The yeast Pichia pastoris is a cost-effective and easily scalable system for recombinant protein production. In this work we compared the conformation of the receptor binding domain (RBD) from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Spike protein expressed in P. pastoris and in the well established HEK-293T mammalian cell system. RBD obtained from both yeast and mammalian cells was properly folded, as indicated by UV-absorption, circular dichroism and tryptophan fluorescence. They also had similar stability, as indicated by temperature-induced unfolding (observed Tm were 50 °C and 52 °C for RBD produced in P. pastoris and HEK-293T cells, respectively). Moreover, the stability of both variants was similarly reduced when the ionic strength was increased, in agreement with a computational analysis predicting that a set of ionic interactions may stabilize RBD structure. Further characterization by high-performance liquid chromatography, size-exclusion chromatography and mass spectrometry revealed a higher heterogeneity of RBD expressed in P. pastoris relative to that produced in HEK-293T cells, which disappeared after enzymatic removal of glycans. The production of RBD in P. pastoris was scaled-up in a bioreactor, with yields above 45 mg/L of 90% pure protein, thus potentially allowing large scale immunizations to produce neutralizing antibodies, as well as the large scale production of serological tests for SARS-CoV-2.

The self-assembly of proteins is an essential process for a variety of cellular functions including cell respiration, mobility and division. On the other hand, protein or peptide misfolding and aggregation is related to the development of Parkinson’s disease and Alzheimer’s disease, among other aggregopathies. As a consequence, significant research efforts are directed towards the understanding of this process. In this review, we are focused on the use of UV-Visible Absorption Spectroscopy, Fluorescence Spectroscopy and Circular Dichroism to evaluate the self-organization of proteins and peptides in solution. These spectroscopic techniques are commonly available in most chemistry and biochemistry research laboratories, and together they are a powerful approach for initial as well as routine evaluation of protein and peptide self-assembly and aggregation under different environmental stimulus. Furthermore, these spectroscopic techniques are even suitable for studying complex systems like those in the food industry or pharmaceutical formulations, providing an overall idea of the folding, self-assembly, and aggregation processes, which is challenging to obtain with high-resolution methods. Here, we compiled and discussed selected examples, together with our results and those that helped us better to understand the process of protein and peptide aggregation. We put particular emphasis on the basic description of the methods as well as on the experimental considerations needed to obtain meaningful information, to help those who are just getting into this exciting area of research. Moreover, this review is particularly useful to those out of the field who would like to improve reproducibility in their cellular and biomedical experiments, especially while working with peptide and protein systems as an external stimulus. Our final aim is to show the power of these low-resolution techniques to improve our understanding of the self-assembly of peptides and proteins and translate this fundamental knowledge in biomedical research or food applications.

The purpose of this work was to obtain information about conformational changes of the plasma membrane Ca 2؉ -pump (PMCA) in the membrane region upon interaction with Ca 2؉ , calmodulin (CaM) and acidic phospholipids. To this end, we have quantified labeling of PMCA with the photoactivatable phosphatidylcholine analog [125 I]TID-PC/16, measuring the shift of conformation E 2 to the auto-inhibited conformation E 1 I and to the activated E 1 A state, titrating the effect of Ca 2؉ under different conditions. Using a similar approach, we also determined the CaM-PMCA dissociation constant. The results indicate that the PMCA possesses a high affinity site for Ca 2؉ regardless of the presence or absence of activators. Modulation of pump activity is exerted through the C-terminal domain, which induces an apparent auto-inhibited conformation for Ca 2؉ transport but does not modify the affinity for Ca 2؉ at the transmembrane domain. The C-terminal domain is affected by CaM and CaM-like treatments driving the auto-inhibited conformation E 1 I to the activated E 1 A conformation and thus modulating the transport of Ca 2؉ . This is reflected in the different apparent constants for Ca 2؉ in the absence of CaM (calculated by Ca 2؉ -ATPase activity) that sharply contrast with the lack of variation of the affinity for the Ca 2؉ site at equilibrium. This is the first time that equilibrium constants for the dissociation of Ca 2؉ and CaM ligands from PMCA complexes are measured through the change of transmembrane conformations of the pump. The data further suggest that the transmembrane domain of the PMCA undergoes major rearrangements resulting in altered lipid accessibility upon Ca 2؉ binding and activation.Detailed structural information about the plasma membrane calcium pump (PMCA) 2 is currently lacking. This pump is an integral part of the Ca 2ϩ signaling mechanism (1) and is thus a crucial component of cell function. It is highly regulated by calmodulin (CaM), which activates this protein by binding to an auto-inhibitory region (2) and changes the conformation of the pump from an inhibited state to an activated one (2, 3).Information about the structure and assembly of the transmembrane domain of an integral membrane protein can be obtained from an analysis of the lipid-protein interactions. In this work, we have used a hydrophobic photolabeling method to study the noncovalent interactions between the membrane domain of the PMCA and surrounding phospholipids under different experimental conditions that lead to known conformations. It has been previously demonstrated that both the conformation and the activity of the pump protein are preserved in either solubilized or reconstituted purified preparations compared with the native pump located in the erythrocyte (4).In recent work, we assessed changes in the overall exposure of PMCA to surrounding lipids by quantifying the extent of protein labeling by the photoactivatable phosphatidylcholine analog [125 I]TID-PC/16 under different conditions (5). This showed that labeling of ...