Search

We have exploited the capability of in-cell NMR to selectively observe flexible regions within folded proteins to carry out a comparative study of two members of the highly conserved frataxin family which are found both in prokaryotes and in eukaryotes. They all contain a globular domain which shares more than 50% identity, which in eukaryotes is preceded by an N-terminal tail containing the mitochondrial import signal. We demonstrate that the NMR spectrum of the bacterial ortholog CyaY cannot be observed in the homologous E. coli system, although it becomes fully observable as soon as the cells are lysed. This behavior has been observed for several other compact globular proteins as seems to be the rule rather than the exception. The NMR spectrum of the yeast ortholog Yfh1 contains instead visible signals from the protein. We demonstrate that they correspond to the flexible N-terminal tail indicating that this is flexible and unfolded. This flexibility of the N-terminus agrees with previous studies of human frataxin, despite the extensive sequence diversity of this region in the two proteins. Interestingly, the residues that we observe in in-cell experiments are not visible in the crystal structure of a Yfh1 mutant designed to destabilize the first helix. More importantly, our results show that, in cell, the protein is predominantly present not as an aggregate but as a monomeric species.

In-cell NMR is a technique developed to study the structure and dynamical behavior of biological macromolecules in their natural environment, circumventing all isolation and purification steps. In principle, the potentialities of the technique are enormous, not only for the possibility of bypassing all purification steps but, even more importantly, for the wealth of information that can be gained from directly monitoring interactions among biological macromolecules in a natural cell. Here, we review critically the promises, successes and limits of this technique as it stands now. Interestingly, many of the problems of NMR in bacterial cells stem from the artificially high concentration of the protein under study whose overexpression is anyway necessary to select it from the background. This has, as a consequence, that when overexpressed, most globular proteins, do not show an NMR spectrum, limiting the applicability of the technique to intrinsically unfolded or specifically behaving proteins. The outlook for in-cell NMR of eukaryotic cells is more promising and is possibly the most attracting aspect for the future.

Protein stability is an important issue for the interpretation of a wide variety of biological problems but its assessment is at times difficult. The most common parameter employed to describe protein stability is the temperature of melting, at which the populations of folded and unfolded species are identical. This parameter may yield ambiguous results. It would always be preferable to measure the whole stability curve. The calculation of this curve is greatly facilitated whenever it is possible to observe cold denaturation. Using Yfh1, one of the few proteins whose cold denaturation occurs at neutral pH and low ionic strength, we could measure the variation of its full stability curve under several environmental conditions. Here we show the advantages of gauging stability as a function of external variables using stability curves.

In vitro studies of biological macromolecules are usually performed in dilute, buffered solutions containing one or just a few different biological macromolecules. Under these conditions, the interactions among molecules are diffusion limited. On the contrary, in living systems, macromolecules of a given type are surrounded by many others, at very high total concentrations. In the last few years, there has been an increasing effort to study biological macromolecules directly in natural crowded environments, as in intact bacterial cells or by mimicking natural crowding by adding proteins, polysaccharides, or even synthetic polymers. Here, we propose the use of hen egg white (HEW) as a simple natural medium, with all features of the media of crowded cells, that could be used by any researcher without difficulty and inexpensively. We present a study of the stability and dynamics behavior of model proteins in HEW, chosen as a prototypical, readily accessible natural medium that can mimic cytosol. We show that two typical globular proteins, dissolved in HEW, give NMR spectra very similar to those obtained in dilute buffers, although dynamic parameters are clearly affected by the crowded medium. The thermal stability of one of these proteins, measured in a range comprising both heat and cold denaturation, is also similar to that in buffer. Our data open new possibilities to the study of proteins in natural crowded media. Proteins 2011. © 2010 Wiley-Liss, Inc.

Understanding the factors that determine protein stability is interesting because it directly reflects the evolutionary pressure coming from function and environment. Here, we have combined experimental and computational methods to study the stability of IscU, a bacterial scaffold protein highly conserved in most organisms and an essential component of the iron-sulfur cluster biogenesis pathway. We demonstrate that the effect of zinc and its consequence strongly depend on the sample history. IscU is a marginally stable protein at low ionic strength to the point that undergoes cold denaturation at around -8°C with a corresponding dramatic decrease of enthalpy, which is consistent with the fluxional nature of the protein. Presence of constitutively bound zinc appreciably stabilizes the IscU fold, whereas it may cause protein aggregation when zinc is added back posthumously. We discuss how zinc coordination can be achieved by different side chains spatially available and all competent for tetrahedral coordination. The individual absence of some of these residues can be largely compensated by small local rearrangements of the others. We discuss the potential importance of our findings in vitro for the function in vivo of the protein.

Although alcohols are well known to be protein denaturants when present at high concentrations, their effect on proteins at low concentrations is much less well characterized. In this paper, we present a study of the effects of alcohols on protein stability using Yfh1, the yeast ortholog of the human protein frataxin. Exploiting the unusual property of this protein of undergoing cold denaturation around 0 °C without any ad hoc destabilization, we determined the stability curve on the basis of both high and low temperature unfolding in the presence of three commonly used alcohols: trifluoroethanol, ethanol and methanol. In all cases, we observed an extended temperature range of protein stability as determined by a modest increase of the high temperature of unfolding but an appreciable decrease in the low temperature of unfolding. Based on simple thermodynamics considerations, we are able to interpret the literature on the effects of alcohols on proteins and to generalise our findings. We suggest that alcohols, at low concentration and physiological pH, stabilize proteins by greatly widening the range of temperatures over which the protein is stable. Our results also clarify the molecular mechanism of the interaction and validate the current theoretical interpretation of the mechanism of cold denaturation.

Although alcohols are well-known to be protein denaturants when present at high concentrations, their effect on proteins at low concentrations is much less well characterized. In this paper, we present a study of the effects of alcohols on protein stability using Yfh1, the yeast ortholog of the human protein frataxin. Exploiting the unusual property of this protein of undergoing cold denaturation around 0 degrees C without any ad hoc destabilization, we determined the stability curve on the basis of both high and low temperature unfolding in the presence of three commonly used alcohols: trifluoroethanol, ethanol, and methanol. In all cases, we observed an extended temperature range of protein stability as determined by a modest increase of the high temperature of unfolding but an appreciable decrease in the low temperature of unfolding. On the basis of simple thermodynamic considerations, we are able to interpret the literature on the effects of alcohols on proteins and to generalize our findings. We suggest that alcohols, at low concentration and physiological pH, stabilize proteins by greatly widening the range of temperatures over which the protein is stable. Our results also clarify the molecular mechanism of the interaction and validate the current theoretical interpretation of the mechanism of cold denaturation.

An increasing family of neurodegenerative disorders such as Alzheimer's, Parkinson's and Huntington's diseases, prion encephalopathies and cystic fibrosis is associated with aggregation of misfolded polypeptide chains which are toxic to the cell. Knowledge of the three-dimensional structure of the proteins implicated is essential for understanding why and how endogenous proteins may adopt a non-native fold. Yet, structural work has been hampered by the difficulty of handling proteins insoluble or prone to aggregation, and at the same time that is why it is interesting to study these molecules. In this review, we compare the structural knowledge accumulated for two paradigmatic misfolding disorders, Alzheimer's disease (AD) and the family of poly-glutamine diseases (poly-Q) and discuss some of the hypotheses suggested for explaining aggregate formation. While a common mechanism between these pathologies remains to be proven, a direct comparison may help in designing new strategies for approaching their study.

The sweet protein MNEI is a construct of 96 amino acid residues engineered by linking, with a Gly-Phe dipeptide, chains B and A of monellin, a sweet protein isolated from Discoreophyllum cuminsii. Here, the solution structure of MNEI was determined on the basis of 1169 nuclear Overhauser enhancement derived distance restraints and 184 dihedral angle restraints obtained from direct measurement of three-bond spin coupling constants. The identification of hydrogen bonded NH groups was obtained by a combination of H/(2)H exchange data and NH resonance temperature coefficients derived from a series of HSQC spectra in the temperature range 278-328 K. The good resolution of the structure is reflected by the Z-score of the quality checking program in WHAT IF (-0.61). The topology of MNEI, like that of natural monellin and of SCM, another single-chain monellin, is typical of the cystatin superfamily: an alpha-helix cradled into the concave side of a five-strand anti-parallel beta-sheet. The high resolution (14 restraints/residue) 3D structure of MNEI shows close similarity to the crystal structures of natural monellin and of SCM but differs from the solution structure of SCM. The structures of SCM in the crystal and in solution differ in some of the secondary structure elements, but most of all in the relative arrangement of the elements: the four main beta-strands that surround the helix in the crystal structure of SCM, are displaced far from the helix in the solution structure of SCM. These differences were attributed to the fact that SCM is a monomer in solution and a dimer in the crystal. This result is at variance with the observation that our solution structure, like that of SCM, corresponds to a monomeric state of the protein, as demonstrated by the insensitivity of HSQC spectra to extreme dilution (down to 20 microM). On the basis of the solution structure of MNEI it is possible to propose that the main glucophores are hosted on loop L34, whereas the N-terminal and C-terminal regions host two other important interaction regions, centered around segments 6-9 and 94-96.

Tuftsin, a linear tetrapeptide (Thr-Lys-Pro-Arg), corresponding to the sequence 289-292 of the heavy chain of leukokinin, has been the object of intensive SAR studies during the past 30 years, owing to its numerous biological activities and to the possibility of generating a novel anticancer drug. A cyclic tuftsin analogue, c-[T-K-P-R-G], has biological activity 50 times higher than that of the parent linear peptide. Here we present a conformational study of c-[T-K-P-R-G] based on NMR data in a cryoprotective DMSO/water mixture. The preferred conformation is a type VIa turn centered on the K-P residues. The orientation of the side chains of the two basic residues (K and R) may represent the essential feature of the bioactive conformation of tuftsin. A possible role of tuftsin as a DNA binding motif is suggested by the similarity of the bioactive conformation of c-[T-K-P-R-G] and of the beta-turn conformation proposed by Suzuki for the [T,S]-P-K-R motif.

beta-Endorphin is the largest natural opioid peptide. The knowledge of its bioactive conformation might be very important for the indirect mapping of the active site of opioid receptors. We have studied beta-endorphin in a variety of solution conditions with the goal of testing the intrinsic tendency of its sequence to assume a regular fold. We ran NMR experiments in water, dimethylsulfoxide and aqueous mixtures of methanol, ethylene glycol, trifluoroethanol, hexafluoracetone trihydrate and dimethylsulfoxide. The solvent in which the peptide is more ordered is the hexafluoracetone trihydrate/water mixture. The helical structure detected for beta-endorphin in this mixture at 300 K extends for the greater part of its address domain, hinting at a possible mechanism of interaction with opioid receptors: a two-point attachment involving an interaction of the helical part of the address domain (PLVTLFKNAIIKNAY) with one of the transmembrane helices and a classical interaction of the message domain (YGGF) with the receptor subsite common to all opioid receptors.

The structural problem posed by ill-defined segments in protein structures is similar to those encountered in the study of most peptide hormones, with terminal tracts resembling linear peptides and loops resembling cyclic peptides. The conformational preferences of short linear peptides in solution can be influenced by the use of solvent mixtures of viscosity higher than that of pure water but comparable to that of cytoplasm. In order to check whether it is possible to use these media in the structural study of proteins, we undertook an exploratory study on BPTI in a mixture of dimethylsulfoxide and water. The complete assignment of BPTI in an 80:20 (by volume) DMSO-d 6/water cryomixture at two temperatures showed that all resonances parallel those in water, hinting at the persistence of the correct protein architecture, which is also confirmed by NOESY experiments. In addition to the NOEs present in the aqueous solution it was possible to detect numerous new cross peaks, in particular from residues belonging to the less-defined regions. The new cross peaks do not originate from spin diffusion and are consistent with the best NMR structure and with the X-ray structures of BPTI.

BACKGROUND: Tyr-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) and Tyr-Tic-Ala were the first peptides with delta opioid antagonist activity lacking Phe, considered essential for opioid activity based on the N-terminal tripeptide sequence (Tyr-D-Xaa-Phe) of amphibian skin opioids. Analogs were then designed to restrain the rotational flexibility of Tyr by the substitution of 2,6-dimethyl-L-tyrosine (Dmt). MATERIALS AND METHODS: Tyr and Dmt peptides were synthesized by solid phase and solution methods using Fmoc technology or condensing Boc-Dmt-OH or Boc-Tyr(But)-OH with H-L-Tic-OBut or H-D-Tic-OBut, respectively. Peptides were purified (> 99%) by HPLC and characteristics determined by 1H-NMR, FAB-MS, melting point, TLC, and amino acid analyses. RESULTS: H-Dmt-Tic-OH had high affinity (Ki delta = 0.022 nM) and extraordinary selectivity (Ki mu/Ki delta = 150,000); H-Dmt-Tic-Ala-OH had a Ki delta = 0.29 nM and delta selectivity = 20,000. Affinity and selectivity increased 8700- and 1000-fold relative to H-Tyr-Tic-OH, respectively. H-Dmt-Tic-OH and H-Dmt-Tic-NH2 fitted one-site receptor binding models (eta = 0.939-0.987), while H-Dmt-Tic-ol, H-Dmt-Tic-Ala-OH and H-Dmt-Tic-Ala-NH2 best fitted two-site models (eta = 0.708-0.801, F 18.9-26.0, p < 0.0001). Amidation increased mu affinity by 10- to 100-fold and acted synergistically with D-Tic2 to reverse selectivity (delta-->mu). Dmt-Tic di- and tripeptides exhibited delta antagonist bioactivity (Ke = 4-66 nM) with mouse vas deferens and lacked agonist mu activity (> 10 microM) in guinea-pig ileum preparations. Dmt-Tic analogs weakly interacted with kappa receptors in the 1 to > 20 microM range. CONCLUSIONS: Dmt-Tic opioidmimetic peptides represent a highly potent class of opioid peptide antagonists with greater potency than the nonopioid delta antagonist naltrindole and have potential application as clinical and therapeutic compounds.

7 p.-2 fig., Taking into account the presence of the crowded environment of a macromolecule has been an important goal of biology over the past 20 years. Molecular crowding affects the motions, stability and the kinetic behaviour of proteins. New powerful approaches have recently been developed to study molecular crowding, some of which make use of the synchrotron radiation light. The meeting “New Frontiers in Molecular Crowding” was organized in July 2022at the European Synchrotron Radiation facility of Grenoble to discuss the new frontiers of molecular crowding. The workshop brought together researchers from different disciplines to highlight the new developments of the field, including areas where new techniques allow the scientists to gain unprecedently expected information. A key conclusion of the meeting was the need to build an international and interdisciplinary research community through enhanced communication, resource-sharing, and educational initiatives that could let the molecular crowding field flourish further., The workshop was funded with support from the European Union's Horizon 2020 Research and Innovation programme under grant agreement No 870313, Streamline which also supports APC.

All globular proteins undergo transitions from their native to unfolded states if exposed either to cold or to heat perturbation. While the heat-induced transition is well described for a large number of proteins, in media compatible with natural environments, the limited number of examples of cold denatured states concern proteins artificially destabilized, for instance, by the presence of denaturants, ad hoc point mutations, or both. Here, we provide a characterization of the low temperature unfolded state of Yfh1, a natural protein that undergoes cold denaturation around water freezing temperature, in the absence of any denaturant. By achieving nearly full assignment of the NMR spectrum, we show that at -1 °C, Yfh1 has all the features of an unfolded protein, although retaining some local, residual secondary structure. The effect is not uniform along the sequence and does not merely reflect the secondary structural features of the folded species. The N-terminus seems to be dynamically more flexible, although retaining some nascent helix character. Interestingly, this region is the one containing functionally important hot-spots. The β-sheet region and the C-terminal helix are completely unfolded, although experiencing some conformational exchange, partly due to the presence of several prolines. Ours is the first step toward a full characterization of the low temperature unfolded state of a natural protein, reached without the aid of any destabilizing agent. We discuss the implications of our findings for understanding cold denatured states.

Confinement of proteins and peptides in a small inert space mimics the natural environment of the cell, allowing structural studies in conditions that stabilize folded conformations. We have previously shown that confinement in polyacrylamide gels (PAGs) is sufficient to induce a change in the viscosity of the aqueous solution without changing the composition and temperature of the solvent. The main limitation of a PAG to run NMR experiments in a confined environment is the need for labelling the peptides. Here we report the use of the agarose gel to run the NMR spectra of proteins and peptides. We show that agarose gels are completely transparent in NMR experiments, relieving the need for labelling. Although it is necessary to expose biomolecules to fairly high temperatures during sample preparation, we believe that this is not generally an obstacle to the study of peptides, and found that the method is also compatible with temperature-resistant proteins. The mesh of agarose gels is too wide for direct effects of confinement on the stability of proteins but confinement can be easily exploited to interact the proteins with other reagents, including crowding macromolecules that can eventually lead to fold stabilization. The use of these gels is ideally suited for low-temperature studies; we show that a very flexible peptide at subzero temperatures is stabilized into a well-folded conformation. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd.

Sweet proteins are the sweetest natural molecules. This aspect prompted several proposals for their use as food additives, mainly because the amounts to be added to food would be very small and safe for people suffering from sucrose-linked diseases. During studies of sweet proteins as food additives we found that their sweetness is affected by water salinity, while there is no influence on protein’s structure. Parallel tasting of small size sweeteners revealed no influence of the water quality. This result is explained by the interference of ionic strength with the mechanism of action of sweet proteins and provides an experimental validation of the wedge model for the interaction of proteins with the sweet receptor.

Proteins are often described in textbooks as being only "marginally stable" but many proteins, specifically those with a high free energy of unfolding are, in fact, so stable that they exist only in the fully folded state except under harsh denaturing conditions. Proteins that are truly only marginally stable, those with a low free energy of unfolding, exist as an equilibrium mixture of folded and unfolded forms under "normal" conditions. To some extent such proteins have some features in common with "intrinsically disordered" proteins. We analyzed the relationship between these marginally stable proteins and intrinsically disordered proteins in order to fully understand the twilight zone that distinguishes the two ensembles in the hope of clarifying the fuzzy borders of the current classification that divides the protein world into folded and intrinsically disordered ones. Our analysis suggests that the division may be too drastic and misleading, because it puts within the same category proteins with very different behaviors. We propose a restricted, albeit operational, definition of "marginally stable proteins", referring by this term only to proteins whose free energy difference between the folded and unfolded states falls in the interval 0-3 kcal/mol. These proteins have special features because they normally exist as equilibrium mixtures of folded and unfolded species or as molten globule states. This coexistence makes marginally stable proteins ideal tools to study even small environmental changes to which they may behave as natural sensors.

Proteins undergo both cold and heat denaturation, but often cold denaturation cannot be detected because it occurs at temperatures below water freezing. Proteins undergoing detectable cold as well as heat denaturation yield a reliable curve of protein stability. Here we use bacterial IscU, an essential and ancient protein involved in iron cluster biogenesis, to show an important example of unbiased cold denaturation, based on electrostatic frustration caused by a dualism between iron–sulfur cluster binding and the presence of a functionally essential electrostatic gate. We explore the structural determinants and the universals that determine cold denaturation with the aid of a coarse grain model. Our results set a firm point in our understanding of cold denaturation and give us general rules to induce and predict protein cold denaturation. The conflict between ligand binding and stability hints at the importance of the structure–function dualism in protein evolution. Proteins can undergo both heat and cold denaturation, and in marginally stable proteins this is often controlled by electrostatic frustration. Here, the authors find that residues essential for protein function are also structural determinants for cold denaturation.

Sweet proteins are a family of proteins with no structural homology, able to elicit a sweet sensation by interacting with the dimeric T1R2-T1R3 sweet receptor [Picone, D. and Temussi, P.A., 2012, Plant science, 195, 135-142]. Our studies focus on MNEI, a single chain derivative of the natural protein monellin, representing one of the sweetest molecules known to date. MNEI stimulates a strong interest for its biological properties, but is also a well accepted model for protein folding and aggregation studies. In the framework of the “wedge model” [Temussi, P.A., 2009, TIBS, 34, 296-302], we have integrated computational and experimental techniques to enhance sweetness and stability of MNEI against physical and chemical agents [Leone S., et al., 2016, Scientific Reports, 6, 34045]. Based on molecular modeling, NMR [Spadaccini, R., et al., 2016, FEBS Lett, (in press)], and X-ray crystallography, we designed the sweetest protein ever obtained, which combines a sweetness threshold of about 25 nM with a very high and pH-independent thermal stability. Docking studies have provided a rationale basis to explain these properties, hinting at a previously unpredicted role of plasticity in the interaction between this protein and the sweet receptor. Additional aspects of protein aggregation and unfolding properties studied by Molecular Dynamics [Leone S. and Picone, D., 2016, PloS one, 11 (6), e0158372], Atomic Force Microscopy and Optic Spectroscopies will be also discussed.

Human kallikrein 6 (KLK6) is highly expressed in the central nervous system and with elevated level in demyelinating disease. KLK6 has a very restricted specificity for arginine (R) and hydrolyses myelin basic protein, protein activator receptors and human ionotropic glutamate receptor subunits. Here we report a previously unreported activity of KLK6 on peptides containing clusters of basic amino acids, as in synthetic fluorogenic peptidyl-Arg-7-amino-4-carbamoylmethylcoumarin (peptidyl-ACC) peptides and FRET peptides in the format of Abz-peptidyl-Q-EDDnp (where Abz=ortho-aminobenzoic acid and Q-EDDnp=glutaminyl-N-(2,4-dinitrophenyl) ethylenediamine), in which pairs or sequences of basic amino acids (R or K) were introduced. Surprisingly, KLK6 hydrolyzed the fluorogenic peptides Bz-A-R(↓)R-ACC and Z-R(↓)R-MCA between the two R groups, resulting in non-fluorescent products. FRET peptides containing furin processing sequences of human MMP-14, nerve growth factor (NGF), Neurotrophin-3 (NT-3) and Neurotrophin-4 (NT-4) were cleaved by KLK6 at the same position expected by furin. Finally, KLK6 cleaved FRET peptides derived from human proenkephalin after the KR, the more frequent basic residues flanking enkephalins in human proenkephalin sequence. This result suggests the ability of KLK6 to release enkephalin from proenkephalin precursors and resembles furin a canonical processing proteolytic enzyme. Molecular models of peptides were built into the KLK6 structure and the marked preference of the cut between the two R of the examined peptides was related to the extended conformation of the substrates.

Sweet proteins are a family of proteins with no structure or sequence homology, able to elicit a sweet sensation in humans through their interaction with the dimeric T1R2-T1R3 sweet receptor. In particular, monellin and its single chain derivative (MNEI) are among the sweetest proteins known to men. Starting from a careful analysis of the surface electrostatic potentials, we have designed new mutants of MNEI with enhanced sweetness. Then, we have included in the most promising variant the stabilising mutation E23Q, obtaining a construct with enhanced performances, which combines extreme sweetness to high, pH-independent, thermal stability. The resulting mutant, with a sweetness threshold of only 0.28 mg/L (25 nM) is the strongest sweetener known to date. All the new proteins have been produced and purified and the structures of the most powerful mutants have been solved by X-ray crystallography. Docking studies have then confirmed the rationale of their interaction with the human sweet receptor, hinting at a previously unpredicted role of plasticity in said interaction.

In addition to many small molecular weight sweeteners there are in nature a few sweet proteins. The molecular volume of sweet proteins is so different from that of common sweeteners that it was difficult to understand how molecules as large as proteins can activate a receptor designed to host small molecules. We have recently shown that sweet proteins can activate the sweet receptor by a mechanism of interaction, called “wedge model”, in which proteins fit a large cavity of the receptor with wedge-shaped surfaces of their structures. In order to substantiate this model we have designed, expressed and characterized seven mutants of MNEI, a single chain monellin. Three uncharged residues of the interaction surface, Met42, Tyr63 and Tyr65, were changed either into acidic or basic residues whereas Asp68, a key acidic residue, was changed into a basic one. As a general trend, we observe that an increase of the negative charge is much more detrimental for sweetness than an increase of positive charge. In addition we show that by a careful choice of a residue at the center of the interface between MNEI and receptor, it is possible even to increase the sweetness of MNEI. These results are fully consistent with the wedge model.

Current views of the role of beta-amyloid (Abeta) peptide fibrils range from regarding them as the cause of Alzheimer's pathology to having a protective function. In the last few years, it has also been suggested that soluble oligomers might be the most important toxic species. In all cases, the study of the conformational properties of Abeta peptides in soluble form constitutes a basic approach to the design of molecules with "antiamyloid" activity. We have experimentally investigated the conformational path that can lead the Abeta-(1-42) peptide from the native state, which is represented by an alpha helix embedded in the membrane, to the final state in the amyloid fibrils, which is characterized by beta-sheet structures. The conformational steps were monitored by using CD and NMR spectroscopy in media of varying polarities. This was achieved by changing the composition of water and hexafluoroisopropanol (HFIP). In the presence of HFIP, beta conformations can be observed in solutions that have very high water content (up to 99 % water; v/v). These can be turned back to alpha helices simply by adding the appropriate amount of HFIP. The transition of Abeta-(1-42) from alpha to beta conformations occurs when the amount of water is higher than 80 % (v/v). The NMR structure solved in HFIP/H2O with high water content showed that, on going from very apolar to polar environments, the long N-terminal helix is essentially retained, whereas the shorter C-terminal helix is lost. The complete conformational path was investigated in detail with the aid of molecular-dynamics simulations in explicit solvent, which led to the localization of residues that might seed beta conformations. The structures obtained might help to find regions that are more affected by environmental conditions in vivo. This could in turn aid the design of molecules able to inhibit fibril deposition or revert oligomerization processes.

What is the mechanism that determines the denaturation of proteins at low temperatures, which is, by now, recognized as a fundamental property of all proteins? We present experimental evidence that clarifies the role of specific interactions that favor the entrance of water into the hydrophobic core, a mechanism originally proposed by Privalov but never proved experimentally. By using a combination of molecular dynamics simulation, molecular biology, and biophysics, we identified a cluster of negatively charged residues that represents a preferential gate for the entrance of water molecules into the core. Even single-residue mutations in this cluster, from acidic to neutral residues, affect cold denaturation much more than heat denaturation, suppressing cold denaturation at temperatures above zero degrees. The molecular mechanism of the cold denaturation of yeast frataxin is intrinsically different from that of heat denaturation. Some like it cold: The molecular mechanism of the cold denaturation of yeast frataxin is intrinsically different from that of heat denaturation. A cluster of negatively charged residues is identified, which acts as a gate for the entrance of water molecules to the core. Single-residue mutations in this cluster affect cold denaturation much more than heat denaturation, suppressing cold denaturation at temperatures above zero degrees.

The major components of neuritic plaques found in Alzheimer disease (AD) are eptides known as amyloid beta-peptides (Abeta), which derive from the proteolitic cleavage of the amyloid precursor proteins. In vitro Abeta may undergo a conformational transition from a soluble form to aggregated, fibrillary beta-sheet structures, which seem to be neurotoxic. Alternatively, it has been suggested that an alpha-helical form can be involved in a process of membrane poration, which would then trigger cellular death. Conformational studies on these eptides in aqueous solution are complicated by their tendency to aggregate, and only recently NMR structures of Abeta-(1-40) and Abeta-(1-42) have been determined in aqueous trifluoroethanol or in SDS micelles. All these studies hint to the presence of two helical regions, connected through a flexible kink, but it proved difficult to determine the length and position of the helical stretches with accuracy and, most of all, to ascertain whether the kink region has a preferred conformation. In the search for a medium which could allow a more accurate structure determination, we performed an exhaustive solvent scan that showed a high propensity of Abeta-(1-42) to adopt helical conformations in aqueous solutions of fluorinated alcohols. The 3D NMR structure of Abeta-(1-42) shows two helical regions encompassing residues 8-25 and 28-38, connected by a regular type I beta-turn. The surprising similarity of this structure, as well as the sequence of the C-terminal moiety, with those of the fusion domain of influenza hemagglutinin suggests a direct mechanism of neurotoxicity.

CCK-15, a peptide derived from the 115-membered CCK preprohormone, was the object of a comparative conformational analysis by NMR spectroscopy and molecular modeling methods. NMR data in several solvents demonstrate that the propensity of the peptide to fold into a helical conformation is intrinsic, not merely a consequence of the interaction with phosphatidylcholine micelles or with a putative receptor, as suggested by a previous study on CCK-8 (Pellegrini, M.; Mierke, D. Biochemistry 1999, 38, 14775-14783.). The prevailing CCK-15 conformer in a mixture 1,1,1,3,3,3-hexafluoroacetone/water reveals that the residues common to CCK-15 and CCK-8 assume very similar conformations. Our CCK-15 structure is consistent with the model of receptor interaction proposed by Pellegrini and Mierke and discloses possible novel interactions that involve a larger area of the putative receptor. The consensus structure between CCK-15 and CCK-8 shows a good superposition of the side chains of residues 12-14 with crucial moieties of two non-peptidic CCK-A antagonists.

Characterization of protein surface accessibility represents a new frontier of structural biology. A surface accessibility investigation for two structurally well-defined proteins, tendamistat and bovine pancreatic trypsin inhibitor, is performed here by a combined analysis of water-protein Overhauser effects and paramagnetic perturbation profiles induced by the soluble spin-label 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl on NMR spectra. This approach seems to be reliable not only for distinguishing between buried and exposed residues but also for finding molecular locations where a network of more ordered waters covers the protein surface. From the presented set of data, an overall picture of the surface accessibility of the two proteins can be inferred. Detailed knowledge of protein accessibility can form the basis for successful design of mutants with increased activity and/or greater specificity.

Peptide T (ASTTTNYT), a fragment corresponding to residues 185-192 of gp120, the coat protein of HIV, is endowed with several biological properties in vitro, notably inhibition of the binding of both isolated gp120 and HIV-1 to the CD4 receptor, and chemotactic activity. Based on previous nuclear magnetic resonance (NMR) studies performed in our laboratory, which were consistent with a regular conformation of the C-terminal pentapeptide, and SAR studies showing that the C-terminal pentapeptide retains most of the biological properties, we designed eight hexapeptides containing in the central part either the TNYT or the TTNY sequence, and charged residues (D/E/R) at the two ends. Conformational analysis based on NMR and torsion angle dynamics showed that all peptides assume folded conformations, albeit with different geometries and stabilities. In particular, peptides carrying an acidic residue at the N-terminus and a basic residue at the C-terminus are characterized by stable helical structures and retain full chemotactic activity. The solution conformation of peptide ETNYTR displays strong structural similarity to the region 19-26 of both bovine pancreatic and bovine seminal ribonuclease, which are endowed with anti-HIV activity. Moreover, the frequent occurrence, in many viral proteins, of TNYT and TTNY, the two core sequences employed in the design of the hexapeptides studied in the present work, hints that the sequence of the C-terminal pentapeptide TTNYT is probably representative of a widespread viral recognition motif. Copyright © 2001 European Peptide Society and John Wiley & Sons, Ltd.

Nocistatin, a new heptadecapeptide encoded in the bPNP-3 gene, has a powerful biological activity connected with the mechanisms of pain transmission. It does not bind to the opioid receptors but to another brain receptor with high affinity. In order to substantiate these novel biological data with a structural basis, we have undertaken a conformational study in solution. Proton nmr data in helicogenic solvents are consistent with a well-defined helical structure that is consistent with the nmr parameters of the C-terminal octapeptide, a shorter fragment that retains allodynia-blocking activity. © 2000 John Wiley & Sons, Inc. Biopoly 53: 257–264, 2000

Frataxins are a family of metal binding proteins associated with the human Friedreich's ataxia disease. Here, we have addressed the effect of non-specifically binding salts on the stability of the yeast ortholog Yfh1. This protein is a sensitive model since its stability is strongly dependent on the environment, in particular on ionic strength. Yfh1 also offers the unique advantage that its cold denaturation can be observed above the freezing point of water, thus allowing the facile construction of the whole protein stability curve and hence the measurement of accurate thermodynamic parameters for unfolding. We systematically measured the effect of several cations and, as a control, of different anions. We show that, while strongly susceptible to ionic strength, as it would be in the cellular environment, Yfh1 stability is sensitive not only to divalent cations, which bind specifically, but also to monovalent cations. We pinpoint the structural bases of the stability and hypothesize that the destabilization induced by an unusual cluster of negatively charged residues favours the entrance of water molecules into the hydrophobic core, consistent with the generally accepted mechanism of cold denaturation.