The folding of a polypeptide chain of a relatively large globular protein into its unique three-dimensional and functionally active structure occurs via folding intermediates. These partly folded states of proteins are difficult to characterize, because they are usually short lived or exist as a distribution of possible conformers. A variety of experimental techniques and approaches have been utilized in recent years in numerous laboratories for characterizing folding intermediates that occur at equilibrium, including spectroscopic techniques, solution X-ray scattering, calorimetry and gel filtration chromatography, as well as genetic methods and theoretical calculations. In this review, we focus on the use of proteolytic enzymes as probes of the structure and dynamics of folding intermediates and we show that this simple biochemical technique can provide useful information, complementing that obtained by other commonly used techniques and approaches. The key result of the proteolysis experiments is that partly folded states (molten globules) of proteins can be sufficiently rigid to prevent extensive proteolysis and appear to maintain significant native-like structure.

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The protein toxin VacA, produced by cytotoxic strains of Helicobacter pylori, causes a vacuolar degeneration of cells, which eventually die. VacA is strongly activated by a short exposure to acidic solutions in the pH 1.5-5.5 range, followed by neutralization. Activated VacA has different CD and fluorescence spectra and a limited proteolysis fragmentation pattern from VacA kept at neutral pH. Moreover, activated VacA is resistant to pH 1.5 and to pepsin. The relevance of these findings to pathogenesis of H. pylori-induced gastrointestinal ulcers is discussed.

We describe here a simple assay that allows the visual detection of a protease. The method takes advantage of the high molar absorptivity of the plasmon band of gold colloids and is based on the color change of their solution when treated with dithiols. We used C-and N-terminal cysteinyl derivatives of a peptide substrate exploiting its selective recognition and cleavage by a specific protease. Contrary to the native ones, cleaved peptides are unable to induce nanoparticles aggregation; hence, the color of the solution does not change. The detection of two proteases is reported: thrombin (involved in blood coagulation and thrombosis) and lethal factor (an enzyme component of the toxin produced by Bacillus anthracis). The sensitivity of this nanoparticle-based assay is in the low nanomolar range.lethal factor ͉ plasmon surface band ͉ thrombin E nzymes analytical detection is a key tool in enzymology, extremely important for the screening of noxious toxins and pathologies associated with their presence, and for the development of effective and selective therapeutics. Among enzymes, proteases (1, 2) are particularly relevant because proteolytic processing is the final step in the expression of the activity of a great variety of proteins (3). Standard assays for proteases include those based on radioisotopes or on fluorogenic substrates. A protease assay system that uses functionalized, supermagnetic nanoparticles as magnetic relaxation switches and bi-biotinylated peptide substrates for particles clustering has been reported (4). All of these techniques require specific instrumentation and hence an equipped laboratory. We report here an assay based on nanometer-size gold colloids. Citrate stabilized gold colloids of Ն4 nm diameter present an absorption band at Ϸ520 nm due to plasmon resonance (5, 6) with a very high molar absorptivity. This band is shifted to longer wavelengths upon clustering of the colloids, thus leading to color changes of the solution, from pink-red to violet-blue (7). Clustering may be induced by physical methods (like the increase of the ionic strength of the solution) (8) or chemically by addition of molecules able to connect one nanoparticle to another (9). By taking advantage of this phenomenon, very sensitive detection procedures have been introduced for analytes ranging from DNA to proteins and metal ions (10). Because thiols interact strongly with gold nanoparticles, a molecule featuring a head and tail thiol causes such a process (11). Indeed, when we treat a gold colloid solution with a peptide of the general formula Cys-(AA) n -Cys (where AA is any amino acid but cysteine), the color of the solution turns from pink-red to violet-blue. No change of color, however, is observed with a peptide lacking one of the terminal cysteines. Accordingly, we reasoned that the cleavage of a Cys-(AA) n -Cys peptide in two fragments, each containing a single cysteine, would result in a system unable to induce aggregation of the gold nanoparticles and, hence, failing to induce the color change of the s...

Limited proteolysis has been used to probe the partially folded state of bovine alpha-lactalbumin (BLA) at acid pH (A-state) or dissolved in aqueous trifluoroethanol (TFE-state). The sites of proteolytic fission have been determined by isolation of the various BLA fragments and comparison of their N-terminal amino acid sequence and amino acid composition after acid hydrolysis, as well as their molecular mass determined by mass spectrometry, with the known sequence of BLA. Incubation of BLA with pepsin at 20-22 degrees C and pH 2.0 in the presence of 0.1 M NaCl results in very rapid cleavage of the 123-residue chain at peptide bond Ala40-Ile41 and subsequently at Leu52-Phe53, leading to a nicked species of BLA constituted by the two fragments 1-40 and 53-123 cross-linked by the four disulfide bridges of the protein. Much slower proteolytic cleavage occurs at Tyr103-Trp104. The highly helical conformational state acquired by BLA when dissolved in aqueous buffer (pH 7.0) containing 50% (v/v) TFE was probed by the TFE-resistant thermolysin. Proteolytic cleavage occurs at the peptide bond Ala40-Ile41 and much more slowly at Phe80-Leu81. Moreover, the peptide bond Gln2-Leu3 at the N-terminus of the chain is partially cleaved by thermolysin. Conversely, native BLA in a pH 7.0 buffer is rather resistant to proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Skeletal muscle is a dynamic organ, characterized by an incredible ability to rapidly increase its rate of energy consumption to sustain activity. Muscle mitochondria provide most of the ATP required for contraction via oxidative phosphorylation. Here we found that skeletal muscle mitochondria express a unique MCU complex containing an alternative splice isoform of MICU1, MICU1.1, characterized by the addition of a micro-exon that is sufficient to greatly modify the properties of the MCU. Indeed, MICU1.1 binds Ca one order of magnitude more efficiently than MICU1 and, when heterodimerized with MICU2, activates MCU current at lower Ca concentrations than MICU1-MICU2 heterodimers. In skeletal muscle in vivo, MICU1.1 is required for sustained mitochondrial Ca uptake and ATP production. These results highlight a novel mechanism of the molecular plasticity of the MCU Ca uptake machinery that allows skeletal muscle mitochondria to be highly responsive to sarcoplasmic [Ca] responses.

3-Nitrotyrosine (NT) is ;103 -fold more acidic than Tyr, and its absorption properties are strongly pH-dependent. NT absorbs radiation in the wavelength range where Tyr and Trp emit fluorescence (300-450 nm), and it is essentially nonfluorescent. Therefore, NT may function as an energy acceptor in resonance energy transfer (FRET) studies for investigating ligand-protein interactions. Here, the potentialities of NT were tested on the hirudin-thrombin system, a well-characterized protease-inhibitor pair of key pharmacological importance. We synthesized two analogs of the N-terminal domain (residues 1-47) of hirudin: Y3NT, in which Tyr3 was replaced by NT, and S2R/Y3NT, containing the substitutions Ser2 ! Arg and Tyr3 ! NT. The binding of these analogs to thrombin was investigated at pH 8 by FRET and UV/Vis-absorption spectroscopy. Upon hirudin binding, the fluorescence of thrombin was reduced by ;50%, due to the energy transfer occurring between the Trp residues of the enzyme (i.e., the donors) and the single NT of the inhibitor (i.e., the acceptor). The changes in the absorption spectra of the enzyme-inhibitor complex indicate that the phenate moiety of NT in the free state becomes protonated to phenol in the thrombin-bound form. Our results indicate that the incorporation of NT can be effectively used to detect protein-protein interactions with sensitivity in the low nanomolar range, to uncover subtle structural features at the ligand-protein interface, and to obtain reliable K d values for structure-activity relationship studies. Furthermore, advances in chemical and genetic methods, useful for incorporating noncoded amino acids into proteins, highlight the broad applicability of NT in biotechnology and pharmacological screening.

In the present work, the effect of Na+ binding on the conformational, stability and molecular recognition properties of thrombin was investigated. The binding of Na+ reduces the CD signal in the far-UV region, while increasing the intensity of the near-UV CD and fluorescence spectra. These spectroscopic changes have been assigned to perturbations in the environment of aromatic residues at the level of the S2 and S3 sites, as a result of global rigidification of the thrombin molecule. Indeed, the Na+-bound form is more stable to urea denaturation than the Na+-free form by approximately 2 kcal/mol (1 cal identical with 4.184 J). Notably, the effects of cation binding on thrombin conformation and stability are specific to Na+ and parallel the affinity order of univalent cations for the enzyme. The Na+-bound form is even more resistant to limited proteolysis by subtilisin, at the level of the 148-loop, which is suggestive of the more rigid conformation this segment assumes in the 'fast' form. Finally, we have used hirudin fragment 1-47 as a molecular probe of the conformation of thrombin recognition sites in the fast and 'slow' form. From the effects of amino acid substitutions on the affinity of fragment 1-47 for the enzyme allosteric forms, we concluded that the specificity sites of thrombin in the Na+-bound form are in a more open and permissible conformation, compared with the more closed structure they assume in the slow form. Taken together, our results indicate that the binding of Na+ to thrombin serves to stabilize the enzyme into a more open and rigid conformation.