Allosteric communication between ligand binding domains modulates substrate inhibition in adenylate kinase.

Enzymes play a vital role in life processes; they control chemical reactions and allow functional cycles to be synchronized. Many enzymes harness large-scale motions of their domains to achieve tremendous catalytic prowess and high selectivity for specific substrates. One outstanding example is provided by the three-domain enzyme adenylate kinase (AK), which catalyzes phosphotransfer between ATP to AMP. Here we study the phenomenon of substrate inhibition by AMP and its correlation with domain motions. Using single-molecule FRET spectroscopy, we show that AMP does not block access to the ATP binding site, neither by competitive binding to the ATP cognate site nor by directly closing the LID domain. Instead, inhibitory concentrations of AMP lead to a faster and more cooperative domain closure by ATP, leading in turn to an increased population of the closed state. The effect of AMP binding can be modulated through mutations throughout the structure of the enzyme, as shown by the screening of an extensive AK mutant library. The mutation of multiple conserved residues reduces substrate inhibition, suggesting that substrate inhibition is an evolutionary well conserved feature in AK. Combining these insights, we developed a model that explains the complex activity of AK, particularly substrate inhibition, based on the experimentally observed opening and closing rates. Notably, the model indicates that the catalytic power is affected by the microsecond balance between the open and closed states of the enzyme. Our findings highlight the crucial role of protein motions in enzymatic activity.

Role of Repeated Conformational Transitions in Substrate Binding of Adenylate Kinase.

The catalytic cycle of the enzyme adenylate kinase involves large conformational motions between open and closed states. A previous single-molecule experiment showed that substrate binding tends to accelerate both the opening and the closing rates and that a single turnover event often involves multiple rounds of conformational switching. In this work, we showed that the repeated conformational transitions of adenylate kinase are essential for the relaxation of incorrectly bound substrates into the catalytically competent conformation by combining all-atom and coarse-grained molecular simulations. In addition, free energy calculations based on all-atom and coarse-grained models demonstrated that the enzyme with incorrectly bound substrates has much a lower free energy barrier for domain opening compared to that with the correct substrate conformation, which may explain the the acceleration of the domain opening rate by substrate binding. The results of this work provide mechanistic understanding to previous experimental observations and shed light onto the interplay between conformational dynamics and enzyme catalysis.

Control of protein activity by photoinduced spin polarized charge reorganization.

Considerable electric fields are present within living cells, and the role of bioelectricity has been well established at the organismal level. Yet much remains to be learned about electric-field effects on protein function. Here, we use phototriggered charge injection from a site-specifically attached ruthenium photosensitizer to directly demonstrate the effect of dynamic charge redistribution within a protein. We find that binding of an antibody to phosphoglycerate kinase (PGK) is increased twofold under illumination. Remarkably, illumination is found to suppress the enzymatic activity of PGK by a factor as large as three. These responses are sensitive to the photosensitizer position on the protein. Surprisingly, left (but not right) circularly polarized light elicits these responses, indicating that the electrons involved in the observed dynamics are spin polarized, due to spin filtration by protein chiral structures. Our results directly establish the contribution of electrical polarization as an allosteric signal within proteins. Future experiments with phototriggered charge injection will allow delineation of charge rearrangement pathways within proteins and will further depict their effects on protein function.

Enhanced Sensitivity to Local Dynamics in Peptides by Use of Temperature-Jump IR Spectroscopy and Isotope Labeling.

Site-specific isotopic labeling of molecules is a widely used approach in IR spectroscopy to resolve local contributions to vibrational modes. The induced frequency shift of the corresponding IR band depends on the substituted masses, as well as on hydrogen bonding and vibrational coupling. The impact of these different factors was analyzed with a designed three-stranded ß-sheet peptide and by use of selected 13 C isotope substitutions at multiple positions in the peptide backbone. Single-strand labels give rise to isotopically shifted bands at different frequencies, depending on the specific sites; this demonstrates sensitivity to the local environment. Cross-strand double- and triple-labeled peptides exhibited two resolved bands that could be uniquely assigned to specific residues, the equilibrium IR spectra of which indicated only weak local-mode coupling. Temperature-jump IR laser spectroscopy was applied to monitor structural dynamics and revealed an impressive enhancement of the isotope sensitivity to both local positions and coupling between them, relative to that of equilibrium FTIR spectroscopy. Site-specific relaxation rates were altered upon the introduction of additional cross-strand isotopes. Likewise, the rates for the global ß-sheet dynamics were affected in a manner dependent on the distinct relaxation behavior of the labeled oscillator. This study reveals that isotope labels provide not only local structural probes, but rather sense the dynamic complexity of the molecular environment.

Isotopically Site-Selected Dynamics of a Three-Stranded ß-Sheet Peptide Detected with Temperature-Jump Infrared-Spectroscopy.

Infrared detected temperature-jump (T-jump) spectroscopy and site-specific isotopic labeling were applied to study a model three-stranded ß-sheet peptide with the goal of individually probing the dynamics of strand and turn structural elements. This peptide had two DPro-Gly (pG) turn sequences to stabilize the two component hairpins, which were labeled with 13C═O on each of the Gly residues to resolve them spectroscopically. Labeling the second turn on the amide preceding the DPro (Xxx-DPro amide) provided an alternate turn label as a control. Placing 13C═O labels on specific in-strand residues gave shifted modes that overlap the Xxx-DPro amide I' modes. Their impact could be separated from the turn dynamics by a novel difference transient analysis approach. Fourier-transform infrared spectra were modeled with density functional theory-computations which showed the local, isotope-selected vibrations were effectively uncoupled from the other amide I modes. Our T-jump dynamics results, combined with nuclear magnetic resonance structures and equilibrium spectral measurements, showed the first turn to be most stable and best formed with the slowest dynamics, whereas the second turn and first strand (N-terminus) had similar dynamics, and the third strand (C-terminus) had the fastest dynamics and was the least structured. The relative dynamics of the strands, Xxx-DPro amides, and 13C-labeled Gly residues on the turns also qualitatively corresponded to molecular dynamics (MD) simulations of turn and strand fluctuations. MD trajectories indicated the turns to be bistable, with the first turn being Type I' and the second turn flipping from I' to II'. The differences in relaxation times for each turn and the separate strands revealed that the folding process of this turn-stabilized ß-sheet structure proceeds in a multistep process.

Role of Aromatic Cross-Links in Structure and Dynamics of Model Three-Stranded ß-Sheet Peptides.

A series of closely related peptide sequences that form triple-strand structures was designed with a variation of cross-strand aromatic interactions and spectroscopically studied as models for ß-sheet formation and stabilities. Structures of the three-strand models were determined with NMR methods and temperature-dependent equilibrium studies performed using circular dichroism and Fourier transform infrared spectroscopies. Our equilibrium data show that the presence of a direct cross-strand aromatic contact in an otherwise folded peptide does not automatically result in an increased thermal stability and can even distort the structure. The effect on the conformational dynamics was studied with infrared-detected temperature-jump relaxation methods and revealed a high sensitivity to the presence and the location of the aromatic cross-links. Aromatic contacts in the three-stranded peptides slow down the dynamics in a site-specific manner, and the impact seems to be related to the distance from the turn. With a Xxx-DPro linkage as a probe with some sensitivity for the turn, small differences were revealed in the relative relaxation of the sheet strands and turn regions. In addition, we analyzed the component hairpins, which showed less uniform dynamics as compared to the parent three-stranded ß-sheet peptides.

Biomolecular dynamics studied with IR-spectroscopy using quantum cascade lasers combined with nanosecond perturbation techniques.

Early events of protein folding can be studied with fast perturbation techniques triggering non-equilibrium relaxation dynamics. A nanosecond laser-excited pH-jump or temperature-jump (T-jump) was applied to initiate helix folding or unfolding of poly-l-glutamic acid (PGA). PGA is a homopolypeptide with titratable carboxyl side-chains whose protonation degree determines the PGA conformation. A pH-jump was realized by the photochemical release of protons and induces PGA folding due to protonation of the side-chains. Otherwise, the helical conformation can be unfolded by a T-jump. We operated under conditions where PGA does not aggregate and temperature and pH are the regulatory properties of its conformation. The experiments were performed in such a manner that the folding/unfolding jump proceeded to the same PGA conformation. We quantified the increase/decrease in helicity induced by the pH-/T-jump and demonstrated that the T-jump results in a relatively small change in helical content in contrast to the pH-jump. This is caused by the strong pH-dependence of the PGA conformation. The conformational changes were detected by time-resolved single wavelength IR-spectroscopy using quantum cascade lasers (QCL). We could independently observe the kinetics for α-helix folding and unfolding in PGA by using different perturbation techniques and demonstrate the high sensitivity of time-resolved IR-spectroscopy to study protein folding mechanisms.

Site-Specific Dynamics of ß-Sheet Peptides with (D) Pro-Gly Turns Probed by Laser-Excited Temperature-Jump Infrared Spectroscopy.

Turn residues and side-chain interactions play an important role for the folding of ß-sheets. We investigated the conformational dynamics of a three-stranded ß-sheet peptide ((D) P(D) P) and a two-stranded ß-hairpin (WVYY-(D) P) by time-resolved temperature-jump (T-jump) infrared spectroscopy. Both peptide sequences contain (D) Pro-Gly residues that favor a tight ß-turn. The three-stranded ß-sheet (Ac-VFITS(D) PGKTYTEV(D) PGOKILQ-NH2 ) is stabilized by the turn sequences, whereas the ß-hairpin (SWTVE(D) PGKYTYK-NH2 ) folding is assisted by both the turn sequence and hydrophobic cross-strand interactions. Relaxation times after the T-jump were monitored as a function of temperature and occur on a sub-microsecond time scale, (D) P(D) P being faster than WVYY-(D) P. The Xxx-(D) Pro tertiary amide provides a detectable IR band, allowing us to probe the dynamics site-specifically. The relative importance of the turn versus the intrastrand stability in ß-sheet formation is discussed.