulation of T-Cell Proliferation in Response to Soluble Anti-CD3 Antibodies through Development of Redirected Cytolytic Activity Eliminating Costimulatory Cells

CD4⁺ T-depleted spleen cells (CD8⁺ T cells) activated by anti-CD3 antibodies (aCD3) suppressed proliferation of CD8⁺ T-depleted spleen cells (CD4⁺ T cells) and fresh normal T cells in response to aCD3. Antigen-nonspecific cytolytic activity was induced in splenic CD8⁺ T cells by stimulation with aCD3 and showed the peak level on day 3, whereas cytolytic activity induced in CD4⁺ T cells was weak. Intact Ig but not F(ab')₂ of aCD3 induced and mediated cytolytic activity. Correspondingly, the cytolytic activity induced by aCD3 was directed against target cells bearing Ig-binding Fc-receptor activity and cytolysis was inhibited by the addition of free Ig into the assay system. We showed that aCD3-activated T cells carried a high level of aCD3 on their surface at the time after the peak proliferation when they attained high cytolytic activity. This raised the possibility that the anti-CD3-induced aCD3-redirected cytolytic activity eliminated Fc-receptor-bearing costimulatory cells in the culture for down-regulation of the T-cell proliferation. This view was supported by partial restoration of anti-CD3-induced low responsiveness of CD8⁺ T cells by the addition of fresh costimulatory cells. These results suggested a new pathway of down-regulation of T-cell proliferation by aCD3-activated cytolytic CD8⁺ T cells.     

Molecular mechanisms of endotoxin activity

Endotoxin (lipopolysaccharide, LPS), a constitutent of the outer membrane of the cell wall of gramnegative bacteria, exerts a wide variety of biological effects in humans. This review focuses on the molecular mechanisms underlying these activities and discusses structure-function relationships of the endotoxin molecule, its interaction with humoral and cellular receptors involved in cell activation, and transmembrane and intra-cellular signal transduction pathways.     

Solvent polarity-dependent structural refolding: A CD and NMR study of a 15 residue peptide

A close association between the HIV surface protein gp120 and the CD4 T cell receptor initiates the viral multiplication cycle. A 15 amino acid peptide (LAV) within the CD4 binding domain of gp 120 has been shown to retain receptor binding ability. The structural behavior of the LAV peptide has been studied by CD and NMR methods in aqueous solution and upon addition of trifluoroethanol (TFE) to emulate the relatively apolar conditions at the membrane bound receptor. Previous work has shown that the LAV peptide folds into a β-pleated structure in more polar buffer/TFE mixtures, while a concerted structural change can be observed at a concentration of 60% TFE (v/v). This abrupt, cooperative refolding from a regular β-sheet to a helical secondary structure is known as “switch” behavior. Former CD experiments with LAV sequence variants have supported the assumption that four amino acids at the N-terminus (LPCR) are indispensable for the “switch.” The tetrad has a strong β-turn forming potential. The suggestion has been formulated that the tetrad can act as a nucleation site governing the refolding. The present NMR study of the LAV peptide in TFE gives evidence for a 3₁₀-helix suggesting that the tetrad adopts a type III β-turn and promotes the formation of a similar bend in the next overlapping tetrad until the sequence is restructured into a 3₁₀-helix at a critical polarity favoring intrachain hydrogen bonds. © 1995 Wiley-Liss, Inc.     

Circular dichroism of ketoprofen complexed to serum albumins: Conformational selection by the protein: A novel optical purity determination technique

Complexation of 2-(3′-benzoylphenyl)propionic acid (ketoprofen), 1 , to bovine serum albumin (BSA) results in an intense negative circular dichroism in the ketonic n → π* band of the benzoylphenyl moiety. This high CD contrasts with the weak CD of 1 -enantiomers dissolved in common solvents. Furthermore, a number of chiral and achiral molecules containing the benzophenone moiety are easily complexed to BSA: all these complexes show an intense CD at the same transition. To account for the observed CD intensities of the above molecules, it appears that BSA complexation markedly shifts the equilibrium between strongly asymmetric, antipodic conformers. Dissymmetry of these conformers is connected to the instability of a structure with phenyl rings coplanar to the carbonyl chromophore, as also indicated by molecular mechanics calculations. The magnification of the Cotton effects of the 1 -antipodes, due to the protein, can be used to measure the optical purity of 1 -samples with excellent precision. In contrast with BSA, human SA is unable to recognize the chirality of 1 -antipodes; oleic acid cocomplexation modifies this fact as well as other features of the binding. © 1995 Wiley-Liss, Inc.     

Conformational studies of chemotactic HCO-Met-Leu-Phe-OMe analogues

Summary In order to investigate the proper peptide backbone conformation able to elicit a biological activity, HCO-Met-Pro-Phe-OMe, HCO-Met-[COO]Leu-Phe-OMe, and HCO-Met-OLeu-Phe-OMe, analogues of the prototypical chemotactic peptide HCO-Met-Leu-Phe-OMe, were studied by CD and IR techniques. The results obtained comparing biological and conformational data evidence the critical presence of (i) the NH group at position 2, (ii) a rather flexible backbone, (iii) the chemical structure of the central residue which can affect the stability of a possible active conformer.     

Establishing isostructural metal substitution in metalloproteins using 1H NMR, circular dichroism, and Fourier transform infrared spectroscopy.

Far-UV CD, 1H-NMR, and Fourier transform infrared (FTIR) spectroscopy are three of the most commonly used methods for the determination of protein secondary structure composition. These methods are compared and evaluated as a means of establishing isostructural metal substitution in metalloproteins, using the crystallographically defined rubredoxin from Desulfovibrio gigas and its well-characterized cadmium derivative as a model system. It is concluded that analysis of the FTIR spectrum of the protein amide I resonance represents the most facile and generally applicable method of determining whether the overall structure of a metalloprotein has been altered upon metal reconstitution. This technique requires relatively little biological material (ca. 300 micrograms total protein) and, unlike either CD or 1H-NMR spectroscopy, is unaffected by the presence of different metal ions, thus allowing the direct comparison of FTIR spectra before and after metal substitution.     

The reaction of cysteine with ceruloplasmin copper

The kinetics of decay in absorbance at 610 nm in the reaction of cysteine with ceruloplasmin was biphasic under anaerobic conditions. Admission of oxygen to the bleached ceruloplasmin restored the blue color to about 75 % of the original value. However, under aerobic or anaerobic conditions an initial bleaching corresponded to a 25 % decrease in blue color. This change was irreversible and remained after removal of excess cysteine from the reaction mixture by dialysis. There was no correlation between transient and steady-state kinetic parameters. Circular dichroism measurements showed a characteristic reduction in the negative band at 450 nm, which is specific for type 1b copper. Isolation and further studies on cysteine-modified ceruloplasmin with a lower A₆₁₀/A₂₈₀ ratio showed < 10% reduction in enzyme activity toward p-phenylenediamine and o-dianisidine. Evidence is also presented that ceruloplasmin catalyzes the oxidation of cysteine with a one-electron reduction of oxygen and the formation of superoxide ion, which is then converted to H₂O₂ by ceruloplasmin. The effect of superoxide dismutase and catalase also confirms the presence of superoxide and H₂O₂. In sum, these data show that a permanent reduction of type 1b copper occurred when cysteine was used as a substrate. We conclude that there is a single electron transfer from cysteine directly to oxygen using one specific copper of ceruloplasmin, type 1b.