Biomedical subjects
Publications and source records attributed to Peter P Borbat.
Helical peptoid oligomers were synthesized in which the positions of nitroxide radical spin probes along the backbone were systematically varied, allowing evaluation of intra-molecular distances and dynamics by electron spin resonance spectroscopy.
In bacterial chemotaxis, an assembly of transmembrane receptors, the CheA histidine kinase and the adaptor protein CheW processes environmental stimuli to regulate motility. The structure of a Thermotoga maritima receptor cytoplasmic domain defines CheA interaction regions and metal ion-coordinating charge centers that undergo chemical modification to tune receptor response. Dimeric CheA-CheW, defined by crystallography and pulsed ESR, positions two CheWs to form a cleft that is lined with residues important for receptor interactions and sized to clamp one receptor dimer. CheW residues involved in kinase activation map to interfaces that orient the CheW clamps. CheA regulatory domains associate in crystals through conserved hydrophobic surfaces. Such CheA self-contacts align the CheW receptor clamps for binding receptor tips. Linking layers of ternary complexes with close-packed receptors generates a lattice with reasonable component ratios, cooperative interactions among receptors and accessible sites for modification enzymes.
Tikhonov regularization (TIKR) has been demonstrated as a powerful and valuable method for the determination of distance distributions of spin-pairs in bi-labeled biomolecules directly from pulsed ESR signals. TIKR is a direct method, which requires no iteration, and, therefore, provides a rapid and unique solution. However, the distribution obtained tends to exhibit oscillatory excursions with negative portions in the presence of finite noise, especially in the peripheral regions of the distribution. The Shannon-Jaynes entropy of a probability distribution provides an intrinsic non-negativity constraint on the probability distribution and an unbiased way of obtaining information from incomplete data. We describe how the maximum entropy regularization method (MEM) may be applied to solve the ill-posed nature of the dipolar signal in pulsed ESR. We make use of it to suppress the negative excursions of the distance distribution and to increase the tolerance to noise in the dipolar signal. Model studies and experimental data are investigated, and they show that, with the initial or "seed" probability distribution that is required for MEM taken as the TIKR result, then MEM is able to provide a regularized solution, subject to the non-negativity constraint, and it is effective in dealing with noise that is problematic for TIKR. In addition we have incorporated into our MEM method the ability to extract the intermolecular dipolar component, which is embedded in the raw experimental data. We find that MEM minimization, which is implemented iteratively, is greatly accelerated using the TIKR result as the seed, and it converges more successfully. Thus we regard the MEM method as a complement to TIKR by securing a positive pair distance distribution and enhancing the accuracy of TIKR.
Pulsed ESR techniques with the aid of site-directed spin labeling have proven useful in providing unique structural information about proteins. The determination of distance distributions in electron spin pairs directly from the dipolar time evolution of the pulsed ESR signals by means of the Tikhonov regularization method is reported. The difficulties connected with numerically inverting this ill-posed mathematical problem are clearly illustrated. The Tikhonov regularization with the regularization parameter determined by the L-curve criterion is then described and tested to confirm its accuracy and reliability. The method is applied to recent experimental results on doubly labeled proteins that have been studied using two pulsed ESR techniques, double quantum coherence (DQC) ESR and double electron-electron resonance (DEER). The extracted distance distributions are able to provide valuable information about the conformational constraints in various partially folded states of proteins. This study supplies a mathematically reliable method for extracting pair distributions from pulsed ESR experimental data and has extended the use of pulsed ESR to provide results of greater value for structural biology.
It is shown how the new technique of double-quantum filtered refocused electron spin-echoes is a significant improvement over double-quantum coherence ESR, since it increases the experimental acquisition time. This enables the measurement of longer distances in bilabeled biomolecules. The method is demonstrated on a long double-stranded A-type RNA, spin labeled at both ends. The measured distance of 72 A is in excellent agreement with molecular modeling.
NMR microscopy is routinely employed in fields of science such as biology, botany, and materials science to observe magnetic parameters and transport phenomena in small scale structures. Despite extensive efforts, the resolution of this method is limited (>10 microm for short acquisition times), and thus cannot answer many key questions in these fields. We show, through theoretical prediction and initial experiments, that ESR microscopy, although much less developed, can improve upon the resolution limits of NMR, and successfully undertake the 1 mum resolution challenge. Our theoretical predictions demonstrate that existing ESR technology, along with advanced imaging probe design (resonator and gradient coils), using solutions of narrow linewidth radicals (the trityl family), should yield 64 x 64 pixels 2D images (with z slice selection) with a resolution of 1 x 1 x 10 microm at approximately 60 GHz in less than 1h of acquisition. Our initial imaging results, conducted by CW ESR at X-band, support these theoretical predictions and already improve upon the previously reported state-of-the-art for 2D ESR image resolution achieving approximately 10 x 10 mum, in just several minutes of acquisition time. We analyze how future progress, which includes improved resonators, increased frequency of measurement, and advanced pulsed techniques, should achieve the goal of micron resolution.