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. In this practical review, we present strategies to efficiently isotopically label proteins, delineate NMR pulse sequences that can be used to determine methyl resonance assignments in the presence and absence of backbone assignments, and outline computational methods for NMR data analysis. We use our experiences from assigning methyl resonances for the aromatic biosynthetic enzymes tryptophan synthase and chorismate mutase to provide advice for all stages of experimental set-up and data analysis.

Amino acid networks in a (ß/α)8 barrel enzyme change during catalytic turnover.

Proteins can be viewed as small-world networks of amino acid residues connected through noncovalent interactions. Nuclear magnetic resonance chemical shift covariance analyses were used to identify long-range amino acid networks in the α subunit of tryptophan synthase both for the resting state (in the absence of substrate and product) and for the working state (during catalytic turnover). The amino acid networks observed stretch from the surface of the protein into the active site and are different between the resting and working states. Modification of surface residues on the network alters the structural dynamics of active-site residues over 25 Å away and leads to changes in catalytic rates. These findings demonstrate that amino acid networks, similar to those studied here, are likely important for coordinating structural changes necessary for enzyme function and regulation.

Distinct conformational dynamics and allosteric networks in alpha tryptophan synthase during active catalysis.

Experimental observations of enzymes under active turnover conditions have brought new insight into the role of protein motions and allosteric networks in catalysis. Many of these studies characterize enzymes under dynamic chemical equilibrium conditions, in which the enzyme is actively catalyzing both the forward and reverse reactions during data acquisition. We have previously analyzed conformational dynamics and allosteric networks of the alpha subunit of tryptophan synthase under such conditions using NMR. We have proposed that this working state represents a four to one ratio of the enzyme bound with the indole-3-glycerol phosphate substrate (E:IGP) to the enzyme bound with the products indole and glyceraldehyde-3-phosphate (E:indole:G3P). Here, we analyze the inactive D60N variant to deconvolute the contributions of the substrate- and products-bound states to the working state. While the D60N substitution itself induces small structural and dynamic changes, the D60N E:IGP and E:indole:G3P states cannot entirely account for the conformational dynamics and allosteric networks present in the working state. The act of chemical bond breakage and/or formation, or possibly the generation of an intermediate, may alter the structure and dynamics present in the working state. As the enzyme transitions from the substrate-bound to the products-bound state, millisecond conformational exchange processes are quenched and new allosteric connections are made between the alpha active site and the surface which interfaces with the beta subunit. The structural ordering of the enzyme and these new allosteric connections may be important in coordinating the channeling of the indole product into the beta subunit.

Substitution of a Surface-Exposed Residue Involved in an Allosteric Network Enhances Tryptophan Synthase Function in Cells.

Networks of noncovalent amino acid interactions propagate allosteric signals throughout proteins. Tryptophan synthase (TS) is an allosterically controlled bienzyme in which the indole product of the alpha subunit (αTS) is transferred through a 25 Å hydrophobic tunnel to the active site of the beta subunit (ßTS). Previous nuclear magnetic resonance and molecular dynamics simulations identified allosteric networks in αTS important for its function. We show here that substitution of a distant, surface-exposed network residue in αTS enhances tryptophan production, not by activating αTS function, but through dynamically controlling the opening of the indole channel and stimulating ßTS activity. While stimulation is modest, the substitution also enhances cell growth in a tryptophan-auxotrophic strain of Escherichia coli compared to complementation with wild-type αTS, emphasizing the biological importance of the network. Surface-exposed networks provide new opportunities in allosteric drug design and protein engineering, and hint at potential information conduits through which the functions of a metabolon or even larger proteome might be coordinated and regulated.

Coordinated Network Changes across the Catalytic Cycle of Alpha Tryptophan Synthase.

Networks of noncovalent interactions are important for protein structural dynamics. We used nuclear magnetic resonance chemical shift covariance analyses on an inactive variant of the alpha subunit of tryptophan synthase to map amino acid interaction networks across its catalytic cycle. Although some network connections were common to every enzyme state, many of the network connections strengthened or weakened over the catalytic cycle; these changes were highly coordinated. These results suggest a higher level of network organization. Our analyses identified periodic, second-order networks that show highly coordinated interaction changes across the catalytic cycle. These periodic networks may help synchronize the sequence of structural transitions necessary for enzyme function. Molecular dynamics simulations identified interaction changes across the catalytic cycle, including those involving the catalytic residue Glu49, which may help drive other interaction changes throughout the enzyme structure. Similar periodic networks may direct structural transitions and allosteric interactions in other proteins.

Engineered control of enzyme structural dynamics and function.

Enzymes undergo a range of internal motions from local, active site fluctuations to large-scale, global conformational changes. These motions are often important for enzyme function, including in ligand binding and dissociation and even preparing the active site for chemical catalysis. Protein engineering efforts have been directed towards manipulating enzyme structural dynamics and conformational changes, including targeting specific amino acid interactions and creation of chimeric enzymes with new regulatory functions. Post-translational covalent modification can provide an additional level of enzyme control. These studies have not only provided insights into the functional role of protein motions, but they offer opportunities to create stimulus-responsive enzymes. These enzymes can be engineered to respond to a number of external stimuli, including light, pH, and the presence of novel allosteric modulators. Altogether, the ability to engineer and control enzyme structural dynamics can provide new tools for biotechnology and medicine.

Millisecond Timescale Motions Connect Amino Acid Interaction Networks in Alpha Tryptophan Synthase.

Tryptophan synthase is a model system for understanding allosteric regulation within enzyme complexes. Amino acid interaction networks were previously delineated in the isolated alpha subunit (αTS) in the absence of the beta subunit (ßTS). The amino acid interaction networks were different between the ligand-free enzyme and the enzyme actively catalyzing turnover. Previous X-ray crystallography studies indicated only minor localized changes when ligands bind αTS, and so, structural changes alone could not explain the changes to the amino acid interaction networks. We hypothesized that the network changes could instead be related to changes in conformational dynamics. As such, we conducted nuclear magnetic resonance relaxation studies on different substrate- and products-bound complexes of αTS. Specifically, we collected 15N R2 relaxation dispersion data that reports on microsecond-to-millisecond timescale motion of backbone amide groups. These experiments indicated that there are conformational exchange events throughout αTS. Substrate and product binding change specific motional pathways throughout the enzyme, and these pathways connect the previously identified network residues. These pathways reach the αTS/ßTS binding interface, suggesting that the identified dynamic networks may also be important for communication with the ßTS subunit.

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. This information can be leveraged towards the design of new allosteric drugs, and the engineering of new protein function and protein regulation strategies.

Severing of a hydrogen bond disrupts amino acid networks in the catalytically active state of the alpha subunit of tryptophan synthase.

Conformational changes in the ß2α2 and ß6α6 loops in the alpha subunit of tryptophan synthase (αTS) are important for enzyme catalysis and coordinating substrate channeling with the beta subunit (ßTS). It was previously shown that disrupting the hydrogen bond interactions between these loops through the T183V substitution on the ß6α6 loop decreases catalytic efficiency and impairs substrate channeling. Results presented here also indicate that the T183V substitution decreases catalytic efficiency in Escherchia coli αTS in the absence of the ßTS subunit. Nuclear magnetic resonance (NMR) experiments indicate that the T183V substitution leads to local changes in the structural dynamics of the ß2α2 and ß6α6 loops. We have also used NMR chemical shift covariance analyses (CHESCA) to map amino acid networks in the presence and absence of the T183V substitution. Under conditions of active catalytic turnover, the T183V substitution disrupts long-range networks connecting the catalytic residue Glu49 to the αTS-ßTS binding interface, which might be important in the coordination of catalytic activities in the tryptophan synthase complex. The approach that we have developed here will likely find general utility in understanding long-range impacts on protein structure and dynamics of amino acid substitutions generated through protein engineering and directed evolution approaches, and provide insight into disease and drug-resistance mutations.

Loop-loop interactions govern multiple steps in indole-3-glycerol phosphate synthase catalysis.

Substrate binding, product release, and likely chemical catalysis in the tryptophan biosynthetic enzyme indole-3-glycerol phosphate synthase (IGPS) are dependent on the structural dynamics of the ß1α1 active-site loop. Statistical coupling analysis and molecular dynamic simulations had previously indicated that covarying residues in the ß1α1 and ß2α2 loops, corresponding to Arg54 and Asn90, respectively, in the Sulfolobus sulfataricus enzyme (ssIGPS), are likely important for coordinating functional motions of these loops. To test this hypothesis, we characterized site mutants at these positions for changes in catalytic function, protein stability and structural dynamics for the thermophilic ssIGPS enzyme. Although there were only modest changes in the overall steady-state kinetic parameters, solvent viscosity and solvent deuterium kinetic isotope effects indicated that these amino acid substitutions change the identity of the rate-determining step across multiple temperatures. Surprisingly, the N90A substitution had a dramatic effect on the general acid/base catalysis of the dehydration step, as indicated by the loss of the descending limb in the pH rate profile, which we had previously assigned to Lys53 on the ß1α1 loop. These changes in enzyme function are accompanied with a quenching of ps-ns and µs-ms timescale motions in the ß1α1 loop as measured by nuclear magnetic resonance studies. Altogether, our studies provide structural, dynamic and functional rationales for the coevolution of residues on the ß1α1 and ß2α2 loops, and highlight the multiple roles that the ß1α1 loop plays in IGPS catalysis. Thus, substitution of covarying residues in the active-site ß1α1 and ß2α2 loops of indole-3-glycerol phosphate synthase results in functional, structural, and dynamic changes, highlighting the multiple roles that the ß1α1 loop plays in enzyme catalysis and the importance of regulating the structural dynamics of this loop through noncovalent interactions with nearby structural elements.