Raffaele Ragone

Simultaneous determination of tyrosine and tryptophan residues in proteins by second-derivative spectroscopy

Abstract The mutual interference between the second-derivative bands of tyrosine and tryptophan in proteins has been evaluated in terms of the ratio r between two peak-to-peak distances. The r values have been found to be well related, although not linearly, to the tyrosine/tryptophan ratio in both model compound mixtures and proteins. A method for the simultaneous determination of the two major protein chromophores at neutral pH has been developed.

The human prion protein α2 helix: A thermodynamic study of its conformational preferences

We have synthesized both free and terminally‐blocked peptide corresponding to the second helical region of the globular domain of normal human prion protein, which has recently gained the attention of structural biologists because of a possible role in the nucleation process and fibrillization of prion protein. The profile of the circular dichroism spectrum of the free peptide was that typical of α‐helix, but was converted to that of β‐structure in about 16 h. Instead, below 2.1 × 10−5 M, the spectrum of the blocked peptide exhibited a single band centered at 200 nm, unequivocally associated to random conformations, which did not evolve even after 24 h. Conformational preferences of this last peptide have been investigated as a function of temperature, using trifluoroethanol or low‐concentration sodium dodecyl sulfate as α‐ or β‐structure inducers, respectively. Extrapolation of free energy data to zero concentration of structuring agent highlighted that the peptide prefers α‐helical to β‐type organization, in spite of results from prediction algorithms. However, the free energy difference between the two forms, as obtained by a thermodynamic cycle, is subtle (roughly 5–8 kJ mol−1 at any temperature from 280 K to 350 K), suggesting conformational ambivalence. This result supports the view that, in the prion protein, the structural behavior of the peptide is governed by the cellular microenvironment. Proteins 2005.

Ionic strength effects on the critical micellar concentration of ionic and nonionic surfactants: the binding model.

We have recently investigated the aggregation behavior of zwitterionic n-dodecyl phosphocholine in the presence of high salt. As double logarithmic Corrin-Harkins plots of the critical micellar concentration versus the salt concentration were not linear, here we re-examine those data in the context of the binding model of surfactant aggregation, as previously developed by us for ionic surfactants. We have also re-examined plenty of data available in the literature on the salt-dependent aggregation of neutral surfactants. The use of double-logarithmic plots allowed us to show that the binding model is of general applicability. Indeed, it permits unified treatment of ionic and uncharged aggregation without requiring the introduction of linear terms in the salt concentration, as needed in the empirical Corrin-Harkins treatment of nonionic surfactants. The use of this model could be of help in a broad range of surfactant-based applications in the presence of high salt.

Modification of Job's method for determining the stoichiometry of protein-protein complexes

These reactions can be easily monitored by spectroscopic techniques, which do not require isolating a stable compound, i.e., physically separating bound and free interacting species, particularly when the amount of complex formed can be selectively measured. A typical protein–ligand titration is usually carried out according to the limiting reagent method, using a concentrated ligand solution to avoid excessive dilution. Namely, several small volumes of ligand solution are added to a protein solution of fixed concentration to monitor signal modifications originated from complex formation [1]. However, this procedure is impracticable when both species are proteins, because a very high concentration of either protein to act as titrant can be difficult to obtain. In such cases, it may be useful to resort to Job s [2] method of continuous variation, originally devised to determine the stoichiometry of inorganic complexes. The purpose of this note is to point out that this method can be successfully employed under conditions more favorable than those traditionally adopted to monitor complex formation. In Job s method different amounts of stock solutions of A and B are mixed, varying the mole ratio of reactants in such a compensatory manner that their total molar concentration is kept constant. Under given circumstances, the maximum amount of complex will form in the solution in which the two species are present in the correct combination ratio, provided that the mixing ratio has been varied from 0 to some value certain to be larger than n=m [2–4]. Usually, this is achieved by preparing a series of mixture solutions such that the total moles of reactants and the total volume across the set of solutions are fixed. It is evident that depending on the number of different mixtures prepared this procedure may require considerable amounts of reagents. Considering, however, that the main requisite of Job s method is that the total molar concentration of interacting species be kept constant [2–4], it can be easily verified that it is sufficient to mix different volumes from two stock solutions of the two species, each with the same concentration C0, to make the total concentration equal C0 throughout the experiment. To satisfy this condition it is not necessary that even the volume of the solutions be unchanged while systematically varying the relative amounts of the two species. In other words, using two such solutions, a titration can be carried out in much the same manner as the limiting reagent method, adding increasing aliquots of one species to any volume of the other species, regardless of the total volume. Incidentally, such a procedure allows us to save some material. As an example, we report here experimental details on the binding between basic fibroblast growth factor (bFGF) and BB homodimer of platelet-derived growth factor (PDGF-BB), as monitored by fluorescence spectroscopy. Concerning this aspect, it is opportune to consider that the primary restraint in the application of Job s method is that the signal used to detect complex formation is suitable to measure the amount of product formed. If, for example, one or both reactants also make Analytical Biochemistry 313 (2003) 170–172

Homocystine solubility and vascular disease

There is evidence that mild elevations of tHcy are associated with an increased risk for occlusive vascular disease, thrombosis, and stroke. It is hypothesized here that cellular toxicity could indirectly result from auto‐oxidation of homocysteine to homocystine. Elevated levels of total plasma homocysteine could be the primary cause of increased vascular risk, causing endothelial damage through a mechanism similar to that of cystine precipitation, which is known to cause stone formation in cystinosis and cystinuria. In fact, only traces of homocysteine circulate in plasma as the free thiol; the remainder is present as oxidation products. Of these, the symmetric disulfide homocystine is scarcely soluble at neutral pH. Its saturation limit is so close to the concentration of homocysteine in normal plasma that a transient increase of homocysteine levels could lead to precipitation of homocystine microcrystals in the bloodstream. These could damage endothelial tissue, acting as a mechanic primer for subsequent prothrombotic blood vessel alterations.—Ragone, R, Homocystine solubility and vascular disease. FASEB J. 16, 401–404 (2002)

The prion protein: structural features and related toxic peptides

Prion diseases are characterized by the conversion of the physiological cellular form of the prion protein (PrPC) into an insoluble, partially protease‐resistant abnormal scrapie form (PrPSc). PrPC is normally expressed in mammalian cell and is highly conserved among species, although its role in cellular function remains elusive. The conversion of PrPC to PrPSc parallels a conformational change of the polypeptide from a predominantly α‐helical to a highly β‐sheet secondary structure. The pathogenesis and molecular basis of the consequent nerve cell loss are not understood. Limited structural information is available on aggregate formation by this protein as the possible cause of these diseases and on its toxicity. This brief overview focuses on the large amount of structure‐activity studies based on the prion fragment approach, hinging on peptides derived from the unstructured N‐terminal and globular C‐terminal domains. It is well documented that most of the fragments with regular secondary structure, with the exception of helices 1 and 3, possess a high β‐sheet propensity and tendency to form β‐sheet‐like aggregates. In this context, helix 2 plays a crucial role because it is able to adopt both misfolded and partially helical conformation. However, only a few mutants are able to display its intrinsic neurotoxicity.

How the protein concentration affects unfolding curves of oligomers

The position of unfolding curves of oligomeric proteins depends on the protein concentration. The extent of this dependence is analyzed here in terms of the midpoint concentration, i.e., the denaturant concentration at which the fractions of folded and unfolded protein are equal. Reexamination of published data highlights that the midpoint concentration decreases as the protein concentration becomes lower, as expected. Moreover, there are differences between urea and guanidine hydrochloride, as well as discrepancies between the linear extrapolation model and the denaturant binding model. These discrepancies could be used to choose the denaturation model that best fits experimental data. The equations used can be applied to any oligomeric system to check the validity of the two-state assumption.

Conformational Diseases and Structure-Toxicity Relationships: Lessons from Prion-Derived Peptides

The physiological form of the prion protein is normally expressed in mammalian cell and is highly conserved among species, although its role in cellular function remains elusive. Available evidence suggests that this protein is essential for neuronal integrity in the brain, possibly with a role in copper metabolism and cellular response to oxidative stress. In prion diseases, the benign cellular form of the protein is converted into an insoluble, protease-resistant abnormal scrapie form. This conversion parallels a conformational change of the polypeptide from a predominantly alpha-helical to a highly beta-sheet secondary structure. The scrapie form accumulates in the central nervous system of affected individuals, and its protease-resistant core aggregates into amyloid fibrils outside the cell. The pathogenesis and molecular basis of the nerve cell loss that accompanies this process are not understood. Limited structural information is available on aggregate formation by this protein as the possible cause of these diseases and on its toxicity. A large amount of structure-activity studies is based on the prion fragment approach, but the resulting information is often difficult to untangle. This overview focuses on the most relevant structural and functional aspects of the prion-induced conformational disease linked to peptides derived from the unstructured N-terminal and globular C-terminal domains.

NMR structure and CD titration with metal cations of human prion alpha2-helix-related peptides.

The 173–195 segment corresponding to the helix 2 of the C-globular prion protein domain could be one of several “spots” of intrinsic conformational flexibility. In fact, it possesses chameleon conformational behaviour and gathers several disease-associated point mutations. We have performed spectroscopic studies on the wild-type fragment 173–195 and on its D178N mutant dissolved in trifluoroethanol to mimic the in vivo system, both in the presence and in the absence of metal cations. NMR data showed that the structure of the D178N mutant is characterized by two short helices separated by a kink, whereas the wild-type peptide is fully helical. Both peptides retained these structural organizations, as monitored by CD, in the presence of metal cations. NMR spectra were however not in favour of the formation of definite ion-peptide complexes. This agrees with previous evidence that other regions of the prion protein are likely the natural target of metal cation binding.

INTERACTION OF TRYPTOPHAN AND N-ACETYLTRYPTOPHANAMIDE WITH DODECYLPENTAOXYETHYLENEGLYCOL ETHER MICELLES

Abstract Through the fluorimetric determination of the critical micellar concentration of dodecylpentaoxyethyleneglycol ether and sodium dodecylsulfate, we have investigated the interaction of the zwitterionic tryptophan and its uncharged analogue N-acetyl- l -tryptophanamide with micelles in terms of binding constants. This could be useful to monitor protein interaction with different membrane-like environments using the intrinsic tryptophan fluorescence. The weakest interaction took place between tryptophan and sodium dodecylsulfate, while the strongest one occurred between N-acetyl- l -tryptophanamide and dodecylpentaoxyethyleneglycol ether. This suggests that micelle head groups control the interaction, but the polar moiety bringing the fluorophore also seems to play a role. The unexpected lack of interaction between tryptophan and dodecylpentaoxyethyleneglycol ether is also discussed.