Peter McPhie

Effect of Dextran on Protein Stability and Conformation Attributed to Macromolecular Crowding

Thermally induced transition curves of hen egg-white lysozyme were measured in the presence of several concentrations of dextran at pH 2.0 by near-UV and far-UV CD. The transition curves were fitted to a two-state model by a non-linear, least-squares method to obtain the transition temperature (T(m)), enthalpy change (deltaH(u)(T(m))), and free energy change (deltaG(u)(T)) of the unfolding transition. An increase in T(m) and almost constant deltaH(u)(T(m)) values were observed in the presence of added dextran at concentrations exceeding ca 100 g l(-1). In addition, dextran-induced conformational changes of fully unfolded protein were investigated by CD spectroscopy. Addition of high concentrations of dextran to solutions of acid-unfolded cytochrome c at pH 2.0 results in a shift of the CD spectrum from that characteristic of the fully unfolded polypeptide to that characteristic of the more compact, salt-induced molten globule state, a result suggesting that the molten globule-like state is stabilized relative to the fully unfolded form in crowded environments. Both observations are in qualitative accord with predictions of a previously proposed model for the effect of intermolecular excluded volume (macromolecular crowding) on protein stability and conformation.

A sequential model of nucleation-dependent protein folding: Kinetic studies of ribonuclease A

A simple sequential model of nucleation-dependent protein folding is presented here, together with some kinetic studies of the reversible thermal unfolding of ribonuclease A. The relevant properties of the simple sequential model are as follows. (1) Nucleation limits the rate of in vitro refolding; (2) there is a definite sequence of steps in the normal pathway of unfolding and refolding; (3) unfolding and refolding are co-operative reactions: the co-operativity depends on the magnitude of the equilibrium constant for nucleation and on the number of steps in folding; (4) by several criteria, unfolding by the simple sequential model approximates a two-state unfolding even when there are sizeable concentrations of stable, partly folded intermediates; and (5) a critical prediction of the simple sequential model (see Appendix) is the approximate division of the kinetics of unfolding into two phases: a rapid transient phase, followed by a slow steady-state unfolding with a single exponential time course. If unfolding is strongly co-operative, the transient phase is predicted to have a small amplitude and to be faster by several orders of magnitude than the steady-state unfolding. The kinetic studies have measured the slow unfolding and refolding reactions of ribonuclease A (—SS— bridges intact) by the change in absorbance at 287 nm, which reflects the exposure to solvent of buried tyrosine groups. The slow reaction follows a single exponential time course at neutral pH, although equilibrium studies by others show significant deviations from two-state behavior in this pH range. This observation can be explained by the model, and provides the chief reason for presenting it. At pH 1.3 some complexity can be detected in the slow unfolding reaction within the first one-third of the thermal transition zone (in agreement with the 1963 results of Scott & Scheraga), although approximate two-state kinetic behavior is found by us in the upper two-thirds of the thermal transition zone. These results are not explained by the simple sequential model, and indicate that more complex models will have to be considered. Equilibrium studies by others have shown a close correspondence to a two-state reaction throughout the thermal transition zone in the pH range 1 to 2.

The functional curli amyloid is not based on in-register parallel beta-sheet structure.

The extracellular curli proteins of Enterobacteriaceae form fibrous structures that are involved in biofilm formation and adhesion to host cells. These curli fibrils are considered a functional amyloid because they are not a consequence of misfolding, but they have many of the properties of protein amyloid. We confirm that fibrils formed by CsgA and CsgB, the primary curli proteins of Escherichia coli, possess many of the hallmarks typical of amyloid. Moreover we demonstrate that curli fibrils possess the cross-β structure that distinguishes protein amyloid. However, solid state NMR experiments indicate that curli structure is not based on an in-register parallel β-sheet architecture, which is common to many human disease-associated amyloids and the yeast prion amyloids. Solid state NMR and electron microscopy data are consistent with a β-helix-like structure but are not sufficient to establish such a structure definitively.

The repeat domain of the melanosome fibril protein Pmel17 forms the amyloid core promoting melanin synthesis

Pmel17 is a melanocyte protein necessary for eumelanin deposition 1 in mammals and found in melanosomes in a filamentous form. The luminal part of human Pmel17 includes a region (RPT) with 10 copies of a partial repeat sequence, pt.e.gttp.qv., known to be essential in vivo for filament formation. We show that this RPT region readily forms amyloid in vitro, but only under the mildly acidic conditions typical of the lysosome-like melanosome lumen, and the filaments quickly become soluble at neutral pH. Under the same mildly acidic conditions, the Pmel filaments promote eumelanin formation. Electron diffraction, circular dichroism, and solid-state NMR studies of Pmel17 filaments show that the structure is rich in beta sheet. We suggest that RPT is the amyloid core domain of the Pmel17 filaments so critical for melanin formation.

Assembly of the Yeast Cell Wall Crh1p AND Crh2p ACT AS TRANSGLYCOSYLASES IN VIVO AND IN VITRO

The cross-linking of polysaccharides to assemble new cell wall in fungi requires mechanisms by which a preexisting linkage is broken for each new one made, to allow for the absence of free energy sources outside the plasma membrane. Previous work showed that Crh1p and Crh2p, putative transglycosylases, are required for the linkage of chitin to β(1–3)glucose branches of β(1–6)glucan in the cell wall of budding yeast. To explore the linking reaction in vivo and in vitro, we used fluorescent sulforhodamine-linked laminari-oligosaccharides as artificial chitin acceptors. In vivo, fluorescence was detected in bud scars and at a lower level in the cell contour, both being dependent on the CRH genes. The linking reaction was also shown in digitonin-permeabilized cells, with UDP-N-acetylglucosamine as the substrate for nascent chitin production. Both the nucleotide and the Crh proteins were required here. A gas1 mutant that overexpresses Crh1p showed very high fluorescence both in intact and permeabilized cells. In the latter, fluorescence was still incorporated in patches in the absence of UDP-GlcNAc. Isolated cell walls of this strain, when incubated with sulforhodamine-oligosaccharide, also showed Crhp-dependent fluorescence in patches, which were identified as bud scars. In all three systems, binding of the fluorescent material to chitin was verified by chitinase digestion. Moreover, the cell wall reaction was inhibited by chitooligosaccharides. These results demonstrate that the Crh proteins act by transferring chitin chains to β(1–6)glucan, with a newly observed high activity in the bud scar. The importance of transglycosylation for cell wall assembly is thus firmly established.

Expression, Purification, and Biochemical Characterization of the Amino-terminal Extracellular Domain of the Human Calcium Receptor

We purified the extracellular domain (ECD) of the human calcium receptor (hCaR) from the medium of HEK-293 cells stably transfected with a hCaR cDNA containing an isoleucine 599 nonsense mutation. A combination of lectin, anion exchange, and gel permeation chromatography yielded milligram quantities of >95% pure protein from 15 liters of starting culture medium. The purified ECD ran as an ∼78-kDa protein on SDS-polyacrylamide gel electrophoresis and was found to be a disulfide-linked dimer. Its NH2-terminal sequence, carbohydrate content, and CD spectrum were defined. Tryptic proteolysis studies showed two major sites accessible to cleavage. These studies provide new insights into the structure of the hCaR ECD. Availability of purified ECD protein should permit further structural studies to help define the mechanism of Ca2+ activation of this G protein-coupled receptor.

ENZYMATIC ASPECTS OF THE PHENOL (ARYL) SULFOTRANSFERASES

The sulfotransferases that are active in the metabolism of xenobiotics represent a large family of enzymes that catalyze the transfer of the sulfuryl group from 3′-phosphoadenosine 5′-phosphosulfate to phenols, to primary and secondary alcohols, to several additional oxygen-containing functional groups, and to amines. Restriction of this review to the catalytic processes of phenol or aryl sulfotransferases does not really narrow the field, because these enzymes have overlapping specificity, not only for specific compounds, but also for multiple functional groups. The presentation aims to provide an overview of the wealth of phenol sulfotransferases that are available for study but concentrates on the enzymology of rat and human enzymes, particularly on the predominant phenol sulfotransferase from rat liver. The kinetics and catalytic mechanism of the rat enzyme is extensively reviewed and is compared with observations from other sulfotransferases.

Purification and partial characterization of an unusual protein of Plasmodium falciparum: histidine-rich protein II

The human malarial parasite Plasmodium falciparum secretes a histidine-rich protein (HRP-II) from infected erythrocytes. HRP-II has a very high content of histidine (H) (34%), alanine (A) (37%) and aspartic acid (D) (10%) and many contiguous repeats of the sequences AHH and AHHAAD. The histidine content of the protein suggested the potential to bind metal ions. We have demonstrated by metal chelate chromatography an extraordinary capacity of HRP-II to bind zinc ions (Zn2+) and employed this characteristic to isolate the extracellular protein. The HRP-II was further purified by antibody affinity chromatography. The identity of the purified protein was verified by relative molecular weight on denaturing polyacrylamide gels, by reactivity with monoclonal antibodies and monospecific rabbit antiserum, and by comparison of the amino-acid analysis with that derived from the cloned gene sequence. Analysis of the sequence for periodicities using the hydrophobic moment method indicated that HRP-II may potentially form a 3/10 helix. Immunoprecipitation of HRP-II from culture supernatants of parasites metabolically labeled with tritiated sugars showed that the extracellular form of HRP-II is a glycoprotein containing galactose.

A turbidimetri milk-clotting assay for pepsin

Abstract The action of pepsin on a dilute solution of skim milk produces a sigmoidal increase in the turbidity of the solution. The time course of the increase depends on the amount of pepsin present. Turbidity changes can be measured in a recording spectrophotometer, providing an automated form of the milk clotting assay, which will easily determine 20 ng of pepsin.

Aegyptin displays high-affinity for the von Willebrand factor binding site (RGQOGVMGF) in collagen and inhibits carotid thrombus formation in vivo.

Aegyptin is a 30 kDa mosquito salivary gland protein that binds to collagen and inhibits platelet aggregation. We have studied the biophysical properties of aegyptin and its mechanism of action. Light‐scattering plot showed that aegyptin has an elongated monomeric form, which explains the apparent molecular mass of 110 kDa estimated by gel‐filtration chromatography. Surface plasmon resonance identified the sequence RGQOGVMGF (where O is hydroxyproline) that mediates collagen interaction with von Willebrand factor (vWF) as a high‐affinity binding site for aegyptin, with a KD of approximately 5 nm. Additionally, aegyptin interacts with the linear peptide RGQPGVMGF and heat‐denatured collagen, indicating that the triple helix and hydroxyproline are not a prerequisite for binding. However, aegyptin does not interact with scrambled RGQPGVMGF peptide. Aegyptin also recognizes the peptides (GPO)10 and GFOGER with low affinity (μm range), which respectively represent glycoprotein VI and integrin α2β1 binding sites in collagen. Truncated forms of aegyptin were engineered, and the C‐terminus fragment was shown to interact with collagen and to attenuate platelet aggregation. In addition, aegyptin prevents laser‐induced carotid thrombus formation in the presence of Rose Bengal in vivo, without significant bleeding in rats. In conclusion, aegyptin interacts with distinct binding sites in collagen, and is useful tool to inhibit platelet–collagen interaction in vitro and in vivo.