Role Of Protein Conformational Change Research Articles

Probing the role of protein conformational changes in the mechanism of prenylated-FMN-dependent phenazine-1-carboxylic acid decarboxylase

Phenazine-1-carboxylic acid decarboxylase (PhdA) is a prenylated-FMN-dependent (prFMN) enzyme belonging to the UbiD family of decarboxylases. Many UbiD-like enzymes catalyze (de)carboxylation reactions on aromatic rings and conjugated double bonds and are potentially valuable industrial catalysts. We have investigated the mechanism of PhdA using a slow turnover substrate, 2,3-dimethylquinoxaline-5-carboxylic acid (DQCA). Detailed analysis of the pH dependence and solvent deuterium isotope effects associated with the reaction uncovered unusual kinetic behavior. At low substrate concentrations, a substantial inverse solvent isotope effect (SIE) is observed on Vmax/KM of ∼ 0.5 when reaction rates of DQCA in H2O and D2O are compared. Under the same conditions, a normal SIE of 4.15 is measured by internal competition for proton transfer to the product. These apparently contradictory results indicate that the SIE values report on different steps in the mechanism. A proton inventory analysis of the reaction under Vmax/KM and Vmax conditions points to a "medium effect" as the source of the inverse SIE. Molecular dynamics simulations of the effect of D2O on PhdA structure support that D2O reduces the conformational lability of the enzyme and results in a more compact structure, akin to the active, "closed" conformer observed in crystal structures of some UbiD-like enzymes. Consistent with the simulations, PhdA was found to be more stable in D2O and to bind DQCA more tightly, leading to the observed rate enhancement under Vmax/KM conditions.

Physical characteristics of complex thermal system and working mechanism of enzymes

This paper discusses the behaviors and thermodynamic principle of complex thermal systems. The equations of enzymatic kinetics are revised to incorporate the thermodynamic parameters of protein conformational state, so that the role of protein conformational change in enzymatic reaction can be analyzed thermodynamically. In addition, the genetic interactions during the informational flow from genome to biological functions are analyzed thermodynamically. Based on these, the working mechanisms of enzyme action and regulation are further interpreted from a thermodynamic point of view and are found to be fundamentally the same. For example, the decrease of activation energy in enzymatic reaction comes from the binding between substrate and enzyme. Finally, we conclude that protein thermodynamics is a theoretical foundation for all biological processes.

The Role of Protein Conformational Changes in Tuning the Fluorescence State of Light-Harvesting Complexes

In a world undergoing rapid changes in sunlight quality and quantity, the successful design of photosynthesis must rely on the ability of plants and algae to quickly and reversibly adapt to the different conditions. A fast response to the fluctuating environment is given by the Light-Harvesting Complexes (LHCs). LHCs are large pigment-protein complexes responsible for the first steps of photosynthesis. LHCs capture light and deliver excitations to Photosystem I and II (PSI and PSII) where photosynthesis starts. Spectroscopy studies showed that LHCs can switch between states of long and short fluorescence lifetime. Quenched fluorescence states help preventing reactions with oxygen eventually leading to oxidative stress. Although postulated for long time, the role of protein conformational changes in tuning optical properties of LHCs remained unconfirmed until only recently. We here present the role of specific protein domains in tuning pigment-pigment interactions in a series of LHCs. A first example is LHCSR3, the trigger of photoprotection in the green alga Chlamydomonas reinhardtii, which possesses a peculiar C-terminus. This domain is pH-sensitive (pH is a stress-indicator in the alga) and causes tuning of fluorescence to quenched states upon acidification [Liguori, N. et al JACS 2013]. Additionally we show that this mechanism is absent in all related LHC proteins from PSII, which indeed do not present the same residue composition at this domain [Liguori, N. et al in preparation]. However LHCII, which is the most abundant LHC in both plants and algae, still undergoes significant conformational changes at a different domain: the N-terminus. It is a highly disordered domain and, as we show, a strong correlation is found between the different conformations of this domain and the different pigment-pigment interactions [Liguori, N. et al Scientific Reports in press].

Two-step mechanism involving active-site conformational changes regulates human telomerase DNA binding.

The ribonucleoprotein enzyme telomerase maintains telomeres and is essential for cellular immortality in most cancers. Insight into the telomerase mechanism can be gained from syndromes such as dyskeratosis congenita, in which mutation of telomerase components manifests in telomere dysfunction. We carried out detailed kinetic and thermodynamic analyses of wild-type telomerase and two disease-associated mutations in the reverse transcriptase domain. Differences in dissociation rates between primers with different 3' ends were independent of DNA affinities, revealing that initial binding of telomerase to telomeric DNA occurs through a previously undescribed two-step mechanism involving enzyme conformational changes. Both mutations affected DNA binding, but through different mechanisms: P704S specifically affected protein conformational changes during DNA binding, whereas R865H showed defects in binding to the 3' region of the DNA. To gain further insight at the structural level, we generated the first homology model of the human telomerase reverse transcriptase domain; the positions of P704S and R865H corroborate their observed mechanistic defects, providing validation for the structural model. Our data reveal the importance of protein interactions with the 3' end of telomeric DNA and the role of protein conformational change in telomerase DNA binding, and highlight naturally occurring disease mutations as a rich source of mechanistic insight.

Relating Protein Motion to Catalysis

This review examines the linkage between protein conformational motions and enzyme catalysis. The fundamental issues related to this linkage are probed in the context of two enzymes that catalyze hydride transfer, namely dihydrofolate reductase and liver alcohol dehydrogenase. The extensive experimental and theoretical studies addressing the role of protein conformational changes in these enzyme reactions are summarized. Evidence is presented for a network of coupled motions throughout the protein fold that facilitate the chemical reaction. This network is comprised of fast thermal motions that are in equilibrium as the reaction progresses along the reaction coordinate and that lead to slower equilibrium conformational changes conducive to the chemical reaction.

Conserved Residues in Domain Ia Are Required for the Reaction of Escherichia coli DNA Ligase with NAD+

NAD(+)-dependent DNA ligases are present in all bacteria and are essential for growth. Their unique substrate specificity compared with ATP-dependent human DNA ligases recommends the NAD(+) ligases as targets for the development of new broad-spectrum antibiotics. A plausible strategy for drug discovery is to identify the structural components of bacterial DNA ligase that interact with NAD(+) and then to isolate small molecules that recognize these components and thereby block the binding of NAD(+) to the ligase. The limitation to this strategy is that the structural determinants of NAD(+) specificity are not known. Here we show that reactivity of Escherichia coli DNA ligase (LigA) with NAD(+) requires N-terminal domain Ia, which is unique to, and conserved among, NAD(+) ligases but absent from ATP-dependent ligases. Deletion of domain Ia abolished the sealing of 3'-OH/5'-PO(4) nicks and the reaction with NAD(+) to form ligase-adenylate but had no effect on phosphodiester formation at a preadenylated nick. Alanine substitutions at conserved residues within domain Ia either reduced (His-23, Tyr-35) or abolished (Tyr-22, Asp-32, Asp-36) sealing of a 5'-PO(4) nick and adenylyl transfer from NAD(+) without affecting ligation of pre-formed DNA-adenylate. We suggest that these five side chains comprise a binding site for the nicotinamide mononucleotide moiety of NAD(+). Structure-activity relationships were clarified by conservative substitutions.

Detection and identification of a chromophoric intermediate during the medium-chain fatty acyl-CoA dehydrogenase-catalyzed reaction via rapid-scanning UV/visible spectroscopy.

We have investigated the medium-chain fatty acyl-CoA dehydrogenase (MCAD)-catalyzed reaction via rapid-scanning stopped-flow (RSSF) UV/vis spectroscopy, combined with the single-wavelength stopped-flow technique, utilizing 3-indolepropionyl-CoA (IPCoA) and trans-3-indoleacryloyl-CoA (IACoA) as chromophoric pseudosubstrates. The RSSF spectral data reveal that formation of an intermediary species with an absorbance maximum at 400 nm and a broad charge-transfer band around 600 nm accompanies the reduction of MCAD-FAD by IPCoA. In the presence of high concentrations of enzyme ([MCAD] >> [IPCoA]) the intermediary spectral band at 400 nm remains unperturbed, whereas in the presence of low concentrations of enzyme ([MCAD] << [IPCoA]) it slowly shifts to an absorption band with an absorbance maximum at 370 nm. Appearance and disappearance of this intermediary species coincides with the appearance and disappearance of the charge-transfer band. Single-wavelength stopped-flow studies, performed under similar high and low enzyme conditions, were consistent with one (1/tau 1) and two (1/tau 1 > 1/tau 2) relaxation rate constants, respectively. These findings, combined with relaxation studies performed in the reverse directions as well as substrate and product binding studies with the oxidized and reduced forms of the enzyme, have allowed us to conclude the following: (1) the intermediary species possesses the properties of reduced flavin and highly conjugated reaction product IACoA (absorbance maximum = 400 nm); (2) this intermediary species collapses into an MCAD-FADH2-IACoA complex (absorbance maximum = 370 nm) in the presence of excessive concentrations of IPCoA; the collapse is being driven by the competitive binding of IPCoA with the reduced form of the enzyme; (3) the 400-nm absorption band and the charge-transfer band are given by the same intermediary species formed during the enzyme-catalyzed reaction pathway. The role of protein conformational changes in modulating the substrate/product structures during the MCAD-catalyzed reaction is discussed.

Role of protein conformational changes, surface approximation and protein cofactors in heparin-accelerated antithrombin-proteinase reactions.

Antithrombin functions as the principal plasma protein inhibitor of most blood coagulation proteinases.1,2 The essential role of this inhibitor in regulating the activity of these proteinases in vivo is indicated from the well-established link between inherited or acquired deficiencies of antithrombin and the tendency to develop thrombotic disease. Antithrombin is a member of the serpin superfamily of protein proteinase inhibitors and its main target enzymes include the blood coagulation factors IXa, Xa and thrombin. This and other serpins are distinguished from other family members in that their reactions with target enzymes are greatly accelerated by the binding of heparin or heparan sulfate glycosaminoglycans. This property is chiefly responsible for the anticoagulant activity of heparin and has suggested a role for endogenous heparin and heparan sulfate in the regulation of blood coagulation proteinases by antithrombin. In this article, we will review our present understanding of the relationship between antithrombin structure and function based on currently available evidence. Our discussion will focus principally on two areas: 1) the mechanism by which antithrombin and other serpins inhibit their target proteinases; and 2) the molecular basis of heparin’s accelerating effect on antithrombin-proteinase reactions.