Conformational Transitions in Yeast Chorismate Mutase Important for Allosteric Regulation as Identified by Nuclear Magnetic Resonance Spectroscopy.

Proteins fluctuate between different conformations in solution, and these conformational fluctuations can be important for protein function and allosteric regulation. The chorismate mutase from Saccharomyces cerevisiae (ScCM), a key enzyme in the biosynthesis of aromatic amino acids, is allosterically activated and inhibited by tryptophan and tyrosine, respectively. It was initially proposed that in the absence of effector, ScCM fluctuates between activated R and inhibited T conformations according to the Monod-Wyman-Changeux (MWC) model, although a more complex regulation pattern was later suggested by mutagenesis and kinetic data.

Phase Separation Mediates NUP98 Fusion Oncoprotein Leukemic Transformation.

Unlabelled: NUP98 fusion oncoproteins (FO) are drivers in pediatric leukemias and many transform hematopoietic cells. Most NUP98 FOs harbor an intrinsically disordered region from NUP98 that is prone to liquid-liquid phase separation (LLPS) in vitro. A predominant class of NUP98 FOs, including NUP98-HOXA9 (NHA9), retains a DNA-binding homeodomain, whereas others harbor other types of DNA- or chromatin-binding domains.

Different Solvent and Conformational Entropy Contributions to the Allosteric Activation and Inhibition Mechanisms of Yeast Chorismate Mutase.

Allosteric regulation is important in many biological processes, including cell signaling, gene regulation, and metabolism. chorismate mutase (ScCM) is a key homodimeric enzyme in the shikimate pathway responsible for the generation of aromatic amino acids, where it is allosterically inhibited and activated by Tyr and Trp, respectively. Our previous studies indicated that binding of both allosteric effectors is negatively cooperative, that is binding at one allosteric binding site discourages binding at the other, due to the entropic penalty of binding the second allosteric effector.

Engineering Allostery into Proteins.

Our ability to engineer protein structure and function has grown dramatically over recent years. Perhaps the next level in protein design is to develop proteins whose function can be regulated in response to various stimuli, including ligand binding, pH changes, and light. Endeavors toward these goals have tested and expanded on our understanding of protein function and allosteric regulation.

Energy and Enzyme Activity Landscapes of Yeast Chorismate Mutase at Cellular Concentrations of Allosteric Effectors.

In solution, proteins fluctuate among many conformational substates, with their relative free energies determining substate populations and energy barriers determining conformational exchange kinetics. It has been suggested that members of the conformational ensemble may be responsible for different protein functions, although it is generally difficult to test such a proposal in most systems. A model protein for deciphering individual substate contributions is the homodimeric chorismate mutase (ScCM) enzyme, which is negatively and positively regulated by tyrosine and tryptophan, respectively.

Assigning methyl resonances for protein solution-state NMR studies.

Solution-state NMR is an important tool for studying protein structure and function. The ability to probe methyl groups has substantially expanded the scope of proteins accessible by NMR spectroscopy, including facilitating study of proteins and complexes greater than 100 kDa in size. While the toolset for studying protein structure and dynamics by NMR continues to grow, a major rate-limiting step in these studies is the initial resonance assignments, especially for larger (>50 kDa) proteins.

Biophysical and computational methods to analyze amino acid interaction networks in proteins.

Globular proteins are held together by interacting networks of amino acid residues. A number of different structural and computational methods have been developed to interrogate these amino acid networks. In this review, we describe some of these methods, including analyses of X-ray crystallographic data and structures, computer simulations, NMR data, and covariation among protein sequences, and indicate the critical insights that such methods provide into protein function.