Marcello Zambonin

Probing the partly folded states of proteins by limited proteolysis

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.

α-Lactalbumin Forms with Oleic Acid a High Molecular Weight Complex Displaying Cytotoxic Activity

α-Lactalbumin (LA) forms with oleic acid (OA) a complex which has been reported to induce the selective death of tumor cells. However, the mechanism by which this complex kills a wide range of tumor cell lines is as yet largely unknown. The difficulty in rationalizing the cytotoxic effects of the LA/OA complex can be due to the fact that the molecular aspects of the interaction between the protein and the fatty acid are still poorly understood, in particular regarding the oligomeric state of the protein and the actual molar ratio of OA over protein in the complex. Here, the effect of LA addition to an OA aqueous solution has been examined by dynamic light scattering measurements and transmission electron microscopy. Upon protein addition, the aggregation state of the rather insoluble OA is dramatically changed, and more water-soluble and smaller aggregates of the fatty acid are formed. A mixture of LA and an excess of OA forms a high molecular weight complex that can be isolated by size-exclusion chromatography and that displays cellular toxicity toward Jurkat cells. On the basis of gel filtration data, cross-linking experiments with glutaraldehyde, and OA titration, we evaluated that the isolated LA/OA complex is given by 4-5 protein molecules that bind 68-85 OA molecules. The protein in the complex adopts a molten globule-like conformation, and it interacts with the fatty acid mostly through its α-helical domain, as indicated by circular dichroism measurements and limited proteolysis experiments. Overall, we interpret our and previous data as indicating that the cellular toxicity of a LA/OA complex is due to the effect of a protein moiety in significantly enhancing the water solubility of the cytotoxic OA and, therefore, that the protein/OA complex can serve mainly as a carrier of the toxic fatty acid in a physiological milieu.

Limited proteolysis of cytochrome c in trifluoroethanol

Horse heart cytochrome c is cleaved by thermolysin in 50% aqueous TFE (v/v) at neutral pH (25°C, 24 h) at the Gly56‐Ile57 peptide bond of the 104‐residue chain of the protein. Additional, but anyway minor, fragmentation at the Gly45‐Phe46 and Met80‐Ile81 peptide bonds is also observed. On the other hand, in buffer only and in the absence of TFE, cytochrome c is digested by thermolysin to numerous small peptides. Considering the broad substrate specificity of the TFE‐resistant thermolysin, clearly the conformational state of the protein substrate dictates the observed selective proteolysis. It is proposed that the highly helical secondary structure acquired by cytochrome c when dissolved in aqueous TFE hampers binding and adaptation of the protein substrate at the active site of the protease and that peptide bond fission occurs at flexible chain segments characterized by a low α‐helix propensity.

Modulation of the structural integrity of helix F in apomyoglobin by single amino acid replacements.

The conformational features of native and mutant forms of sperm‐whale apomyoglobin (apoMb) at neutral pH were probed by limited proteolysis experiments utilizing up to eight proteases of different substrate specificities. It was shown that all proteases selectively cleave apoMb at the level of chain segment 82–94 (HEAELKPLAQSHA), encompassing helix F in the X‐ray structure of the holo form of the native protein; for example, thermolysin cleaves the Pro 88–Leu 89 peptide bond. These results indicate that helix F is highly flexible or largely disrupted in apoMb. Because helix F contains the helix‐breaking Pro 88 residue, we propose that helix F is kept in place in the native holo protein by a variety of helix–heme stabilizing interactions. To modulate the stability of helix F, the Pro88Ala and Pro88Gly mutants were prepared by site‐directed mutagenesis, and their conformational properties investigated by both far‐UV circular dichroism spectroscopy and limited proteolysis. The helix content of the Pro88Ala mutant was somewhat enhanced with respect to that of both native and Pro88Gly mutant, as expected from the fact that Ala is the strongest helix inducer among the 20 amino acid residues. The rate of limited proteolysis of the three apoMb variants by thermolysin and proteinase K was in the order native > Pro88Gly >> Pro88Ala, in agreement with the scale of helix propensity of Ala, Gly, and Pro. The possible role of the flexible/unfolded chain segment 82–94 for the function and fate of apoMb at the cellular level is discussed.

Limited Proteolysis in the Study of Protein Conformation

The methods of choice for determining the three-dimensional structure of globular proteins are X-ray crystallography and two-dimensional NMR (Lecomte, 1991; Mac Arthur et al, 1994). Currently we are observing a flow of reports describing the structural analysis of proteins, since significant theoretical and methodological advances have been made recently in utilizing both X-ray and NMR techniques. However, every experimental technique for protein structure determination has strengths and weaknesses and, in particular, we need protein crystals for X-ray crystallography and a nonaggregating protein solution at a millimolar concentration for NMR. Since these requirements are not always fulfilled, perhaps alternative methods can be employed, even if these will provide structural information at a lower level resolution. In this chapter we will show that a classical biochemical method such as limited proteolysis can be used to probe structure, and dynamics of proteins in solution, providing experimental results which are easy to obtain and well complement those derived from the use of other more classical physicochemical methods and approaches.

Rigidity of Thermophilic Enzymes

Enzymes and proteins isolated from thermophilic microorganisms are not only unusually stable to heat and protein denaturants, but also display enhanced protein rigidity in respect to that of their mesophilic counterparts. The molecular rigidity of thermophilic enzymes appears to explain why their specific activity at room temperature often is less than that of the corresponding mesophilic enzymes, considering that an appropriate level of protein mobility is required for catalysis. Evidence of protein rigidity can be obtained from hydrogen exchange measurements, molecular dynamics simulations, by computing flexibility indices based on crystallographic data, as well as by proteolysis experiments. Although the structural and functional complexity of proteins likely does not allow firm generalizations, it can be proposed that thermophilic enzymes are rigid molecules, but not optimally active at ambient temperature. Considering that extremophiles appeared earlier on hearth in a hotter environment, it can be suggested that present-day mesophilic enzymes evolved to be more flexible, and thus more labile, in order to optimize their catalytic function.

Limited Proteolysis as a Tool to Detect Structure and Dynamic Features of Globular Proteins: Studies on Thermolysin

In analogy to all enzymatic reactions, the proteolytic cleavage of a polypeptide chain occurs only if the site of cleavage can bind and adapt itself in a specific way to the stereochemistry of the active site of the protease. This is difficult to achieve with native globular proteins, whereas denatured proteins are much more susceptible to proteolysis. In a number of cases, an extraordinary lability to enzymatic hydrolysis of a very small number of specific bonds in a native globular protein has been observed and this selective peptide bond fission has been termed “limited proteolysis”. It is conceivable to suggest that the sites of limited proteolysis in a native globular protein are dictated solely by the stereochemistry of the protein substrate, if a protease of low specificity is employed. In addition, some motility of the substrate protein at the site of cleavage would be required for a proper adaptation to the active site of the protease (Neurath and Walsh 1976; Neurath 1980, 1986).

Limited Proteolysis of Proteins by Thermolysin in Trifluoroethanol

Abstract We have examined the proteolysis of model proteins by thermolysin when dissolved in aqueous buffer at neutral pH in the presence of 50% (by vol.) trifluoroethanol (TFE). Under these solvent conditions, proteins acquire a new conformational state characterized by enhanced helical secondary structure, but lacking the specific tertiary interactions of the native species. It was found that the TFE-state of proteins dictates very selective peptide bond fissions by the TFE-resistant thermolysin, which otherwise shows broad substrate specificity. Nicked protein species with a single peptide bond hydrolyzed have been prepared and isolated to homogeneity in the case of bovine ribonuclease A (cleavage at Asn34-Leu35), hen lysozyme (Lys97-Ile98), bovine α-lactalbumin (Ala40-Ile41) and horse cytochrome c (Gly56-Ile57).