Heme-regulated eukaryotic initiation factor 2␣ (eIF2␣) kinase (HRI) functions in response to the heme iron concentration. At the appropriate heme iron concentrations under normal conditions, HRI function is suppressed by binding of the heme iron. Conversely, upon heme iron shortage, HRI autophosphorylates and subsequently phosphorylates the substrate, eIF2␣, leading to the termination of protein synthesis. The molecular mechanism of heme sensing by HRI, including identification of the specific binding site, remains to be established. In the present study we demonstrate that His-119/His-120 and Cys-409 are the axial ligands for the Fe(III)-protoporphyrin IX complex (hemin) in HRI, based on spectral data on site-directed mutant proteins. Cys-409 is part of the heme-regulatory CysPro motif in the kinase domain. A P410A full-length mutant protein displayed loss of heme iron affinity. Surprisingly, inhibitory effects of the heme iron on catalysis and changes in the heme dissociation rate constants in full-length His-119/His-120 and Cys-409 mutant proteins were marginally different to wild type. In contrast, heme-induced inhibition of Cys-409 mutants of the isolated kinase domain and N-terminal-truncated proteins was substantially weaker than that of the full-length enzyme. A pulldown assay disclosed heme-dependent interactions between the N-terminal and kinase domains. Accordingly, we propose that heme regulation is induced by interactions between heme and the catalytic domain in conjunction with global tertiary structural changes at the N-terminal domain that accompany heme coordination and not merely by coordination of the heme iron with amino acids on the protein surface.Eukaryotic cells decrease their overall rates of protein synthesis for survival in response to a variety of stress conditions, such as shortage of amino acids, UV light illumination, virus infection, and accumulation of denatured proteins. Much of the decrease in protein synthesis is caused by phosphorylation of eukaryotic initiation factor 2␣ (eIF2␣) 4 at Ser-51 by eIF2␣ kinases that respond specifically to stress (1-4). Heme-regulated eIF2␣ kinase or heme-regulated inhibitor (HRI) is a member of the eIF2␣ kinase family that controls globin synthesis in response to the heme concentration in reticulocytes (5-8). HRI is inactive at normal heme concentrations. Under conditions of heme deficiency, the enzyme is activated by autophosphorylation and subsequently phosphorylates eIF2␣ at Ser-51. In addition to globin, HRI controls the synthesis of tryptophan 2,3-dioxygenase and cytochrome P450 2B in liver upon acute porphyria (9, 10). Thus, HRI is possibly one of the most important existing heme sensor protein families for eukaryote survival in response to cell emergency states.Heme-responsive/sensing proteins, also known as "heme sensor proteins" are a current focus of investigation. In these proteins heme association (or dissociation) per se regulates various important physiological functions, such as transcription, proteolysis, and kinase activity (11...

The effect of fluorine substitution on the aromaticity of polycyclic hydrocarbons (PAH) is investigated. Magnetically induced current densities, current pathways, and current strengths, which can be used to assess molecular aromaticity, are calculated using the gauge-including magnetically induced current method (GIMIC). The degree of aromaticity of the individual rings is compared to those obtained using calculated nucleus-independent chemical shifts at the ring centers (NICS(0) and NICS(0)(zz)). Calculations of explicitly integrated current strengths for selected bonds show that the aromatic character of the investigated polycyclic hydrocarbons is weakened upon fluorination. In contrast, the NICS(0) values for the fluorinated benzenes increase noteworthy upon fluorination, predicting a strong strengthening of the aromatic character of the arene rings. The integrated current strengths also yield explicit current pathways for the studied molecules. The current pathways of the investigated linear polyacenes, pyrene, anthanthrene, coronene, ovalene, and phenanthro-ovalene are not significantly affected by fluorination. NISC(0) and NICS(0)(zz) calculations provide contradictory degrees of aromaticity of the fused individual ring. Obtained NICS values do not correlate with the current strengths circling around the individual rings.

Molecular recognition plays a central role in many biological processes. For enzymatic reactions and slow protein-protein recognition events, turn-over rates and on-rates in the millisecond-to-second time scale have been connected to internal protein dynamics detected with atomic resolution by NMR spectroscopy, and in particular conformational sampling could be established as a mechanism for enzyme-substrate and protein-protein recognition. [1][2][3][4][5] Recent theoretical studies indicate that faster rates of conformational interconversion in the microsecond time scale might limit on-rates for protein-protein recognition. [6,7] However experimental proofs were lacking so far, mainly because such rates could not be determined accurately enough and kinetic experiments in the microsecond time range are difficult to perform.Nevertheless, for proteins and TAR-RNA, [8][9][10] recent studies based on residual dipolar couplings (RDCs) and other NMR spectroscopy techniques [11,12] have detected substantial internal dynamics in a time window from the rotational correlation time t c (one-digit nanoseconds) to approximately 50 ms, [8,[13][14][15] called the supra-t c window in the following. However, the exact rates of internal dynamics within this four orders of magnitude wide time window could not be determined.Supra-t c dynamics in ubiquitin [9] and TAR-RNA [16] could be connected to the conformational sampling required for molecular recognition. While the amplitudes of motions have been indirectly detected by RDCs and characterized in great detail, it has so far been impossible to directly observe these motions and to determine the exact rate of these supra-t c motions. In contrast, conformational sampling in enzymes occurs on a time scale that is 100 to 1000 times slower than supra-t c dynamics and therefore NMR relaxation dispersion (RD) techniques have been able to establish the functional link to enzyme kinetics with atomic resolution at physiological conditions.[1, 2, 5] However, for technical reasons, RD is not sensitive to motion faster than approximately 50 ms (RD window) and therefore does not access motion in the supra-t c window at room temperature.Here we determine the rate of interconversion between conformers of free ubiquitin by a combination of NMR RD experiments in super-cooled solution and dielectric relaxation spectroscopy (DR). Furthermore, we corroborate the motional amplitudes in the RDC-derived ensembles quantitatively with the observed amplitudes of RD and DR. The methods utilized herein can be used to directly study protein dynamics in a time range that was previously inaccessible.Significant motional amplitude in the supra-t c window has been observed using RDC measurements, and was connected to the conformational sampling for a protein in the ground

The aromatic pathways and the degree of aromaticity of expanded porphyrins have been determined by explicit calculations of the routes and strengths of the magnetically induced currents using the gauge-including magnetically induced current (GIMIC) approach. Density functional theory calculations show that the doubly twisted hexaphyrins fulfilling Hückel's (4n + 2) pi-electron rule for aromaticity and those obeying the 4n pi-electron rule for antiaromaticity are aromatic and antiaromatic, respectively. The investigated [26]hexaphyrin (2) and (3) and [30]hexaphyrin (5) isomers are aromatic, and [28]hexaphyrin (4) is antiaromatic. The formally antiaromatic [24]hexaphyrin (1) does not sustain any strong ring current and must be considered nonaromatic. A detailed analysis of the current pathways of the hexaphyrins is presented. It was found that the current pathways of the investigated aromatic hexaphyrins are not always dominated by the flow along the inner route through the non-hydrogenated C-N-C moieties, as previously proposed. The current flow is often split into two branches at the pyrrole rings, but sometimes it takes the outer route via the C=C bond of the pyrrole. The current pathway of the weak paratropic ring current of [24]hexaphyrin is dominated by the outer C=C route. The calculations show that the routes of the current transport cannot be assessed merely by inspection or from nucleus independent chemical shifts; explicit calculations of the current pathways are compulsory. The current-density studies also show that the pyrrole rings do not sustain any strong ring currents of their own.