Deuteration of nonexchangeable protons on proteins affects their thermal stability, side-chain dynamics, and hydrophobicity.

We have investigated the effect of deuteration of non-exchangeable protons on protein global thermal stability, hydrophobicity, and local flexibility using well-known thermostable model systems such as the villin headpiece subdomain (HP36) and the third immunoglobulin G-binding domain of protein G (GB3). Reversed-phase high-performance liquid chromatography (RP-HPLC) measurements as a function of temperature probe global thermal stability in the presence of acetonitrile, while differential scanning calorimetry determines thermal stability in solution. Both indicate small but measurable changes in the order of several degrees.

Design of Novel Amphipathic α-Helical Antimicrobial Peptides with No Toxicity as Therapeutics against the Antibiotic-Resistant Gram-Negative Bacterial Pathogen, .

We designed and synthesized two series of five 26-residue amphipathic α-helical cationic antimicrobial peptides (AMPs) with five or six positively charged residues (D-Lys, L-Dab (2,4-diaminobutyric acid) or L-Dap (2,3-diaminopropionic acid)) on the polar face where all other residues are in the D-conformation. Hemolytic activity against human red blood cells was determined using the most stringent conditions for the hemolysis assay, 18h at 37°C, 1% human erythrocytes and peptide concentrations up to 1000 μg/mL (~380 μM). Antimicrobial activity was determined against 7 strains, resistant to polymyxin B and colistin (antibiotics of last resort) to show the effect of positively charged residues in two different locations on the polar face (positions 3, 7, 11, 18, 22 and 26 positions 3, 7, 14, 15, 22 and 26).

Separation of highly charged (+5 to +10) amphipathic α-helical peptide standards by cation-exchange and reversed-phase high-performance liquid chromatography.

We are currently examining the potential of amphipathic cationic α-helical peptides as a new generation of peptide standards for both cation-exchange high-performance liquid chromatography and reversed-phase chromatography. Thus, amphipathic peptides are particularly suitable for high-performance liquid chromatography standards due to the preferred binding of the non-polar face to the hydrophobic stationary phase of reversed-phase packings or the preferred binding of the polar face to the charged/hydrophilic stationary phase of cation-exchange packings. The ability of different reversed-phase or cation-exchange matrices to separate mixtures of peptide standards with only subtle hydrophilicity/hydrophobicity variations in both the non-polar and polar face of the peptides can then be assessed.

Optimized purification of a fusion protein by reversed-phase high performance liquid chromatography informed by the linear solvent strength model.

Fusion protein systems are commonly used for expression of small proteins and peptides. An important criterion for a fusion protein system to be useful is the ability to separate the protein of interest from the tag. Additionally, because no protease cleaves fusion proteins with 100% efficiency, the ability to separate the desired peptide from any remaining uncleaved protein is also necessary.

Role of positively charged residues on the polar and non-polar faces of amphipathic α-helical antimicrobial peptides on specificity and selectivity for Gram-negative pathogens.

We have designed de novo and synthesized eight 26-residue all D-conformation amphipathic α-helical cationic antimicrobial peptides (AMPs), four with "specificity determinants" which provide specificity for prokaryotic cells over eukaryotic cells and four AMPs without specificity determinants. The eight AMPs contain six positively charged Lys residues on the polar face in four different arrangements to understand the role of these residues have on antimicrobial activity against 14 Acinetobacter baumannii strains, seven of which were resistant to polymyxin B and colistin; six diverse Pseudomonas aeruginosa strains and 17 Staphylococcus aureus strains, nine of which were methicillin-sensitive, and eight of which were methicillin-resistant. The four AMPs without specificity determinants are extremely hemolytic.

Separation of Peptides on HALO 2-Micron Particles.

Reversed-phase high-performance liquid chromatography (RP-HPLC) is of fundamental importance to the isolation and separation of peptides, proteins, and other biomolecules. Hence, there is a continuing high demand for the development of RP-HPLC stationary-phase materials with enhanced separation efficiency. HALO packing materials began the revolution in "core-shell" technology with the advantages of faster separations, higher resolution and peak capacity, high temperature stability, and rugged reliable performance compared to traditional HPLC and UHPLC.

Platform technology to generate broadly cross-reactive antibodies to α-helical epitopes in hemagglutinin proteins from influenza A viruses.

We have utilized a de novo designed two-stranded α-helical coiled-coil template to display conserved α-helical epitopes from the stem region of hemagglutinin (HA) glycoproteins of influenza A. The immunogens have all the surface-exposed residues of the native α-helix in the native HA protein of interest displayed on the surface of the two-stranded α-helical coiled-coil template. This template when used as an immunogen elicits polyclonal antibodies which bind to the α-helix in the native protein.

Opposing activity changes in AMP deaminase and AMP-activated protein kinase in the hibernating ground squirrel.

Hibernating animals develop fatty liver when active in summertime and undergo a switch to a fat oxidation state in the winter. We hypothesized that this switch might be determined by AMP and the dominance of opposing effects: metabolism through AMP deaminase (AMPD2) (summer) and activation of AMP-activated protein kinase (AMPK) (winter). Liver samples were obtained from 13-lined ground squirrels at different times during the year, including summer and multiples stages of winter hibernation, and fat synthesis and β-fatty acid oxidation were evaluated.

Structure-activity relationships of the antimicrobial peptide gramicidin S and its analogs: aqueous solubility, self-association, conformation, antimicrobial activity and interaction with model lipid membranes.

GS10 [cyclo-(VKLdYPVKLdYP)] is a synthetic analog of the naturally occurring antimicrobial peptide gramicidin (GS) in which the two positively charged ornithine (Orn) residues are replaced by two positively charged lysine (Lys) residues and the two less polar aromatic phenylalanine (Phe) residues are replaced by the more polar tyrosine (Tyr) residues. In this study, we examine the effects of these seemingly conservative modifications to the parent GS molecule on the physical properties of the peptide, and on its interactions with lipid bilayer model and biological membranes, by a variety of biophysical techniques. We show that although GS10 retains the largely β-sheet conformation characteristic of GS, it is less structured in both water and membrane-mimetic solvents.

Structure-guided RP-HPLC chromatography of diastereomeric α-helical peptide analogs substituted with single amino acid stereoisomers.

An α-helical model peptide (Ac-EAEKAAKE-X-EKAAKEAEK-amide) was used as a template to examine the efficacy of conventional reversed-phase high-performance liquid chromatography (RP-HPLC) in separating peptide analogs with single substitutions (at position X) of diasteromeric amino acids Ile, allo-Ile, d-Ile and d-allo-Ile. We compared differences in peptide retention behavior on a C8 column and a C18 column at different temperatures. We demonstrated how subtle differences in peptide secondary structure affected by the different substitutions of amino acids with identical overall hydrophobicity enabled effective resolution of these peptide analogs.

An improved approach to hydrophilic interaction chromatography of peptides: salt gradients in the presence of high isocratic acetonitrile concentrations.

Hydrophilic interaction chromatography (HILIC) for separations of peptides has been employed infrequently, particularly considering that this technique was introduced over 20 years ago. The present manuscript describes a radical departure from the traditional HILIC elution approach, where separations are achieved via increasing salt (sodium perchlorate) gradients in the presence of high isocratic concentrations (>80%) of acetonitrile, denoted HILIC/SALT. This initial study compared to reversed-phase chromatography (RPC), HILIC and HILIC/SALT for the separation of mixtures of synthetic peptide standards varying in structure (amphipathic α-helix, random coil), length (10-26 residues), number of positively charged residues (+1 to +11) and hydrophilicity/hydrophobicity.

Counteracting roles of AMP deaminase and AMP kinase in the development of fatty liver.

Fatty liver (hepatic steatosis) is associated with nucleotide turnover, loss of ATP and generation of adenosine monophosphate (AMP). It is well known that in fatty liver, activity of the AMP-activated kinase (AMPK) is reduced and that its stimulation can prevent hepatic steatosis by both enhancing fat oxidation and reducing lipogenesis. Here we show that another AMP dependent enzyme, AMPD2, has opposing effects on fatty acid oxidation when compared to AMPK.

Purification and structural characterization of siderophore (corynebactin) from Corynebacterium diphtheriae.

During infection, Corynebacterium diphtheriae must compete with host iron-sequestering mechanisms for iron. C. diphtheriae can acquire iron by a siderophore-dependent iron-uptake pathway, by uptake and degradation of heme, or both.

Design of peptide standards with the same composition and minimal sequence variation to monitor performance/selectivity of reversed-phase matrices.

The present manuscript extends our de novo peptide design approach to the synthesis and evaluation of a new generation of reversed-phase HPLC peptide standards with the same composition and minimal sequence variation (SCMSV). Thus, we have designed and synthesized four series of peptide standards with the sequences Gly-X-Leu-Gly-Leu-Ala-Leu-Gly-Gly-Leu-Lys-Lys-amide, where the N-terminal is either N(α)-acetylated (Series 1) or contains a free α-amino group (Series 3); and Gly-Gly-Leu-Gly-Gly-Ala-Leu-Gly-X-Leu-Lys-Lys-amide, where the N-terminal is either N(α)-acetylated (Series 2) or contains a free α-amino group (Series 4). In this initial study, the single substitution position, X, was substituted with alkyl side-chains (Ala View Article and Find Full Text PDF

Reversed-phase HPLC of peptides: Assessing column and solvent selectivity on standard, polar-embedded and polar endcapped columns.

We desired to evaluate the chromatographic selectivity for peptides of silica-based RP high-performance liquid chromatography stationary phases with various modifications (polar embedding and polar endcapping on C(18) columns; ether-linked phenyl column with polar endcapping) compared with n-alkyl (C(18), C(8)) and aromatic phenylhexyl columns. Thus, we have designed and synthesized two series of synthetic peptide standards with the sequence Gly-Gly-Leu-Gly-Gly-Ala-Leu-Gly-X-Leu-Lys-Lys-amide, where the N-terminal either contains a free α-amino group (AmC series) or is N(α)-acetylated (AcC series) and where position X is substituted by Gly, Ala, Val, Ile, Phe or Tyr. These represent series of peptides with single substitutions of n-alkyl (Gly View Article and Find Full Text PDF

Intrinsic amino acid side-chain hydrophilicity/hydrophobicity coefficients determined by reversed-phase high-performance liquid chromatography of model peptides: comparison with other hydrophilicity/hydrophobicity scales.

An accurate determination of the intrinsic hydrophilicity/hydrophobicity of amino acid side-chains in peptides and proteins is fundamental in understanding many area of research, including protein folding and stability, peptide and protein function, protein-protein interactions and peptide/protein oligomerization, as well as the design of protocols for purification and characterization of peptides and proteins. Our definition of intrinsic hydrophilicity/hydrophobicity of side-chains is the maximum possible hydrophilicity/hydrophobicity of side-chains in the absence of any nearest-neighbor effects and/or any conformational effects of the polypeptide chain that prevent full expression of side-chain hydrophilicity/hydrophobicity. In this review, we have compared an experimentally derived intrinsic side-chain hydrophilicity/hydrophobicity scale generated from RP-HPLC retention behavior of de novo designed synthetic model peptides at pH 2 and pH 7 with other RP-HPLC-derived scales, as well as scales generated from classic experimental and calculation-based methods of octanol/water partitioning of Nalpha-acetyl-amino-acid amides or free energy of transfer of free amino acids.

Effects of hydrophobicity on the antifungal activity of alpha-helical antimicrobial peptides.

We utilized a series of analogs of D-V13K (a 26-residue amphipathic alpha-helical antimicrobial peptide, denoted D1) to compare and contrast the role of hydrophobicity on antifungal and antibacterial activity to the results obtained previously with Pseudomonas aeruginosa strains. Antifungal activity for zygomycota fungi decreased with increasing hydrophobicity (D-V13K/A12L/A20L/A23L, denoted D4, the most hydrophobic analog was sixfold less active than D1, the least hydrophobic analog). In contrast, antifungal activity for ascomycota fungi increased with increasing hydrophobicity (D4, the most hydrophobic analog was fivefold more active than D1).

Mixed-mode hydrophilic interaction/cation-exchange chromatography (HILIC/CEX) of peptides and proteins.

This review represents a summary of the development and application of a novel mixed-mode HPLC approach to the separation and analysis of peptides and proteins termed hydrophilic interaction/cation-exchange chromatography (HILIC/CEX). This approach combines the most advantageous aspects of two widely different separation mechanisms, i.e.

HPLC analysis and purification of peptides.

High-performance liquid chromatography (HPLC) has proved extremely versatile over the past 25 yr for the isolation and purification of peptides varying widely in their sources, quantity and complexity. This article covers the major modes of HPLC utilized for peptides (size-exclusion, ion-exchange, and reversed-phase), as well as demonstrating the potential of a novel mixed-mode hydrophilic interaction/cation-exchange approach developed in this laboratory. In addition to the value of these HPLC modes for peptide separations, the value of various HPLC techniques for structural characterization of peptides and proteins will be addressed, e.

Mixed-mode hydrophilic interaction/cation-exchange chromatography: separation of complex mixtures of peptides of varying charge and hydrophobicity.

Mixed-mode hydrophilic interaction/cation-exchange chromatography (HILIC/CEX) was applied to the separation of two mixtures of synthetic peptide standards: (i) a 27-peptide mixture containing three groups of peptides (each group containing nine peptides of the same net charge of +1, +2 or +3), where the hydrophilicity/hydrophobicity of adjacent peptides within the groups varied only subtly (generally by only a single carbon atom); and (ii) peptide pairs with the same composition but different sequences, where the sole difference between the peptides was the position of a single amino acid substitution. HILIC/CEX is essentially CEX chromatography in the presence of high levels of organic modifier (generally ACN). The present study demonstrated the dramatic effect of increasing ACN concentration (optimum levels of 60-80%, depending on the application) on the separation of both mixtures of peptides.

Ion-interaction CZE: the presence of high concentrations of ion-pairing reagents demonstrates the complex mechanisms involved in peptide separations.

We have furthered our understanding of the separative mechanism of a novel CE approach, termed ion-interaction CZE (II-CZE), developed in our laboratory for the resolution of mixtures of cationic peptides. Thus, II-CZE and RP-HPLC were applied to the separation of peptides differing by a single amino acid substitution in 10- and 12-residue synthetic model peptide sequences. Substitutions differed by a wide range of properties or side-chain type (e.

Requirements for prediction of peptide retention time in reversed-phase high-performance liquid chromatography: hydrophilicity/hydrophobicity of side-chains at the N- and C-termini of peptides are dramatically affected by the end-groups and location.

The value of reversed-phase high-performance liquid chromatography (RP-HPLC) and the field of proteomics would be greatly enhanced by accurate prediction of retention times of peptides of known composition. The present study investigates the hydrophilicity/hydrophobicity of amino acid side-chains at the N- and C-termini of peptides while varying the functional end-groups at the termini. We substituted all 20 naturally occurring amino acids at the N- and C-termini of a model peptide sequence, where the functional end-groups were N(alpha)-acetyl-X- and N(alpha)-amino-X- at the N-terminus and -X-C(alpha)-carboxyl and -X-C(alpha)-amide at the C-terminus.

Role of peptide hydrophobicity in the mechanism of action of alpha-helical antimicrobial peptides.

In the present study, the 26-residue amphipathic alpha-helical antimicrobial peptide V13KL (Y. Chen et al., J.

Preparative reversed-phase high-performance liquid chromatography collection efficiency for an antimicrobial peptide on columns of varying diameters (1mm to 9.4mm I.D.).

The present study examines the effect of reversed-phase high-performance liquid chromatography (RP-HPLC) column diameter (1mm to 9.4mm I.D.