Symmetry breaking meets multisite modification

Multisite modification is a basic way of conferring functionality to proteins, and a key component of post-translational modification networks. Additional interest in multisite modification stems from its capability of acting as complex information processors. In this paper we connect two seemingly disparate themes: symmetry and multisite modification. We examine different classes of random modification networks of substrates involving separate or common enzymes. We demonstrate that under different instances of symmetry of the modification network (invoked explicitly or implicitly and discussed in the literature), the biochemistry of multisite modification can lead to the symmetry being broken. This is shown computationally and consolidated analytically, revealing parameter regions where this can (and in fact does) happen, and characteristics of the symmetry broken state. We discuss the relevance of these results in situations where exact symmetry is not present. Overall, through our study we show how symmetry breaking (i) can confer new capabilities to protein networks, including concentration robustness of different combinations of species (in conjunction with multiple steady states) (ii) could have been the basis for ordering of multisite modification, which is widely observed in cells (iii) can significantly impact information processing in multisite modification and in cell signalling networks/pathways where multisite modification is present (iv) can be a fruitful new angle for engineering in synthetic biology and chemistry. All in all, the emerging conceptual synthesis provides a new vantage point for the elucidation and the engineering of molecular systems at the junction of chemical and biological systems.

Systematic identification of engineered methionines and oxaziridines for efficient, stable, and site-specific antibody bioconjugation

The field of chemical modification of proteins has been dominated by random modification of lysines or more site-specific labeling of cysteines, each with attendant challenges. Recently, we have developed oxaziridine chemistry for highly selective modification of methionine called redox-activated chemical tagging (ReACT) but have not broadly tested the molecular parameters for efficient and stable protein modification. Here we systematically scanned methionines throughout one of the most popular antibody scaffolds, trastuzumab, used for antibody engineering and drug conjugation. We tested the expression, reactivities, and stabilities of 123 single engineered methionines distributed over the surface of the antibody when reacted with oxaziridine. We found uniformly high expression for these mutants and excellent reaction efficiencies with a panel of oxaziridines. Remarkably, the stability to hydrolysis of the sulfimide varied more than 10-fold depending on temperature and the site of the engineered methionine. Interestingly, the most stable and reactive sites were those that were partially buried, presumably because of their reduced access to water. There was also a 10-fold variation in stability depending on the nature of the oxaziridine, which was determined to be inversely correlated with the electrophilic nature of the sulfimide. Importantly, the stabilities of the best analogs were sufficient to support their use as antibody drug conjugates and potent in a breast cancer mouse xenograft model over a month. These studies provide key parameters for broad application of ReACT for efficient, stable, and site-specific antibody and protein bioconjugation to native or engineered methionines.

Systematic identification of engineered methionines and oxaziridines for efficient, stable, and site-specific antibody bioconjugation

Chemical modification of antibodies is one of the most important bioconjugations utilized by biologists and biotechnology. To date, the field has been dominated by random modification of lysines or more site-specific labeling of cysteines, each with attendant challenges. Recently we have developed oxaziridine chemistry for highly selective and efficient sulfimide modification of methionine called redox-activated chemical tagging (ReACT). Here, we systematically scanned methionines throughout one of the most popular antibody scaffolds, trastuzumab, for antibody engineering and drug conjugation. We tested the expression, reactivities, and stabilities of 123 single engineered methionines distributed over the surface of the antibody when reacted with oxaziridine. We found uniformly high expression for these mutants and generally good reaction efficiencies with the panel of oxaziridines. Remarkably, the stability to hydrolysis of the sulfimide varied more than ten-fold depending on temperature and the site of the engineered methionine. Interestingly, the most stable and reactive sites were those that were partially buried, likely because of their reduced access to water. There was also a ten-fold variation in stability depending on the nature of the oxaziridine, which we determined was inversely correlated with the electrophilic nature of the sulfimide. Importantly, the stabilities of the best analogs and antibody drug conjugate potencies were comparable to those reported for cysteine-maleimide modifications of trastuzumab. We also found our antibody drug conjugates to be potent in a breast cancer mouse xenograft model. These studies provide a roadmap for broad application of ReACT for efficient, stable, and site-specific antibody and protein bioconjugation.

Use of Stored Energy Distribution in Stochastic Vertex Model

The stored energy distribution versus crystal orientation in polycrystalline copper was determined using synchrotron radiation. This distribution is an important input data for recrystallization models. The stochastic vertex model of recrystallization was used in the present work. It is a mixture of the classical vertex model and the Monte Carlo algorithm. Both grain boundary energy and stored energy are taken into account in the calculations. In each elementary step, a reasonably small, random modification of a given vertex position is generated and a corresponding total energy change of a system is calculated. A new vertex position is retained with a probability proportional to the Boltzmann factor. In such a way one avoids solving a complex system of equations. This approach is also closer to the stochastic nature of recrystallization process. The inclusion of the stored energy distribution in the above model enables a good explanation of the recrystallization process. The recrystallization textures for polycrystalline copper rolled to low and high reductions were predicted in agreement with experimental results.

Ductile Separation and Its Role in the Evolution of Gold Contacts

In this paper, we study the role of ductile separation on the evolution of gold-on-gold micro-contacts. A specially designed SPM contact test station has been used to conduct the cycling tests. The evolution of contacts is studied by monitoring the characteristics of the pull-off force. The magnitude of the pull-off force, the force vs. displacement curves, and the rate-dependent pull-off force are sampled during cycling. It is found that ductile separation causes significant and random modification of the contact surfaces. The magnitude of the pull-off force also changes due to the variation of surface morphology. Significant plastic deformation during ductile separation can form a plateau region in the force-displacement curve which is characteristic of ductile separation. This deformation can also contribute to a higher pull-off force when the contacts are cycled at 300Hz compared with cycling at 0.5Hz. The difference between these rate-dependent pull-off forces can be used to indicate the degree of plastic dissipation during each separation.