SummaryIn protein and RNA macromolecules, only a limited number of different side-chain chemical groups are available to function as catalysts. The myriad of enzyme-catalyzed reactions results from the ability of most of these groups to function either as nucleophilic, electrophilic, or general acid-base catalysts, and the key to their adapted chemical function lies in their states of protonation. Ionization is determined by the intrinsic pK a of the group and the microenvironment created around the group by the protein or RNA structure, which perturbs its intrinsic pK a to its functional or apparent pK a . These pK a shifts result from interactions of the catalytic group with other fully or partially charged groups as well as the polarity or dielectric of the medium that surrounds it. The electro- Abbreviations: AAD, acetoacetate decarboxylase; AbAld, antibody aldolase; Ala-Race, alanine racemase; hdvAR, hepatitis delta virus antigenomic ribozyme; ArsC, arsenate reductase; AspAT, aspartate aminotransferase; BCX, Bacillus circulans xylanase; BR, ground-state bacteriorhodopsin with all trans retinal, protonated D96, protonated Schiff base, and unprotonated D85; BR-M, excited M-state bacteriorhodopsin with 13-cis retinal, protonated D96, unprotonated Schiff base, and protonated D85; BR-N, excited N-state bacteriorhodopsin with 13-cis retinal, unprotonated D96, protonated Schiff base, and protonated D85; Chy-TFKs, chymotrypsin complexed with various peptidyl trifluoroketones; hmCK, human muscle creatine kinase; DsbA and DsbC, disulfide bond enzymes A and C in E. coli; GalE, UDP-galactose 4-epimerase; GRX, glutaredoxin; HB, hydrogen bond; HPE, hydrophobic environment; KSI, ketosteroid isomerase; LBHB, low-barrier hydrogen bond; NMR, nuclear magnetic resonance; NTα, N-terminus of an α-helix; Nuc/Lv, nucleophile/leaving group; 4-OT, 4-oxalocrotonate tautomerase; PDI, protein disulfide isomerase; PLP, pyridoxal 5 -phosphate; PMP, pyridoxamine 5 -phosphate; blmPTP, bovine liver low molecular weight protein tyrosine phosphatase; hPTP1, human protein tyrosine phosphatase; yPTP, Yersenia protein tyrosine phosphatase; rPTC, ribosomal peptidyl transferase center; RNaseH1, ribonuclease H1; TIM, triosephosphate isomerase; bTRX, bacterial thioredoxin; hTRX, human thioredoxin.
Abstract. We have used monolayers of control 3T3 cells and 3T3 cells expressing transfected human L1 as a culture substrate for rat PC12 cells and rat cerebellar neurons. PC12 cells and cerebellar neurons extended longer neurites on human L1 expressing cells. Neurons isolated from the cerebellum at postnatal day 9 responded equally as well as those isolated at postnatal day 1-4, and this contrasts with the failure of these older neurons to respond to the transfected human neural cell adhesion molecule (NCAM). Human Ll-dependent neurite outgrowth could be blocked by antibodies that bound to rat L1 and, additionally, the response could be fully inhibited by pertussis toxin and substantially inhibited by antagonists of L-and N-type calcium channels. Calcium influx into neurons induced by K + depolarization fully mimics the L1 response. Furthermore, we show that L1-and K+-dependent neurite outgrowth can be specifically inhibited by a reduction in extraceUular calcium to 0.25 ttM, and by pretreatment of cerebellar neurons with the intracellular calcium chelator BAPTA/AM. In contrast, the response was not inhibited by heparin or by removal of polysialic acid from neuronal NCAM both of which substantially inhibit NCAM-dependent neurite outgrowth. These data demonstrate that whereas NCAM and L1 promote neurite outgrowth via activation of a common CAM-specific second messenger pathway in neurons, neuronal responsiveness to NCAM and L1 is not coordinately regulated via posttranslational processing of NCAM. The fact that NCAM-and L1-dependent neurite outgrowth, but not adhesion, are calcium dependent provides further evidence that adhesion per se does not directly contribute to neurite outgrowth.
Free energies of oxygen-linked subunit assembly and cooperative interaction have been determined for 34 molecular species of human hemoglobin, which differ by amino acid alterations as a result of mutation or chemical modification at specific sites. These studies required the development of extensions to our earlier methodology. In combination with previous results they comprise a data base of 60 hemoglobin species, characterized under the same conditions. The data base was analyzed in terms of the five following issues. (1) Range and sensitivity to site modifications. Deoxy tetramers showed greater average energetic response to structural modifications than the oxy species, but the ranges are similar for the two ligation forms. (2) Structural localization of cooperative free energy. Difference free energies of dimer-tetramer assembly (oxy minus deoxy) yielded delta Gc for each hemoglobin, i.e., the free energy used for modulation of oxygen affinity over all four binding steps. A structure-energy map constructed from these results shows that the alpha 1 beta 2 interface is a unique structural location of the noncovalent bonding interactions that are energetically coupled to cooperativity. (3) Relationship of cooperativity to intrinsic binding. Oxygen binding energetics for dissociated dimers of mutants strongly indicates that cooperativity and intrinsic binding are completely decoupled by tetramer to dimer dissociation. (4) Additivity, site-site coupling and adventitious perturbations. All these are exhibited by individual-site modifications of this study. Large nonadditivity may be correlated with global (quaternary) structure change. (5) Residue position vs. chemical nature. Functional response is solely dictated by structural location for a subset of the sites, but varies with side-chain type at other sites. The current data base provides a unique framework for further analyses and modeling of fundamental issues in the structural chemistry of proteins and allosteric mechanisms.
Ground-state absorbance measurements show that BR from Halobacterium halobium containing asparagine at residue 85 (D85N) exists as three distinct chromophoric states in equilibrium. In the pH range 6-12 the absorbance spectra of the three states are demonstrated to be similar to flash-induced spectral intermediates which comprise the latter portion of the wild-type BR photocycle. One of the states absorbs maximally at 405 nm, has a deprotonated Schiff base, and contains predominantly the 13-cis form of retinal, identifying it as a close homologue of the M intermediate in the BR photocycle. The other species possess absorbance maxima with correspondence to those of the wild-type N (570 nm) and O (615 nm) photointermediates. The retinal composition of the O-like form was found to be dominated by all-trans isomer. The pH dependence of the concentrations of the equilibrium species corresponds closely with the pH dependence of the M, N, and O photointermediates. These data support kinetic models which emphasize the role of back-reactions during the photocycle of bacteriorhodopsin. Energetic and spectral characterization of the D85N ground-state equilibrium supports its use as a model for elucidating molecular transitions comprising the latter portion of the BR photocycle.
The inability to localize and measure the free energy of protein structure and structure change severely limits protein structure-function investigations. The local unfolding model for protein hydrogen exchange quantitatively related the free energy of local structural stability with the hydrogen exchange rate of concerted sets of structurally related protons. In tests with a number of modified hemoglobin forms, the loss in structural free energy obtained locally from hydrogen exchange results matches the loss in allosteric free energy measured globally by oxygen-binding and subunit dissociation experiments.
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