Peter B. Crowley

Cation–π interactions in protein–protein interfaces

Arginine is an abundant residue in protein–protein interfaces. The importance of this residue relates to the versatility of its side chain in intermolecular interactions. Different classes of protein–protein interfaces were surveyed for cation–π interactions. Approximately half of the protein complexes and one‐third of the homodimers analyzed were found to contain at least one intermolecular cation–π pair. Interactions between arginine and tyrosine were found to be the most abundant. The electrostatic interaction energy was calculated to be ∼3 kcal/mol, on average. A distance‐based search of guanidinium:aromatic interactions was also performed using the Macromolecular Structure Database (MSD). This search revealed that half of the guanidinium:aromatic pairs pack in a coplanar manner. Furthermore, it was found that the cationic group of the cation–π pair is frequently involved in intermolecular hydrogen bonds. In this manner the arginine side chain can participate in multiple interactions, providing a mechanism for inter‐protein specificity. Thus, the cation–π interaction is established as an important contributor to protein–protein interfaces. Proteins 2005.

Physicochemical Properties of Cells and Their Effects on Intrinsically Disordered Proteins (IDPs)

It has long been axiomatic that a protein’s structure determines its function. Intrinsically disordered proteins (IDPs) and disordered protein regions (IDRs) defy this structure–function paradigm. They do not exhibit stable secondary and/or tertiary structures and exist as dynamic ensembles of interconverting conformers with preferred, nonrandom orientations.1−4 The concept of IDPs and IDRs as functional biological units was initially met with skepticism. For a long time, disorder, intuitively implying chaos, had no place in our perception of orchestrated molecular events controlling cell biology. Over the past years, however, this notion has changed. Aided by findings that structural disorder constitutes an ubiquitous and abundant biological phenomenon in organisms of all phyla,5−7 and that it is often synonymous with function,8−11 disorder has become an integral part of modern protein biochemistry. Disorder thrives in eukaryotic signaling pathways12 and functions as a prominent player in many regulatory processes.13−15 Disordered proteins and protein regions determine the underlying causes of many neurodegenerative disorders and constitute the main components of amyloid fibrils.16 They further contribute to many forms of cancer, diabetes and to cardiovascular and metabolic diseases.17,18 Research into disordered proteins produced significant findings and established important new concepts. On the structural side, novel experimental and computational approaches identified and described disordered protein ensembles3,19,20 and led to terms such as secondary structure propensities, residual structural features, and transient long-range contacts.1,21 The discovery of coupled folding-and-binding reactions defined the paradigm of disorder-to-order transitions22 and high-resolution insights into the architectures of amyloid fibrils were obtained.23,24 On the biological side, we learned about the unexpected intracellular stability of disordered proteins, their roles in integrating post-translational protein modifications in cell signaling and about their functions in regulatory processes ranging from transcription to cell fate decisions.15,25,26 One open question remaining to be addressed is how these in vitro structural insights relate to biological in vivo effects. How do complex intracellular environments modulate the in vivo properties of disordered proteins and what are the implications for their biological functions (Figure ​(Figure11)?27−29 Figure 1 Intracellular complexity. (A) Left: Cryo-electron tomography slice of a mammalian cell. Middle: Close-up view of cellular structures colored according to their identities: Right: Three-dimensional surface representation of the same region. Yellow, endoplasmic ...

Protein camouflage in cytochrome c–calixarene complexes

Small molecules that recognize protein surfaces are important tools for modifying protein interaction properties. Since the 1980s, several thousand studies concerning calixarenes and host–guest interactions have been published. Although there is growing interest in protein–calixarene interactions, only limited structural information has been available to date. We now report the crystal structure of a protein–calixarene complex. The water-soluble p-sulfonatocalix[4]arene is shown to bind the lysine-rich cytochrome c at three different sites. Binding curves obtained from NMR titrations reveal an interaction process that involves two or more binding sites. Together, the data indicate a dynamic complex in which the calixarene explores the surface of cytochrome c. In addition to providing valuable information on protein recognition, the data also indicate that the calixarene is a mediator of protein–protein interactions, with potential applications in generating assemblies and promoting crystallization. A calixarene–protein host–guest complex has been characterized in detail by using a combination of NMR spectroscopy and X-ray crystallography. The water-soluble sulfonato-calix[4]arene binds to cytochrome c at various lysine residues to yield a dynamic complex. This interaction may serve to facilitate crystallization by mediating protein–protein contacts.

The architecture of the binding site in redox protein complexes: Implications for fast dissociation

Interprotein electron transfer is characterized by protein interactions on the millisecond time scale. Such transient encounters are ensured by extremely high rates of complex dissociation. Computational analysis of the available crystal structures of redox protein complexes reveals features of the binding site that favor fast dissociation. In particular, the complex interface is shown to have low geometric complementarity and poor packing. These features are consistent with the necessity for fast dissociation since the absence of close packing facilitates solvation of the interface and disruption of the complex. Proteins 2004;55:000–000.

Influence of Additives and Mixing Time on Crumb Grain Characteristics of Wheat Bread

ABSTRACT The effect of additives and processing parameters on wheat bread were investigated objectively using image analysis (IA). Five different bread types were produced by varying the ingredients (standard, standard with fat, standard with emulsifiers) and changing the mixing times (90, 150, and 240 sec). A digital IA system for wheat bread was developed from generic commercial software. The system yielded reproducible results for a variety of bread crumb grain image features. Bread slices were scanned and evaluated using the IA system. Image characteristics were determined for each bread type. All data was statistically evaluated to detect significant differences between bread types. It was shown quantitatively that inclusion of fat or emulsifiers or extension of mixing time had a significant effect on crumb grain features such as mean cell area, total cell area, and number of cells/cm2. The five bread types could be distinguished solely by crumb grain characteristics.

Protein interactions in the Escherichia coli cytosol: an impediment to in-cell NMR spectroscopy.

Protein science is shifting towards experiments performed under native or native‐like conditions. In‐cell NMR spectroscopy for instance has the potential to reveal protein structure and dynamics inside cells. However, not all proteins can be studied by this technique. 15N‐labelled cytochrome c (cyt c) over‐expressed in Escherichia coli was undetectable by in‐cell NMR spectroscopy. When whole‐cell lysates were subjected to size‐exclusion chromatography (SEC) cyt c was found to elute with an apparent molecular weight of >150 kDa. The presence of high molecular weight species is indicative of complex formation between cyt c and E. coli cytosolic proteins. These interactions were disrupted by charge‐inverted mutants in cyt c and by elevated concentrations of NaCl. The physiologically relevant salt, KGlu, was less efficient at disrupting complex formation. Notably, a triple mutant of cyt c could be detected in cell lysates by NMR spectroscopy. The protein, GB1, yields high quality in‐cell spectra and SEC analysis of lysates containing GB1 revealed a lack of interaction between GB1 and E. coli proteins. Together these data suggest that protein “stickiness” is a limiting factor in the application of in‐cell NMR spectroscopy.

NMR spectroscopy reveals cytochrome c-poly(ethylene glycol) interactions.

In vitro protein studies are typically performed on samples that are composed almost entirely of water. However, the cell interior is a heterogeneous “crowded” solution of small molecules, proteins, nucleic acids and membranes. At a concentration of 300–400 gL , the macromolecular content of the cell influences the kinetics and thermodynamics of protein folding, ligand binding and protein–protein interactions through excluded volume effects. Therefore, in order to build realistic models of protein structure and function, it is necessary to study proteins in vivo or under “crowded” conditions that mimic the cellular environment. The necessity for in vivo protein characterisation is being addressed by the development of in-cell NMR spectroscopy. While “biologically inert” proteins are largely unaffected by the crowded cell interior, the disordered protein FlgM was shown to gain structure inside Escherichia coli cells. A similar gain in structure occurred in vitro in the presence of crowding agents. Artificially crowded environments can be created by using sugars, proteins or polymers such as Ficoll, dextran and poly(ethylene glycol) (PEG). Such sample conditions are accessible by NMR spectroscopy, and the effects of macromolecular crowding on protein structure and dynamics have been investigated. Related NMR studies of macromolecular confinement have been performed by using polyacrylamide gels, reverse micelles, sol–gels and agarose gels. Generally, crowding/confinement tends to accelerate protein folding, promotes self-association and stabilises protein structure. 5,8–13] Given that macromolecular crowding can enhance protein association, the use of crowding agents is likely to facilitate the structural characterisation of weak protein interactions. We are interested in using NMR spectroscopy to study the effects of macromolecular crowding on the transient interactions of redox proteins. Saccharomyces cerevisiae cytochrome c (cyt c) in PEG-containing solutions was chosen for initial studies. PEG– protein interactions are usually repulsive, and volume exclusion results in preferential hydration of the protein surface. The repulsive interactions can be reduced by minimising (through conformation changes, precipitation or crystallisation) the protein surface area exposed to the solvent. The effect of PEG on protein solutions is not limited to volume exclusion. Although highly water soluble, PEG is hydrophobic in nature and can interact with hydrophobic proteins. An interesting example of this type of interaction is found in the crystal structure of the odorant-binding protein from Anopheles, in which a hydrophobic channel is occupied by a PEG molecule (PDB code: 2erb). The study of protein–PEG mixtures is further underlined by the growing importance of PEGylated-protein therapeutics. When modified by the covalent attachment of a PEG chain, proteins are less susceptible to proteolysis and have reduced immunogenicity. We report here the interaction of cyt c with PEG as revealed by H,N correlation spectroscopy. For comparison, experiments were performed on cyt c embedded in agarose gels. Similar PEG-induced effects were observed for both reduced and oxidised cyt c, and therefore this report focuses on the ACHTUNGTRENNUNGresults for reduced cyt c.N-labelled cyt c was studied in the presence of different sizes and concentrations of PEG. Samples containing up to 300 gL 1 of PEG were used to mimic the ACHTUNGTRENNUNGintracellular macromolecular content. Figure 1A illustrates a region of the H,N correlation spectrum of cyt c and the spectral changes associated with the presence of increasing concentrations of PEG 8000. The majority of cyt c resonances demonstrated small changes in line width, increasing on average by 25–35% at 200 and 300 gL 1 PEG. Compared to the approximately twofold line-width increases for cyt c bound to cyt c peroxidase, and cyt b5 encapsulated in sol–gel, [14] this indicates that the rotational correlation time (tc) of cyt c is weakly influenced by PEG. Resonance broadening was greater for a number of amides found in flexible loops, including Gly34, which was broadened beyond detection. Considering that loops are prone to conformation changes, the resonance broadening suggests that, in the presence of PEG, the exchange between different conformations is slow on the NMR timescale. In addition to line broadening, concentration-dependent chemical-shift perturbations of the order of 0.1 (H) and 0.3 (N) ppm were observed. Figure S1 in the Supporting Information gives a plot of the averaged H and N shifts for each backbone amide. Mapping these perturbations onto the crystal structure of cyt c reveals that the majority of the shifts surround the exposed haem edge (Figure 1B) with Gln16 and Lys79 standing out as most strongly affected. Note that Lys79 lies flat on the protein surface and thus contributes to the hydrophobic patch around the haem (Figure S2). Similar results were found for PEG 3350, 8000 and 20000; this indicates that the molecular weight of PEG does not affect its propensity to bind cyt c. Surprisingly, the chemical-shift-perturbation map of cyt c in the presence of PEG is qualitatively similar to the binding maps for cyt c in complex with cyt c peroxidase, and the nonphysiological partner cyt f. In particular, the down-field [a] Dr. P. B. Crowley, K. Brett UCD School of Biomolecular and Biomedical Science Conway Institute, University College Dublin Belfield, Dublin 4 (Ireland) Fax: (+ 353) 1-716 6701 E-mail : peter.crowley@ucd.ie [b] Dr. J. Muldoon UCD Centre for Synthesis and Chemical Biology University College Dublin Belfield, Dublin 4 (Ireland) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.

Co-operative ortho-effects on the Wittig reaction. Interpretation of stereoselectivity in the reaction of ortho -halo-substituted benzaldehydes and benzylidenetriphenylphosphoranes

Abstract The E/Z ratios of the stilbenes 1 formed in the Wittig reaction of ortho -halo substituted benzyltriphenylphosphonium salts 2 and benzaldehydes 3 were determined. It was found that there is a co-operative effect of one ortho -halo group on each of the two reacting partners which increases Z -selectivity, but two such groups on the same reactant gives high E -selectivity. The effects are strong enough to be preparatively significant in certain cases and can be interpreted within the modern framework of the Wittig mechanism established by Vedejs and co-workers.

Structure of a PEGylated protein reveals a highly porous double-helical assembly

PEGylated proteins are a mainstay of the biopharmaceutical industry. Although the use of poly(ethylene glycol) (PEG) to increase particle size, stability and solubility is well-established, questions remain as to the structure of PEG-protein conjugates. Here we report the structural characterization of a model β-sheet protein (plastocyanin, 11.5 kDa) modified with a single PEG 5,000. An NMR spectroscopy study of the PEGylated conjugate indicated that the protein and PEG behaved as independent domains. A crystal structure revealed an extraordinary double-helical assembly of the conjugate, with the helices arranged orthogonally to yield a highly porous architecture. Electron density was not observed for the PEG chain, which indicates that it was disordered. The volume available per PEG chain in the crystal was within 10% of the calculated random coil volume. Together, these data support a minimal interaction between the protein and the synthetic polymer. Our work provides new possibilities for understanding this important class of protein-polymer hybrids and suggests a novel approach to engineering protein assemblies.

The Ternary Complex of Cytochrome f and Cytochrome c: Identification of a Second Binding Site and Competition for Plastocyanin Binding

The complex of yeast cytochrome c and cytochrome f from the cyanobacterium Phormidium laminosum was investigated by NMR spectroscopy. Chemical shift perturbation analysis reveals that residues around the haem edge of cytochrome c are involved in the complex interface. Binding curves derived from an NMR spectroscopy titration at 10 mM ionic strength indicate that there are two sites for cytochrome c with binding constants of approximately 2×104 M−1 and 4×103 M−1. A protein docking simulation with NMR‐derived constraints identifies two sites, at the front (Site I) and back faces (Site II) of the haem region of cytochrome f. Site I is homologous to the binding site previously determined for the natural cytochrome f partner plastocyanin. Site II may represent the binding site for the Rieske protein in the cytochrome bf complex. Cytochrome c and plastocyanin are shown to compete for binding at Site I. The competition appears to involve electrostatic screening rather than simple steric occlusion of the binding site.