A multi-angular view on the impact of protein unfolding on biophysical structural data.

The performance of biophysical methods used for the characterization of protein higher order structure (HOS) is key to ensure reliable structural data for drug applications, as these methods are not routinely validated. To assess the analytical performance characteristics, the impact of increasing amounts of heat-denatured material (HDM) on HOS data obtained for a monoclonal antibody (mAb) and its cysteine-conjugated antibody-drug conjugate (ADC) by a set of biophysical methods routinely used in the pharmaceutical industry was evaluated. Relationships between structural data generated by these methods were established using statistical correlation analysis. Most individual methods revealed a linear correlation with increasing amounts of HDM, in the presence of intact mAb or ADC. Overall, Pearson correlation analysis showed strong correlations between the biophysical data obtained. Moreover, biophysical methods that are generally claimed to be orthogonal, were confirmed to provide similar structural insights based on the data obtained. Some methods were capable of differentiating the impact of structural change and/or onset of protein aggregation between the mAb and the ADC. Our results underline the capabilities and performance of the biophysical characterization methods investigated, thereby substantiating these are 'scientifically sound' and 'fit for purpose': the interrogation of protein HOS as part of pharmaceutical development.

Comparison of antioxidant activities of bovine whey proteins before and after simulated gastrointestinal digestion.

Oxidative stress caused by free radicals has been implicated in several human disorders. Dietary antioxidants can help the body to counteract those reactive species and reduce oxidative stress. Antioxidant activity is one of the multiple health-promoting attributes assigned to bovine whey products. The present study investigated whether this activity was retained during upper gut transit using a static simulated in vitro gastrointestinal digestion (SGID) model. The capacity to scavenge free radicals and reduce ferric ion of whey protein isolate (WPI), individual whey proteins, and hydrolysates pre- and post-SGID were measured and compared using various antioxidant assays. In addition, the free AA released from individual protein fractions in physiological gut conditions were characterized. Our results indicated that the antioxidant activity of WPI after exposure to the harsh conditions of the upper gut significantly increased compared with intact WPI. From an antioxidant bioactivity viewpoint, this exposure negates the need for prior hydrolysis of WPI. The whey protein α-lactalbumin showed the highest antioxidant properties post-SGID (oxygen radical absorbance capacity = 1,825.94 ± 50.21 µmol of Trolox equivalents/g of powder) of the 4 major whey proteins tested with the release of the highest amount of the antioxidant AA tryptophan, 6.955 µmol of tryptophan/g of protein. Therefore, α-lactalbumin should be the preferred whey protein in food formulations to boost antioxidant defenses.

Changes in protein conformation and surface hydrophobicity upon peroxidase-catalyzed cross-linking of apo-α-lactalbumin.

In this study, we explore the effect of peroxidase-catalyzed cross-linking on the molecular conformation of apo-α-lactalbumin (apo-α-LA) and the resulting changes in protein surface hydrophobicity. In studying conformational changes, we distinguish between early stages of the reaction ("partial cross-linking"), in which only protein oligomers (10(6) Da > Mw ≥ 10(4) Da) are formed, and a later stage ("full cross-linking"), in which larger protein particles (Mw ≥ 10(6) Da) are formed. Partial cross-linking induces a moderate loss of α-helical content. Surprisingly, further cross-linking leads to a partial return of α-helices that are lost upon early cross-linking. At the same time, for partially and fully cross-linked apo-α-LA, almost all tertiary structure is lost. The protein surface hydrophobicity first increases for partial cross-linking, but then decreases again at full cross-linking. Our results highlight the subtle changes in protein conformation and surface hydrophobicity of apo-α-LA upon peroxidase-catalyzed cross-linking.

Single mutation in Shine-Dalgarno-like sequence present in the amino terminal of lactate dehydrogenase of Plasmodium effects the production of an eukaryotic protein expressed in a prokaryotic system.

One of the most important step in structure-based drug design studies is obtaining the protein in active form after cloning the target gene. In one of our previous study, it was determined that an internal Shine-Dalgarno-like sequence present just before the third methionine at N-terminus of wild type lactate dehydrogenase enzyme of Plasmodium falciparum prevent the translation of full length protein. Inspection of the same region in P. vivax LDH, which was overproduced as an active enzyme, indicated that the codon preference in the same region was slightly different than the codon preference of wild type PfLDH. In this study, 5'-GGAGGC-3' sequence of P. vivax that codes for two glycine residues just before the third methionine was exchanged to 5'-GGAGGA-3', by mimicking P. falciparum LDH, to prove the possible effects of having an internal SD-like sequence when expressing an eukaryotic protein in a prokaryotic system. Exchange was made by site-directed mutagenesis. Results indicated that having two glycine residues with an internal SD-like sequence (GGAGGA) just before the third methionine abolishes the enzyme activity due to the preference of the prokaryotic system used for the expression. This study emphasizes the awareness of use of a prokaryotic system to overproduce an eukaryotic protein.

Protein cluster formation during enzymatic cross-linking of globular proteins.

Work on enzymatic cross-linking of globular food proteins has mainly focused on food functional effects such as improvements of gelation and enhanced stabilization of emulsions and foams, and on the detailed biochemical characterization of the cross-linking chemistry. What is still lacking is a physical characterization of cluster formation and gelation, as has been done for example, for cluster formation and gelation during heat-induced protein aggregation. Here we present preliminary results along these lines. We propose that enzymatic cross-linking of apo-alpha-lactalbumin is a good model system for studying the problem of cluster formation and gelation during enzymatic cross-linking of globular proteins. We present initial results on cluster sizes produced when crosslinking dilute solutions of apo-alpha-lactalbumin with a range of cross-linking enzymes: microbial transglutaminase, horseradish peroxidase, and mushroom tyrosinase. These results are used to highlight similarities and differences between different enzymes, when acting on the same substrate. Next we consider cluster growth and gelation in somewhat more detail for the specific case of cross-linking by horseradish peroxidase, under the periodic addition of H2O2. Upon increasing the initial concentration of apo-alpha-lactalbumin, at a fixed enzyme-to-substrate ratio and fixed reaction time, the size of the clusters at the end of the reaction increases rapidly, and above a critical concentration, gelation occurs. For the conditions that we have used, gelation occurred at very low initial apo-alpha-lactalbumin concentrations of 34% (w/v), indicating a very dilute cross-linked protein network, with a low average number of cross-links per protein. It is found that reactive protein monomers are first rapidly (1-2 h) incorporated into small covalent clusters. This is followed by a much slower phase (up to about 12 h) in which the small clusters are coupled together to form much larger covalent protein clusters. Consistent with this two-step mechanism, atomic force microscopy shows that the covalent protein clusters are very heterogeneous and seem to consist of smaller subclusters.