1984
Electrochemical and structural characterization of Azotobacter vinelandii flavodoxin II
Helen M. Segal, Thomas Spatzal, Michael G. Hill, Andrew K. Udit, and Douglas C. Rees
Nitrogenase catalyzes the reduction of atmospheric dinitrogen to the bioavailable form of ammonia in a key step of the global nitrogen cycle. In the bacteria Azotobacter vinelandii, the electron transfer protein flavodoxin II can serve as the electron donor to nitrogenase, funneling metabolically generated reducing equivalents into nitrogen fixation. Using a novel electrochemical method and by determining the 1.17 Å resolution crystal structure, properties of Azotobacter flavodoxin II relevant to its role in nitrogen fixation are examined. With this structure, the native nitrogenase electron transfer proteins isolated from A. vinelandii have all been structurally characterized.
2021
Computational design of a specific heavy chain/κ light chain interface for expressing fully IgG bispecific antibodies
K. J. Froning, A. Leaver‐Fay, X. Wu, S. Phan, L. Gao, F. Huang, A. Pustilnik, M. Bacica, K. Houlihan, Q. Chai, J. R. Fitchett, J. Hendle, B. Kuhlman, and S. J. Demarest
Naturally occurring IgG monoclonal antibodies contain two “arms”, each of which bind the same antigen. Bispecific antibodies are engineered proteins that can bind simultaneously to two separate antigens and provide new approaches for treating human disease. For instance, they can be used to co‐localize immune cells to tumor cells to fight cancer. Here, we develop a new approach for creating fully IgG bispecific antibodies by designing an orthogonal interface between the kappa light chain and heavy chain of an antibody. By combining with other reengineered interfaces, these mutations allow the proper assembly of a bispecific IgG antibody that can bind different antigens with each arm. One advantage of this approach is that it avoids the use of antibody fragments, such as single chain variable domains, which can be unstable and are commonly used in many bispecific antibody platforms.
1932
Peptide backbone circularization enhances antifreeze protein thermostability
Corey A. Stevens, Joanna Semrau, Dragos Chiriac, Morgan Litschko, Robert L. Campbell, David N. Langelaan, Steven P. Smith, Peter L. Davies, and John S. Allingham
Antifreeze proteins (AFPs) are a class of ice‐binding proteins that enhance survival of freeze‐intolerant organisms in sub‐zero environments by decreasing the freezing temperature of body fluids. Many AFPs are seasonally produced in these organisms during cold periods of the year, and tend to unfold or aggregate at warmer temperatures. Applications of AFPs for controlling ice in cryo‐medicine, agriculture, biotechnology and the food industry could be greatly expanded with improved resistance of the AFP to denaturation. In this study, we showed that intein‐mediated backbone cyclization produced an AFP that is significantly more stable against heat‐induced unfolding and irreversible inactivation than the non‐cyclized native form.
2098
Neutron crystallographic studies of T4 lysozyme at cryogenic temperature
Le Li, Shantanu Shukla, Flora Meilleur, Robert F. Standaert, Josh Pierce, Dean A. A. Myles, and Matthew J. Cuneo
Hydrogen atoms account for over half of the total atoms in a protein, but are invisible in most protein structures determined by X‐ray crystallography. Bacteriophage T4 lysozyme (T4L) has served as a paradigm for many seminal biophysical studies on protein structure, dynamics and stability, with the positions of interacting polar hydrogen atoms geometrically inferred from the available X‐ray structures. In contrast, neutron crystallography can directly resolve hydrogen atoms in protein structures, even at moderate resolution (∼2.5 Å). The neutron structure of perdeuterated T4L reveals the positions of 1737 hydrogen atoms, providing explicit details of hydrogen bonding interactions in the protein and the protonation states of several important residues. This detailed information may aid future biochemical and computational experiments on this archetypal protein.
