Mikael Akke

Chemical exchange saturation transfer (CEST) has become an indispensable NMR method to characterize slow exchange affecting biomacromolecules, especially for cases involving exchange between a major state and a minor state, the latter of which is often invisible in the spectrum. The CEST method is based on successive irradiation of selective regions of the NMR spectrum using a weak radiofrequency field, B ₁ , while observing the effect on the visible major state when the B ₁ field saturates the invisible minor state. The need for selective saturation of narrow spectral regions has to date required acquisition of many tens of two-dimensional CEST spectra to sample the entire spectrum with sufficient resolution. Here we present the ACCEST method which measures an entire CEST profile from a single two-dimensional accordion-CEST spectrum plus a reference spectrum. ACCEST is based on the concept of accordion spectroscopy, where in the present implementation the carrier frequency of the weak saturating B ₁ field is stepped in synchrony with the dwell-time incrementation in the indirect dimension of the two-dimensional spectrum. We benchmarked ACCEST against conventional CEST, resulting in excellent agreement for both backbone ¹⁵ N and methyl ¹³ C CEST profiles. ACCEST offers substantial time savings that scale linearly with the number of spectra required in the corresponding conventional CEST experiment. Thus, ACCEST can dramatically speed up lengthy serial experiments, such as ligand titrations or temperature-dependent studies, and enable studies of non-equilibrium systems or samples with limited lifetimes.

Conformational transitions are critical for protein function and often involve substantial rearrangements of the interior packing of amino acid residues. Rotations of aromatic side chains—so called “ring flips”—require transient expansion of the surrounding protein core and represent hallmark examples of transient conformational fluctuations. We recently demonstrated that activation volume and isothermal compressibility provide unique insights into ring flip transition states. Here, we report transition‐state compressibilities for four aromatic side chains in bovine pancreatic trypsin inhibitor (BPTI). The compressibilities vary widely, ranging from values typical of unfolded proteins to those of the fully folded state. Notably, transition‐state compressibility correlates negatively with activation energy, indicating that the likelihood of a ring flip scales with the degree of expansion in the transition state. Our results show that different structural regions, even within a small protein such as BPTI, display distinct dynamic transitions and volume fluctuations, reflecting varying extents of structural rearrangement along the transition pathways. Thus, isothermal compressibility emerges as a powerful parameter yielding insights into the relationships between ground‐state structure and the transition‐state expansion during conformational transitions.

Metabolically engineered baker’s yeast can be used to produce chiral amines through whole-cell bioconversion of prochiral ketones. This study investigates the modulation of the alanine-pyruvate metabolic node to enhance reductive amination, using the stereoselective conversion of benzylacetone to (S)-1-methyl-3-phenylpropylamine (MPPA) as a model reaction. Chromosomal integration of multiple copies of the promiscuous omega transaminase from Chromobacterium violaceum (cv-ATA) resulted in an active yeast catalyst. Physiological characterization in bioreactors under aerobic batch cultivation revealed that amine production occurred only under post-diauxic growth on ethanol. To reduce native alanine utilization, the endogenous alanine aminotransferase (ALT1) was knocked out and replaced with cv-ATA. To rapidly employ this strategy in other strains, a simple CRISPR/cas9 method for universal gene replacement was developed. The replacement of ALT1 with cv-ATA improved the reaction by 2.6-fold compared to the control strain with intact ALT1. NMR measurements of metabolites originating from ¹⁵N L-alanine and ¹³C glucose indicated that pyruvate formation during growth on glucose inhibited amine production. Under optimal conditions, the biocatalytic bioconversion of benzylacetone to MPPA reached a yield of 58%.

Histidine is a key amino-acid residue in proteins with unique properties engendered by its imidazole side chain that can exist in three different states: two different neutral tautomeric forms and a protonated, positively charged one with a pK_a value close to physiological pH. Commonly, two or all three states coexist and interchange rapidly, enabling histidine to act as both donor and acceptor of hydrogen bonds, coordinate metal ions, and engage in acid/base catalysis. Understanding the exchange dynamics among the three states is critical for assessing histidine's mechanistic role in catalysis, where the rate of proton exchange and interconversion among tautomers might be rate limiting for turnover. Here, we determine the exchange kinetics of histidine residues with pK_a values representative of the accessible range from 5 to 9 by measuring pH-dependent ¹⁵N, ¹³C, and ¹H transverse relaxation rate constants for 5 nuclei in each imidazole. Proton exchange between the imidazole and the solvent is mediated by hydronium ions at acidic and neutral pH, whereas hydroxide mediated exchange becomes the dominant mechanism at basic pH. Proton transfer is very fast and reaches the diffusion limit for pK_a values near neutral pH. We identify a direct pathway between the two tautomeric forms, likely mediated by a bridging water molecule or, in the case of high pH, hydroxide ion. For histidines with pK_a 7, we determine all rate constants (lifetimes) involving protonation over the entire pH range. Our approach should enable critical insights into enzymatic acid/base catalyzed reactions involving histidines in proteins.

Structural characterization of protein-ligand binding interfaces at atomic resolution is essential for improving the design of specific and potent inhibitors. Herein, we explored fast ¹⁹F- and ¹H-detected magic angle spinning NMR spectroscopy to investigate the interaction between two fluorinated ligand diastereomers with the microcrystalline galectin-3 carbohydrate recognition domain. The detailed environment around the fluorine atoms was mapped by 2D ¹³C-¹⁹F and ¹H-¹⁹F dipolar correlation experiments and permitted characterization of the binding interface. Our results demonstrate that ¹⁹F MAS NMR is a powerful tool for detailed characterization of protein-ligand interfaces and protein interactions at the atomic level.

Protein–ligand complex formation is fundamental to biological function. A central question is whether proteins spontaneously adopt binding-competent conformations to which ligands bind conformational selection (CS) or whether ligands induce the binding-competent conformation induced fit (IF). Here, we resolve the CS and IF binding pathways by characterizing protein conformational dynamics over a wide range of ligand concentrations using NMR relaxation dispersion. We determined the relative flux through the two pathways using a four-state binding model that includes both CS and IF. Experiments conducted without ligand show that galectin-3 exchanges between the ground-state conformation and a high-energy conformation similar to the ligand-bound conformation, demonstrating that CS is a plausible pathway. Near-identical crystal structures of the apo and ligand-bound states suggest that the high-energy conformation in solution corresponds to the apo crystal structure. Stepwise additions of the ligand lactose induce progressive changes in the relaxation dispersions that we fit collectively to the four-state model, yielding all microscopic rate constants and binding affinities. The ligand affinity is higher for the bound-like conformation than for the ground state, as expected for CS. Nonetheless, the IF pathway contributes greater than 70% of the total flux even at low ligand concentrations. The higher flux through the IF pathway is explained by considerably higher rates of exchange between the two protein conformations in the ligand-associated state. Thus, the ligand acts to decrease the activation barrier between protein conformations in a manner reciprocal to enzymatic transition-state stabilization of reactions involving ligand transformation.

β-Mannanases hydrolyze β-mannans, important components of plant and microalgae cell walls. Retaining β-mannanases can also catalyze transglycosylation, forming new β-mannosidic bonds that are applicable for synthesis. This study focused on the blue mussel (Mytilus edulis) GH5_10 β-mannanase MeMan5A, which contains two semi-conserved tryptophans (W240 and W281) in the distal subsite +2 of its active site cleft. Variants of MeMan5A were generated by replacing one or both tryptophans with alanines. The substitutions reduced the enzyme’s catalytic efficiency (k_cat/K_m using galactomannan) by three-fold (W281A), five-fold (W240A), or 20-fold (W240A/W281A). Productive binding modes were analyzed by ¹⁸O labeling of hydrolysis products and mass spectrometry. Results show that the substitution of both tryptophans was required to shift away from the dominant binding mode of mannopentaose (spanning subsites −3 to +2), suggesting that both tryptophans contribute to glycan binding. NMR spectroscopy and molecular dynamics simulations were conducted to analyze protein flexibility and glycan binding. We suggest that W240 is rigid and contributes to +2 subsite mannosyl specificity, while W281 is flexible, which enables stacking interactions in the +2 subsite by loop movement to facilitate binding. The substitutions significantly reduced or eliminated transglycosylation with saccharides as glycosyl acceptors but had no significant effect on reactions with alcohols.

Plant hemoglobins, often referred to as phytoglobins, play important roles in abiotic stress tolerance. Several essential small physiological metabolites can be bound to these heme proteins. In addition, phytoglobins can catalyze a range of different oxidative reactions in vivo. These proteins are often oligomeric, but the degree and relevance of subunit interactions are largely unknown. In this study, we delineate which residues are involved in dimer formation of a sugar beet phytoglobin type 1.2 (BvPgb1.2) using NMR relaxation experiments. E. coli cells harboring a phytoglobin expression vector were cultivated in isotope-labeled (²H, ¹³C and ¹⁵N) M9 medium. The triple-labeled protein was purified to homogeneity using two chromatographic steps. Two forms of BvPgb1.2 were examined, the oxy-form and the more stable cyanide-form. Using three-dimensional triple-resonance NMR experiments, sequence-specific assignments for CN-bound BvPgb1.2 were achieved for 137 backbone amide cross-peaks in the ¹H-¹⁵N TROSY spectrum, which amounts to 83% of the total number of 165 expected cross-peaks. A large proportion of the non-assigned residues are located in α-helixes G and H, which are proposed to be involved in protein dimerization. Such knowledge around dimer formation will be instrumental for developing a better understanding of phytoglobins’ roles in planta.

Aromatic residues form a significant part of the protein core, where they make tight interactions with multiple surrounding side chains. Despite the dense packing of internal side chains, the aromatic rings of phenylalanine and tyrosine residues undergo 180° rotations, or flips, which are mediated by transient and large-scale “breathing” motions that generate sufficient void volume around the aromatic ring. Forty years after the seminal work by Wagner and Wüthrich, NMR studies of aromatic ring flips are now undergoing a renaissance as a powerful means of probing fundamental dynamic properties of proteins. Recent developments of improved NMR methods and isotope labeling schemes have enabled a number of advances in addressing the mechanisms and energetics of aromatic ring flips. The nature of the transition states associated with ring flips can be described by thermodynamic activation parameters, including the activation enthalpy, activation entropy, activation volume, and also the isothermal volume compressibility of activation. Consequently, it is of great interest to study how ring flip rate constants and activation parameters might vary with protein structure and external conditions like temperature and pressure. The field is beginning to gather such data for aromatic residues in a variety of environments, ranging from surface exposed to buried. In the future, the combination of solution and solid-state NMR spectroscopy together with molecular dynamics simulations and other computational approaches is likely to provide detailed information about the coupled dynamics of aromatic rings and neighboring residues. In this Perspective, we highlight recent developments and provide an outlook toward the future.