Submillisecond folding of monomeric lambda repressor.

The folding kinetics of a truncated form of the N-terminal domain of phage lambda repressor [lambda 6-85] has been investigated by using the technique of dynamic NMR. lambda 6-85 has been shown previously to fold in a purely two-state fashion. This allows the determination of folding and unfolding rates from simulation of the exchange-broadened aromatic resonances of Tyr-22. The folding kinetics were determined over a range of 1.35 to 3.14 M urea. The urea dependence of both folding and unfolding rate constants is exponential, suggesting that the rate-determining step is invariant at the urea concentrations studied. The folding and unfolding rates extrapolated to 0 M urea at 37 degrees C are 3600 +/- 400 s-1 and 27 +/- 6 s-1, respectively. The observed lambda 6-85 folding rate constant exceeds that of other fast-folding globular proteins by a factor of 14-54. The urea dependence of the folding and unfolding rate constants suggests that the transition state of the rate-determining step is considerably more exposed to solvent than previously studied protein-folding transition states. The surprising rapidity of lambda 6-85 folding and unfolding may be the consequence of its all-helical secondary structure. These kinetic results clearly demonstrate that all of the fundamental events of protein folding can occur on the submillisecond time scale.

A variant of lambda repressor with an altered pattern of cooperative binding to DNA sites.

The bacteriophage lambda repressor binds cooperatively to pairs of adjacent sites in the lambda chromosome, one repressor dimer binding to each site. The repressor's amino domain (that which mediates DNA binding) is connected to its carboxyl domain (that which mediates dimerization and the interaction between dimers) by a protease-sensitive linker region. We have generated a variant lambda repressor that lacks this linker region. We show that dimers of the variant protein are deficient in cooperative binding to sites at certain, but not all, distances. The linker region thus extends the range over which carboxyl domains of DNA-bound dimers can interact. In particular, the linker is required for cooperative binding to a pair of sites as found in the lambda chromosome, and thus is essential for the repressor's physiological function.

Operator binding by lambda repressor heterodimers with one or two N-terminal arms.

The first 6 amino acids (NH2-Ser1-Thr2-Lys3-Lys4-Lys5-Pro6) of bacteriophage lambda cI repressor form a flexible arm that wraps around the operator DNA. Homodimeric lambda repressor has two arms. To determine whether both arms are necessary or only one arm is sufficient for operator binding, we constructed heterodimeric repressors with two, one, or no arms by fusing the DNA binding domain of lambda repressor to leucine zippers from Fos and Jun. Although only one arm is visible in the cocrystal structure of the N-domain-operator complex, our results indicate that both arms are required for optimal operator binding and normal site discrimination.

Configuration-dependent diffusion can shift the kinetic transition state and barrier height of protein folding

We show that diffusion can play an important role in protein-folding kinetics. We explicitly calculate the diffusion coefficient of protein folding in a lattice model. We found that diffusion typically is configuration- or reaction coordinate-dependent. The diffusion coefficient is found to be decreasing with respect to the progression of folding toward the native state, which is caused by the collapse to a compact state constraining the configurational space for exploration. The configuration- or position-dependent diffusion coefficient has a significant contribution to the kinetics in addition to the thermodynamic free-energy barrier. It effectively changes (increases in this case) the kinetic barrier height as well as the position of the corresponding transition state and therefore modifies the folding kinetic rates as well as the kinetic routes. The resulting folding time, by considering both kinetic diffusion and the thermodynamic folding free-energy profile, thus is slower than the estimation from the thermodynamic free-energy barrier with constant diffusion but is consistent with the results from kinetic simulations. The configuration- or coordinate-dependent diffusion is especially important with respect to fast folding, when there is a small or no free-energy barrier and kinetics is controlled by diffusion.Including the configurational dependence will challenge the transition state theory of protein folding.

Epitope-specific tolerance induction with an engineered immunoglobulin.

Isologous and heterologous immunoglobulins have been shown to be extremely effective as tolerogenic carriers for nearly 30 years. The efficacy of these proteins is due in part to their long half-life in vivo, as well as their ability to crosslink surface IgM with Fc receptors. The concept of using IgG as a carrier molecule to induce unresponsiveness in the adult immune system has been exploited for simple haptens, such as nucleosides, as well as for peptides. To further evaluate the in vivo potential of these molecules for inducing tolerance to a defined epitope, we have engineered a fusion protein of mouse IgG1 with the immunodominant epitope 12-26 from bacteriophage lambda cI repressor protein. This 15-mer, which contains both a B-cell and T-cell epitope, has been fused in-frame to the N terminus of a mouse heavy chain IgG1 construct, thus creating a "genetic hapten-carrier" system. We describe a novel in vitro and in vivo experimental system for studying the feasibility of engineered tolerogens, consisting of a recombinant flagellin challenge antigen and a murine IgG1 tolerogen, both expressing the lambda repressor epitope 12-26. Herein, we show that peptide-grafted IgG molecules injected i.v., or expressed by transfected, autologous B cells, can efficiently modulate the cellular and humoral immune responses to immunodominant epitopes. This model displays the feasibility of "tailor-designing" immune responses to whole antigens by selecting epitopes for either tolerance or immunity.

Formation of linear plasmid multimers promoted by the phage lambda Red-system in lon mutants of Escherichia coli

We report here the formation of plasmid linear multimers promoted by the Red-system of phage lambda using a multicopy plasmid comprised of lambda red alpha and red beta genes, under the control of the lambda cI857 repressor. Our observations have revealed that the multimerization of plasmid DNA is dependent on the red beta and recA genes, suggesting a concerted role for these functions in the formation of plasmid multimers. The formation of multimers occurred in a recBCD+ sbcB+ xthA+ lon genetic background at a higher frequency than in the isogenic lon+ host cells. The multimers comprised tandem repeats of monomer plasmid DNA. Treatment of purified plasmid DNA with exonuclease III revealed the presence of free double-chain ends in the molecules. Determination of the size of multimeric DNA, by pulse field gel electrophoresis, revealed that the bulk of the DNA was in the range 50-240 kb, representing approximately 5-24 unit lengths of monomeric plasmid DNA. We provide a conceptual framework for Red-system-promoted formation and enhanced accumulation of plasmid linear multimers in lon mutants of E. coli.

Corepressor-induced organization and assembly of the biotin repressor: A model for allosteric activation of a transcriptional regulator

The Escherichia coli biotin repressor binds to the biotin operator to repress transcription of the biotin biosynthetic operon. In this work, a structure determined by x-ray crystallography of a complex of the repressor bound to biotin, which also functions as an activator of DNA binding by the biotin repressor (BirA), is described. In contrast to the monomeric aporepressor, the complex is dimeric with an interface composed in part of an extended β-sheet. Model building, coupled with biochemical data, suggests that this is the dimeric form of BirA that binds DNA. Segments of three surface loops that are disordered in the aporepressor structure are located in the interface region of the dimer and exhibit greater order than was observed in the aporepressor structure. The results suggest that the corepressor of BirA causes a disorder-to-order transition that is a prerequisite to repressor dimerization and DNA binding.

Transition metal ion activation of DNA binding by the diphtheria tox repressor requires the formation of stable homodimers.

The diphtheria tox repressor (DtxR) is a transition metal ion-dependent regulatory element that controls the expression of diphtheria toxin and several genes involved in the synthesis of siderophores in Corynebacterium diphtheriae. In the presence of transition metal ions apo-DtxR becomes activated and specifically binds to its target DNA sequences. We demonstrate by glutaraldehyde cross-linking that monomeric apo-DtxR is in weak equilibrium with a dimeric form and that upon addition of activating metal ions to the reaction mixture a dimeric complex is stabilized. Addition of the DNA-binding-defective mutant apo-DtxR(delta 1-47) to apo-DtxR in the absence of transition metal ions inhibits conversion of the apo-repressor to its activated DNA-binding form. We also show that the binding of Ni2+ to both apo-DtxR and apo-DtxR(delta 1-47) is cooperative and that upon ion binding there is a conformational change in the environment of the indole ring moiety of Trp-104. For the wild-type repressor the consequences of this conformational change include a shift in equilibrium toward dimer formation and activation of target DNA binding by the repressor. We conclude that the formation of DtxR homodimers is mediated through a protein-protein interaction domain that is also activated on metal ion binding.

Bistability and switching in the lysis/lysogeny genetic regulatory network of bacteriophage lambda

Bistability and switching are two important aspects of the genetic regulatory network of phage. Positive and negative feedbacks are key regulatory mechanisms in this network. By the introduction of threshold values, the developmental pathway of A phage is divided into different stages. If the protein level reaches a threshold value, positive or negative feedback will be effective and regulate the process of development. Using this regulatory mechanism, we present a quantitative model to realize bistability and switching of phage based on experimental data. This model gives descriptions of decisive mechanisms for different pathways in induction. A stochastic model is also introduced for describing statistical properties of switching in induction. A stochastic degradation rate is used to represent intrinsic noise in induction for switching the system from the lysogenic pathway to the lysis pathway. The approach in this paper represents an attempt to describe the regulatory mechanism in genetic regulatory network under the influence of intrinsic noise in the framework of continuous models. (C) 2003 Elsevier Ltd. All rights reserved.

meso-Porphyrinylphosphine Oxides: Mono- and Bidentate Ligands for Supramolecular Chemistry and the Crystal Structures of Monomeric [10,20-Diphenylporphyrinatonickel(II)-5,15-diyl]-bis-[P(O)Ph2] and Polymeric Self-Coordinated ([10,20-Diphenylporphyrinatozinc(II)-5,15-diyl]-bis-[P(O)Ph2])

A series of porphyrins substituted in one or two meso-positions by diphenylphosphine oxide groups has been prepared by the palladium catalysed reaction of diphenylphosphine or its oxide with the corresponding bromoporphyrins. Compounds {MDPP-[P(O)Ph2]n} (M = H2, Ni, Zn; H2DPP = 5,15-diphenylporphyrin; n = 1, 2) were isolated in yields of 60-95%. The reaction is believed to proceed via the conventional oxidative addition, phosphination and reductive elimination steps, as the stoichiometric reaction of η1-palladio(II) porphyrin [PdBr(H2DPP)(dppe)] (H2DPP = 5,15-diphenylporphyrin; dppe = 1,2-bis(diphenylphosphino)ethane) with diphenylphosphine oxide also results in the desired mono-porphyrinylphosphine oxide [H2DPP-P(O)Ph2]. Attempts to isolate the tertiary phosphines failed due to their extreme air-sensitivity. Variable temperature 1H NMR studies of [H2DPP-P(O)Ph2] revealed an intrinsic lack of symmetry, while fluorescence spectroscopy showed that the phosphine oxide group does not behave as a "heavy atom" quencher. The electron withdrawing effect of the phosphine oxide group was confirmed by voltammetry. The ligands were characterised by multinuclear NMR and UV-visible spectroscopy as well as mass spectrometry. Single crystal X-ray crystallography showed that the bis(phosphine oxide) nickel(II) complex {[NiDPP-[P(O)Ph2]2} is monomeric in the solid state, with a ruffled porphyrin core and the two P=O fragments on the same side of the average plane of the molecule. On the other hand, the corresponding zinc(II) complex formed infinite chains through coordination of one Ph2PO substituent to the neighbouring zinc porphyrin through an almost linear P=O---Zn unit, leaving the other Ph2PO group facing into a parallel channel filled with disordered water molecules. These new phosphine oxides are attractive ligands for supramolecular porphyrin chemistry.

Cyclin-dependent kinase-mediated phosphorylation of RBP1 and pRb promotes their dissociation to mediate release of the SAP30·mSin3·HDAC transcriptional repressor complex

Eukaryotic cell cycle progression is mediated by phosphorylation of protein substrates by cyclin-dependent kinases (CDKs). A critical substrate of CDKs is the product of the retinoblastoma tumor suppressor gene, pRb, which inhibits G1-S phase cell cycle progression by binding and repressing E2F transcription factors. CDK-mediated phosphorylation of pRb alleviates this inhibitory effect to promote G1-S phase cell cycle progression. pRb represses transcription by binding to the E2F transactivation domain and recruiting the mSin3·histone deacetylase (HDAC) transcriptional repressor complex via the retinoblastoma-binding protein 1 (RBP1). RBP1 binds to the pocket region of pRb via an LXCXE motif and to the SAP30 subunit of the mSin3·HDAC complex and, thus, acts as a bridging protein in this multisubunit complex. In the present study we identified RBP1 as a novel CDK substrate. RBP1 is phosphorylated by CDK2 on serines 864 and 1007, which are N- and C-terminal to the LXCXE motif, respectively. CDK2-mediated phosphorylation of RBP1 or pRb destabilizes their interaction in vitro, with concurrent phosphorylation of both proteins leading to their dissociation. Consistent with these findings, RBP1 phosphorylation is increased during progression from G 1 into S-phase, with a concurrent decrease in its association with pRb in MCF-7 breast cancer cells. These studies provide new mechanistic insights into CDK-mediated regulation of the pRb tumor suppressor during cell cycle progression, demonstrating that CDK-mediated phosphorylation of both RBP1 and pRb induces their dissociation to mediate release of the mSin3·HDAC transcriptional repressor complex from pRb to alleviate transcriptional repression of E2F.

Krüppel-associated box (KRAB)-associated co-repressor (KAP-1) Ser-473 phosphorylation regulates heterochromatin protein 1 (HP1- ) mobilization and DNA repair in heterochromatin

The DNA damage response encompasses a complex series of signaling pathways that function to regulate and facilitate the repair of damaged DNA. Recent studies have shown that the repair of transcriptionally inactive chromatin, named heterochromatin, is dependent upon the phosphorylation of the co-repressor, Krüppel-associated box (KRAB) domain-associated protein (KAP-1), by the ataxia telangiectasia-mutated (ATM) kinase. Co-repressors, such as KAP-1, function to regulate the rigid structure of heterochromatin by recruiting histone-modifying enzymes, such HDAC1/2, SETDB1, and nucleosome-remodeling complexes such as CHD3. Here, we have characterized a phosphorylation site in the HP1-binding domain of KAP-1, Ser-473, which is phosphorylated by the cell cycle checkpoint kinase Chk2. Expression of a nonphosphorylatable S473A mutant conferred cellular sensitivity to DNA-damaging agents and led to defective repair of DNA double-strand breaks in heterochromatin. In addition, cells expressing S473A also displayed defective mobilization of the HP1-β chromodomain protein. The DNA repair defect observed in cells expressing S473A was alleviated by depletion of HP1-β, suggesting that phosphorylation of KAP-1 on Ser-473 promotes the mobilization of HP1-β from heterochromatin and subsequent DNA repair. These results suggest a novel mechanism of KAP-1-mediated chromatin restructuring via Chk2-regulated HP1-β exchange from heterochromatin, promoting DNA repair.

Defining the E-Cadherin repressor interactome in epithelial-mesenchymal transition : the PMC42 model as a case study

Epithelial-mesenchymal transition (EMT) is a feature of migratory cellular processes in all stages of life, including embryonic development and wound healing. Importantly, EMT features cluster with disease states such as chronic fibrosis and cancer. The dissolution of the E-cadherin-mediated adherens junction (AJ) is a key preliminary step in EMT and may occur early or late in the growing epithelial tumour. This is a first step for tumour cells towards stromal invasion, intravasation, extravasation and distant metastasis. The AJ may be inactivated in EMT by directed E-cadherin cleavage; however, it is increasingly evident that the majority of AJ changes are transcriptional and mediated by an expanding group of transcription factors acting directly or indirectly to repress E-cadherin expression. A review of the current literature has revealed that these factors may regulate each other in a hierarchical pattern where Snail1 (formerly Snail) and Snail2 (formerly Slug) are initially induced, leading to the activation of Zeb family members, TCF3, TCF4, Twist, Goosecoid and FOXC2. Within this general pathway, many inter-regulatory relationships have been defined which may be important in maintaining the EMT phenotype. This may be important given the short half-life of Snail1 protein. We have investigated these inter-regulatory relationships in the mesenchymal breast carcinoma cell line PMC42 (also known as PMC42ET) and its epithelial derivative, PMC42LA. This review also discusses several newly described regulators of E-cadherin repressors including oestrogen receptor-α and new discoveries in hypoxia- and growth factor-induced EMT. Finally, we evaluated how these findings may influence approaches to current cancer treatment.

Detection of the protein dimers, multiple monomeric states and hydrated forms of Plasmodium falciparum triosephosphate isomerase in the gas phase

Dimeric and monomeric forms of the enzyme triosephosphate isomerase (TIM) from Plasmodium falciparum (Pf) have been detected under conditions of nanoflow by electrospray mass spectrometry. The dimer (M = 55 663 Da) exhibits a narrow charge state distribution with intense peaks limited to values of 18(+) to 21(+), maximal intensity being observed for charge states 19(+) and 20(+). A monomeric species with a charge state distribution ranging from 11(+) to 16(+) is also observed, which may be assigned to folded dissociated subunits. Complete dimer dissociation results under normal electrospray condition. The effects of solution pH and source temperature have been investigated. The observation of four distinct charge state distributions which may be assigned to a dimer, folded monomer, partially folded monomer and unfolded monomer is reported. Circular dichromism and fluorescence studies of Pf TIM at low pH support the retention of substantial secondary and tertiary structures. Satellite peaks in mass spectra corresponding to hydrated species are also observed and isotope shift upon deuteration is demonstrated. The analysis of all available independent crystal structures of Pf TIM and TIMs from other organisms permits identification of structurally conserved water molecules. Hydration observed in the dimer and folded monomeric forms in the gas phase may correspond to these conserved sites.