James M. Kovacs

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 Nα‐acetyl‐amino‐acid amides or free energy of transfer of free amino acids. Generally poor correlation was found with previous RP‐HPLC‐derived scales, likely due to the random nature of the peptide mixtures in terms of varying peptide size, conformation and frequency of particular amino acids. In addition, generally poor correlation with the classical approaches served to underline the importance of the presence of a polypeptide backbone when generating intrinsic values. We have shown that the intrinsic scale determined here is in full agreement with the structural characteristics of amino acid side‐chains.

Detection of complement activation using monoclonal antibodies against C3d

During complement activation the C3 protein is cleaved, and C3 activation fragments are covalently fixed to tissues. Tissue-bound C3 fragments are a durable biomarker of tissue inflammation, and these fragments have been exploited as addressable binding ligands for targeted therapeutics and diagnostic agents. We have generated cross-reactive murine monoclonal antibodies against human and mouse C3d, the final C3 degradation fragment generated during complement activation. We developed 3 monoclonal antibodies (3d8b, 3d9a, and 3d29) that preferentially bind to the iC3b, C3dg, and C3d fragments in solution, but do not bind to intact C3 or C3b. The same 3 clones also bind to tissue-bound C3 activation fragments when injected systemically. Using mouse models of renal and ocular disease, we confirmed that, following systemic injection, the antibodies accumulated at sites of C3 fragment deposition within the glomerulus, the renal tubulointerstitium, and the posterior pole of the eye. To detect antibodies bound within the eye, we used optical imaging and observed accumulation of the antibodies within retinal lesions in a model of choroidal neovascularization (CNV). Our results demonstrate that imaging methods that use these antibodies may provide a sensitive means of detecting and monitoring complement activation-associated tissue inflammation.

Molecular Mechanism of Membrane Docking by the Vam7p PX Domain

The Vam7p t-SNARE is an essential component of the vacuole fusion machinery that mediates membrane trafficking and protein sorting in yeast. Vam7p is recruited to vacuoles by its N-terminal PX domain that specifically recognizes PtdIns(3)P in the bilayers, however the precise mechanism of membrane anchoring remains unclear. Here we describe a molecular basis for membrane targeting and penetration by the Vam7p PX domain based on structural and quantitative analysis of its interactions with lipids and micelles. Our results derived from in vitro binding measurements using NMR, monolayer surface tension experiments and mutagenesis reveal a multivalent membrane docking mechanism involving specific PtdIns(3)P recognition that is facilitated by electrostatic interactions and accompanying hydrophobic insertion. Both the hydrophobic and electrostatic components enhance the Vam7p PX domain association with PtdIns(3)P-containing membranes. The inserting Val70, Leu71, and Trp75 residues located next to the PtdIns(3)P binding pocket are surrounded by a basic patch, which is involved in nonspecific electrostatic contacts with acidic lipids, such as PtdSer. Substitution of the insertion residues significantly reduces the binding and penetrating power of the Vam7p PX domain and leads to cytoplasmic redistribution of the EGFP-tagged protein. The affinities of the PX domain for PtdIns(3)P and other lipids reveal a remarkable synergy within the multivalent complex that stably anchors Vam7p at the vacuolar membrane.

Mapping of the C3d Ligand Binding Site on Complement Receptor 2 (CR2/CD21) Using Nuclear Magnetic Resonance and Chemical Shift Analysis

Complement receptor 2 (CR2, CD21) is a cell membrane protein, with 15 or 16 extracellular short consensus repeats (SCRs), that promotes B lymphocyte responses and bridges innate and acquired immunity. The most distally located SCRs (SCR1-2) mediate the interaction of CR2 with its four known ligands (C3d, Epstein-Barr virus gp350, interferon-α, and CD23). Inhibitory monoclonal antibodies against SCR1-2 block binding of all ligands. To develop ligand-specific inhibitors that would also assist in identifying residues unique to each receptor-ligand interaction, phage were selected from randomly generated libraries by panning with recombinant SCR1-2, followed by specific ligand-driven elution. Derived peptides were tested by competition ELISA. One peptide, C3dp1 (APQHLSSQYSRT) exhibited ligand-specific inhibition at midmicromolar IC50. C3d was titrated into 15N-labeled SCR1-2, which revealed chemical shift changes indicative of specific intermolecular interactions. With backbone assignments made, the chemical shift changes were mapped onto the crystal structure of SCR1-2. With regard to C3d, the binding surface includes regions of SCR1, SCR2, and the inter-SCR linker, specifically residues Arg13, Tyr16, Arg28, Tyr29, Ser32, Thr34, Lys48, Asp56, Lys57, Tyr68, Arg83, Gly84, Asn101, Asn105, and Ser109. SCR1 and SCR2 demonstrated distinct binding modes. The CR2 binding surface incorporating SCR1 is inconsistent with a previous x-ray CR2-C3d co-crystal analysis but consistent with mutagenesis, x-ray neutron scattering, and inhibitory monoclonal antibody epitope mapping. Titration with C3dp1 yielded chemical shift changes (Arg13, Tyr16, Thr34, Lys48, Asp56, Lys57, Tyr68, Arg83, Gly84, Asn105, and Ser109) overlapping with C3d, indicating that C3dp1 interacts at the same CR2 site as C3d.

Biophysical Investigations of Complement Receptor 2 (CD21 and CR2)-Ligand Interactions Reveal Amino Acid Contacts Unique to Each Receptor-Ligand Pair

Human complement receptor type 2 (CR2 and CD21) is a cell membrane receptor, with 15 or 16 extracellular short consensus repeats (SCRs), that promotes B lymphocyte responses and bridges innate and acquired immunity. The most distally located SCRs, SCR1–2, mediate the interaction of CR2 with its four known ligands (C3d, EBV gp350, IFNα, and CD23). To ascertain specific interacting residues on CR2, we utilized NMR studies wherein gp350 and IFNα were titrated into 15N-labeled SCR1–2, and chemical shift changes indicative of specific inter-molecular interactions were identified. With backbone assignments made, the chemical shift changes were mapped onto the crystal structure of SCR1–2. With regard to gp350, the binding region of CR2 is primarily focused on SCR1 and the inter-SCR linker, specifically residues Asn11, Arg13, Ala22, Arg28, Ser32, Arg36, Lys41, Lys57, Tyr64, Lys67, Tyr68, Arg83, Gly84, and Arg89. With regard to IFNα, the binding is similar to the CR2-C3d interaction with specific residues being Arg13, Tyr16, Arg28, Ser42, Lys48, Lys50, Tyr68, Arg83, Gly84, and Arg89. We also report thermodynamic properties of each ligand-receptor pair determined using isothermal titration calorimetry. The CR2-C3d interaction was characterized as a two-mode binding interaction with Kd values of 0.13 and 160 μm, whereas the CR2-gp350 and CR2-IFNα interactions were characterized as single site binding events with affinities of 0.014 and 0.035 μm, respectively. The compilation of chemical binding maps suggests specific residues on CR2 that are uniquely important in each of these three binding interactions.

HPLC analysis and purification of peptides.

Summary 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.g., assessment of oligomerization state of peptides/proteins by size-exclusion chromatography and monitoring the hydrophilicity/hydrophobicity of amphipathic α-helical peptides, a vital precursor for the development of novel antimicrobial peptides. The value of capillary electrophoresis for peptide separations is also demonstrated. Preparative reversed-phase chromatography purification protocols for sample loads of up to 200 mg on analytical columns and instrumentation are introduced for both peptides and recombinant proteins.