Elena Scaramella

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.

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.

Trifluoroethanol-assisted protein folding: fragment 53–103 of bovine α-lactalbumin

Fragment 53--103 of bovine alpha-lactalbumin, prepared by limited peptic digestion of the protein at low pH, is a 51-residue polypeptide chain crosslinked by two disulfide bonds encompassing helix C (residues 86--98) of the native protein. Refolding of the fully reduced fragment (four--SH groups) is expected to lead to three fully oxidized isomers, the native (61--77, 73--91) and the two misfolded species named ribbon (61--91, 73--77) and beads (61--73, 77--91) isomers. The fragment with correct disulfide bonds was formed in approx. 30% yield when refolding was conducted in aqueous solution at neutral pH in the presence of the redox system constituted by reduced and oxidized glutathione. On the other hand, when the reaction was conducted in 30% (v/v) trifluoroethanol (TFE), the oxidative refolding to the native isomer was almost quantitative. To provide an explanation of the beneficial effect of TFE in promoting the correct oxidative folding, the conformational features of the various fragment species were analyzed by far-UV circular dichroism measurements. The fully reduced fragment is largely unfolded in water, but it becomes helical in aqueous TFE. Correctly refolded fragment is produced most when the helical contents of the reduced and oxidized fragment in aqueous TFE are roughly equal. It is proposed that 30% TFE promotes a native-like format of the fragment and thus an efficient and correct pairing of disulfides. Higher concentrations of TFE, instead, promote some non-native helical secondary structure in the fragment species, thus hampering correct folding.

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).