Conformational changes relevant to channel activity and folding within the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator.

Deletion of Phe-508 (F508del) in the first nucleotide binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to defects in folding and channel gating. NMR data on human F508del NBD1 indicate that an H620Q mutant, shown to increase channel open probability, and the dual corrector/potentiator CFFT-001 similarly disrupt interactions between β-strands S3, S9, and S10 and the C-terminal helices H8 and H9, shifting a preexisting conformational equilibrium from helix to coil. CFFT-001 appears to interact with β-strands S3/S9/S10, consistent with docking simulations.

A gene optimization strategy that enhances production of fully functional P-glycoprotein in Pichia pastoris.

Background: Structural and biochemical studies of mammalian membrane proteins remain hampered by inefficient production of pure protein. We explored codon optimization based on highly expressed Pichia pastoris genes to enhance co-translational folding and production of P-glycoprotein (Pgp), an ATP-dependent drug efflux pump involved in multidrug resistance of cancers.

Methodology/principal Findings: Codon-optimized "Opti-Pgp" and wild-type Pgp, identical in primary protein sequence, were rigorously analyzed for differences in function or solution structure.

Structures of a minimal human CFTR first nucleotide-binding domain as a monomer, head-to-tail homodimer, and pathogenic mutant.

Upon removal of the regulatory insert (RI), the first nucleotide binding domain (NBD1) of human cystic fibrosis transmembrane conductance regulator (CFTR) can be heterologously expressed and purified in a form that remains stable without solubilizing mutations, stabilizing agents or the regulatory extension (RE). This protein, NBD1 387-646(Delta405-436), crystallizes as a homodimer with a head-to-tail association equivalent to the active conformation observed for NBDs from symmetric ATP transporters. The 1.

Structural adaptation of an interacting non-native C-terminal helical extension revealed in the crystal structure of NAD+ synthetase from Bacillus anthracis.

The crystal structures of NH(3)-dependent NAD+ synthetase from Bacillus anthracis as the apoenzyme (1.9 A), in complex with the natural catalytic products AMP and pyrophosphate (2.4 A) and in complex with the substrate analog adenosine 5'-(alpha,beta-methylene)triphosphate (2.

Structure of a complex of human lactoferrin N-lobe with pneumococcal surface protein a provides insight into microbial defense mechanism.

Human lactoferrin, a component of the innate immune system, kills a wide variety of microorganisms including the Gram positive bacteria Streptococcus pneumoniae. Pneumococcal surface protein A (PspA) efficiently inhibits this bactericidal action. The crystal structure of a complex of the lactoferrin-binding domain of PspA with the N-lobe of human lactoferrin reveals direct and specific interactions between the negatively charged surface of PspA helices and the highly cationic lactoferricin moiety of lactoferrin.

Förster resonance energy transfer measurements are consistent with a helical bundle model for lipid-free apolipoprotein A-I.

Apolipoprotein (apo) A-I mutants were constructed for FRET studies to distinguish between two possible lipid-free conformers, a globular helix bundle and an elongated helical hairpin. Mutants containing a single Trp at position 50 were prepared by replacing Trps at positions 8, 72, and 108 with Phe (W@50). Two mutants were constructed from W@50 by incorporating Cys at Arg83 (W@50R83C) or Arg173 (W@50R173C) for attachment of the fluorescent probe AEDANS.

Role of inhibitor aliphatic chain in the thermodynamics of inhibitor binding to Escherichia coli enoyl-ACP reductase and the Phe203Leu mutant: a proposed mechanism for drug resistance.

The antibacterial target enoyl-acyl carrier protein (ACP) reductase is a homotetrameric enzyme that catalyzes the last reductive step of fatty acid biosynthesis. In the present paper, four 2-(2-hydroxyphenoxy)phenol inhibitors, wherein the 4-position substituent varied from H to n-propyl, were studied to determine the contribution of the aliphatic chain to the binding to the wild-type (wt) enoyl-ACP reductase from Escherichia coli (FabI) and a drug-resistant mutant, (F203L)FabI, in which phenylalanine 203 is mutated to leucine. Thermodynamic parameters of ternary complex formation (enzyme-NAD(+)-inhibitor) were determined by isothermal titration calorimetry.

Deletion of DnaK's lid strengthens binding to the nucleotide exchange factor, GrpE: a kinetic and thermodynamic analysis.

In this study, we have used surface plasmon resonance (SPR) and isothermal microtitration calorimetry (ITC) to study the mechanism of complex formation between the Hsp70 molecular chaperone, DnaK, and its cochaperone, GrpE, which is a nucleotide exchange factor. Experiments were geared toward understanding the influence of DnaK's three domains, the ATPase (residues 1-388), substrate-binding (residues 393-507), and lid (residues 508-638) domains, on complex formation with GrpE. We show that the equilibrium dissociation constants for the interaction of GrpE with wtDnaK, lidless DnaK(2-517), the ATPase domain (2-388), and the substrate-binding fragment (393-507) are 64 (+/-16) nM, 4.

Increased stability of human growth hormone with reduced lactogenic potency.

Human growth hormone (hGH), whose main function is the somatic growth stimulation, induces diverse effects including lactation. We examined the possibility of hGH stabilization by elimination of its lactogenic activity. Chimeric GHs were constructed by replacement of different segments of hGH with sequences derived from non-lactogenic porcine GH.