Molecular dynamics investigation of psalmopeotoxin I. Probing the relationship between 3D structure, anti-malarial activity and thermal stability.

PcFK1 is a member of the cysteine knot inhibitor family that displays anti-malarial properties. The naturally occurring molecule is ∼ 40 amino acids in length and forms a highly constrained 3D structure due to the presence of 3 disulfide and multiple intra-molecular H-bonds. Recent experimental studies on PcFK1 wild-type and mutants, where the cystiene residues of each disulfide bond were mutated into serine residues, suggest that alterations to these structural constraints can give rise to sizeable differences in SAR.

Synthesis, biophysical, and biological studies of wild-type and mutant psalmopeotoxins--anti-malarial cysteine knot peptides from Psalmopoeus cambridgei.

Psalmopeotoxin I and II (PcFK1 and PcFK2), an anti-malarial peptide first extracted from Psalmopoeus cambridgei was synthesized and characterized. Both peptides belong to the Inhibitor Cystine Knot (ICK) superfamily, containing three disulfide bridges. The six cysteine residues are conserved similar to other members of the ICK superfamily, suggesting their critical role for either folding or function.

Physical and biophysical effects of polysorbate 20 and 80 on darbepoetin alfa.

We studied the physical and biophysical affects of the nonionic surfactants polysorbate 20 and 80 and their mechanism of interaction using darbepoetin alfa, a 4-helix bundle protein, as the exemplary protein. Differences were observed between the abilities of the polysorbates to prevent surface loss/aggregation and correlated with each polysorbates initiation of micelle formation prior to the critical micelle concentration (CMC). The biophysical properties monitored by far-UV circular dichroism (CD) and tryptophan (Trp) fluorescence showed effects due to polysorbates, but were not correlated with their CMC.

Understanding the mechanism of beta-sheet folding from a chemical and biological perspective.

Perturbing the structure of the Pin1 WW domain, a 34-residue protein comprised of three beta-strands and two intervening loops has provided significant insight into the structural and energetic basis of beta-sheet folding. We will review our current perspective on how structure acquisition is influenced by the sequence, which determines local conformational propensities and mediates the hydrophobic effect, hydrogen bonding, and analogous intramolecular interactions. We have utilized both traditional site-directed mutagenesis and backbone mutagenesis approaches to alter the primary structure of this beta-sheet protein.

Structural and functional characterization of disulfide isoforms of the human IgG2 subclass.

In the accompanying report ( Wypych, J., Li, M., Guo, A.

Biophysical comparability of the same protein from different manufacturers: a case study using Epoetin alfa from Epogen and Eprex.

This study focuses on the development and application of biophysical methodology to characterize conformations of Epogen and Eprex, the injectable formulations of recombinant human Epoetin alfa produced by different manufacturers and commonly used for the treatment of renal anemia. In these studies Eprex, from prefilled syringes, and Epogen bulk product formulated in a buffer similar to the Eprex formulation, were purified by anion-exchange chromatography. Analytical ultracentrifugation studies of the purified main peak from each sample demonstrated that Epogen contains a single component with an s value of 2.

Backbone-Backbone H-Bonds Make Context-Dependent Contributions to Protein Folding Kinetics and Thermodynamics: Lessons from Amide-to-Ester Mutations.

The contribution of backbone-backbone hydrogen bonds (H-bonds) to protein folding energetics has been controversial. This is due, at least in part, to the inability to perturb backbone-backbone H-bonds by traditional methods of protein mutagenesis. Recently, however, protein backbone mutagenesis has become possible with the development of chemical and biological methods to replace individual amides in the protein backbone with esters.

Beta-sheet folding mechanisms from perturbation energetics.

Amide backbone and sidechain mutagenesis data can be used in combination with kinetic and thermodynamic measurements to understand the energetic contributions of backbone hydrogen bonding and the hydrophobic effect to the acquisition of beta-sheet structure. For example, it has been revealed that loop 1 of the WW domain forms in the transition state, consistent with the emerging theme that reverse turn formation is rate limiting in beta-sheet folding. A distinct subset of WW domain residues principally influences thermodynamic stability by forming hydrogen bonds and hydrophobic interactions that stabilize the native state.

Controlling the morphology of cross beta-sheet assemblies by rational design.

Low molecular weight peptidomimetics with simple amphiphilic sequences can help to elucidate the structures of cross beta-sheet assemblies, such as amyloid fibrils. The peptidomimetics described herein comprise a dibenzofuran template, two peptide strands made up of alternating hydrophilic and hydrophobic residues, and carboxyl termini, each of which can be varied to probe the structural requirements for beta-sheet self-assembly processes. The dibenzofuran template positions the strands approximately 10 A apart, allowing corresponding hydrophobic side chains in the strands to pack into a collapsed U-shaped structure.

Toward assessing the position-dependent contributions of backbone hydrogen bonding to beta-sheet folding thermodynamics employing amide-to-ester perturbations.

An amide-to-ester backbone substitution in a protein is accomplished by replacing an alpha-amino acid residue with the corresponding alpha-hydroxy acid, preserving stereochemistry, and conformation of the backbone and the structure of the side chain. This substitution replaces the amide NH (a hydrogen bond donor) with an ester O (which is not a hydrogen bond donor) and the amide carbonyl (a strong hydrogen bond acceptor) with an ester carbonyl (a weaker hydrogen bond acceptor), thus perturbing folding energetics. Amide-to-ester perturbations were used to evaluate the thermodynamic contribution of each hydrogen bond in the PIN WW domain, a three-stranded beta-sheet protein.

Solid-phase synthesis and stereochemical assignments of tenuecyclamides A-D employing heterocyclic amino acids derived from commercially available Fmoc alpha-amino acids.

[reaction: see text] The solid-phase assembly of heterocyclic amino acids enabled the total synthesis of numerous diastereoisomers of tenuecyclamides A-D, establishing or correcting the stereochemistry of each natural product. This strategy provides a very efficient route to synthesize thiazole- and oxazole-containing macrolactams from heterocyclic amino acids that are readily prepared from Fmoc-alpha-amino acids. This methodology appears to be broadly applicable to the synthesis of natural product libraries incorporating unnatural heterocyclic amino acid residues for the purpose of drug discovery.

Context-dependent contributions of backbone hydrogen bonding to beta-sheet folding energetics.

Backbone hydrogen bonds (H-bonds) are prominent features of protein structures; however, their role in protein folding remains controversial because they cannot be selectively perturbed by traditional methods of protein mutagenesis. Here we have assessed the contribution of backbone H-bonds to the folding kinetics and thermodynamics of the PIN WW domain, a small beta-sheet protein, by individually replacing its backbone amides with esters. Amide-to-ester mutations site-specifically perturb backbone H-bonds in two ways: a H-bond donor is eliminated by replacing an amide NH with an ester oxygen, and a H-bond acceptor is weakened by replacing an amide carbonyl with an ester carbonyl.

Tissue damage in the amyloidoses: Transthyretin monomers and nonnative oligomers are the major cytotoxic species in tissue culture.

The transthyretin (TTR) amyloidoses are human diseases in which the misfolded TTR protein aggregates in tissues with subsequent visceral, peripheral, and autonomic nerve dysfunction. Recent reports have stressed the importance of oligomeric intermediates as major cytotoxic species in various forms of amyloidogenesis. We have examined the cytotoxic effects of several quaternary structural states of wild-type and variant TTR proteins on cells of neural lineage.

Synthesis of all nineteen appropriately protected chiral alpha-hydroxy acid equivalents of the alpha-amino acids for Boc solid-phase depsi-peptide synthesis.

[reaction: see text] The preparation of depsi-peptides, amide-to-ester-substituted peptides used to probe the role of hydrogen bonding in protein folding energetics, is accomplished by replacing specific l-alpha-amino acid residues by their alpha-hydroxy acid counterparts in a solid-phase synthesis employing a t-Boc strategy. Herein we describe the efficient stereoselective synthesis of all 19 appropriately protected alpha-hydroxy acid equivalents of the l-alpha-amino acids, employing commercially available materials, expanding the number of available alpha-hydroxy acids from 9 to 19.

Packing, specificity, and mutability at the binding interface between the p160 coactivator and CREB-binding protein.

Among the most common interaction motifs between nuclear proteins is the recognition of one or more amphipathic helices. In an effort to determine principles behind this recognition, we have investigated the interaction between the p160 coactivator protein ACTR and the ACTR-binding domain of the CREB-binding protein, CBP. The two proteins use relatively small portions of their primary sequences to form a single synergistically folded domain consisting of six intertwined alpha-helices, three from each protein.

Recombinant expression, purification, and comparative characterization of torsinA and its torsion dystonia-associated variant Delta E-torsinA.

Early-onset torsion dystonia is an autosomal dominant movement disorder that has been linked to the deletion of one of a pair of glutamic acid residues in the protein torsinA (E(302/303); DeltaE-torsinA). In transfected cells, DeltaE-torsinA exhibits similar biochemical properties to wild type (WT)-torsinA, but displays a distinct subcellular localization. Primary structural analysis of torsinA suggests that this protein is a membrane-associated member of the AAA family of ATP-binding proteins.

Antibody domain exchange is an immunological solution to carbohydrate cluster recognition.

Human antibody 2G12 neutralizes a broad range of human immunodeficiency virus type 1 (HIV-1) isolates by binding an unusually dense cluster of carbohydrate moieties on the "silent" face of the gp120 envelope glycoprotein. Crystal structures of Fab 2G12 and its complexes with the disaccharide Manalpha1-2Man and with the oligosaccharide Man9GlcNAc2 revealed that two Fabs assemble into an interlocked VH domain-swapped dimer. Further biochemical, biophysical, and mutagenesis data strongly support a Fab-dimerized antibody as the prevalent form that recognizes gp120.

Synthesis of a negatively charged dibenzofuran-based beta-turn mimetic and its incorporation into the WW miniprotein-enhanced solubility without a loss of thermodynamic stability.

A versatile synthesis has been developed to functionalize the 4-(2-aminoethyl)-6-dibenzofuran propionic acid residue (1a) at the 2 and 8 positions with a variety of different substructures. The unfunctionalized version of this peptidomimetic (1a) is known to facilitate beta-hairpin formation in a variety of small peptides and proteins in aqueous solution when incorporated in place of the i + 1 and i + 2 residues of a beta-turn. In this study, we append propionate substituents on 1a at the 2 and 8 positions to successfully overcome solubility problems encountered with the incorporation of 1a in place of the i + 1 and i + 2 residues of the beta-turn in loop 1 of the WW domain.

The effect of backbone cyclization on the thermodynamics of beta-sheet unfolding: stability optimization of the PIN WW domain.

Backbone cyclization is often used in attempts to enhance protein stability, but is not always successful as it is possible to remove stabilizing or introduce destabilizing interactions in the process. Cyclization of the PIN1 WW domain, a 34-residue three-stranded beta-sheet structure, removes a favorable electrostatic interaction between its termini. Nevertheless, optimization of the linker connecting the N- and C-termini using information based on the previously determined ensemble of NMR structures leads to beta-sheets that are more stable than those derived from the linear sequence.