Giacomo Parigi - Academia.edu (original) (raw)

Papers by Giacomo Parigi

NMR in Biomedicine, 2021

Several fruit juices are used as oral contrast agents to improve the quality of images in magneti... more Several fruit juices are used as oral contrast agents to improve the quality of images in magnetic resonance cholangiopancreatography. They are often preferred to conventional synthetic contrast agents because of their very low cost, natural origin, intrinsic safety, and comparable image qualities. Pineapple and blueberry juices are the most employed in clinical practice due to their higher content of manganese(II) ions. The interest of pharmaceutical companies in these products is testified by the appearance in the market of fruit juice derivatives with improved contrast efficacy. Here, we investigate the origin of the contrast of blueberry juice, analyze the parameters that can effect it, and elucidate the differences with pineapple juice and manganese(II) solutions. It appears that, although manganese(II) is the paramagnetic ion responsible for the contrast, it is the interaction of manganese(II) with other juice components that modulates the efficiency of the juice as a magnetic resonance contrast agent. On these grounds, we conclude that blueberry juice concentrated to the same manganese concentration of pineapple juice would prove a more efficient contrast agent than pineapple juice.

Magnetochemistry, 2021

Nuclear Magnetic Resonance is particularly sensitive to the electronic structure of matter and is... more Nuclear Magnetic Resonance is particularly sensitive to the electronic structure of matter and is thus a powerful tool to characterize in-depth the magnetic properties of a system. NMR is indeed increasingly recognized as an ideal tool to add precious structural information for the development of Single Ion Magnets, small complexes that are recently gaining much popularity due to their quantum computing and spintronics applications. In this review, we recall the theoretical principles of paramagnetic NMR, with particular attention to lanthanoids, and we give an overview of the recent advances in this field.

Emerging Topics in Life Sciences, 2018

NMR (nuclear magnetic resonance) investigation through the exploitation of paramagnetic effects i... more NMR (nuclear magnetic resonance) investigation through the exploitation of paramagnetic effects is passing from an approach limited to few specialists in the field to a generally applicable method that must be considered, especially for the characterization of systems hardly affordable with other techniques. This is mostly due to the fact that paramagnetic data are long range in nature, thus providing information for the structural and dynamic characterization of complex biomolecular architectures in their native environment. On the other hand, this information usually needs to be complemented by data from other sources. Integration of paramagnetic NMR with other techniques, and the development of protocols for a joint analysis of all available data, is fundamental for achieving a comprehensive characterization of complex biological systems. We describe here a few examples of the new possibilities offered by paramagnetic data used in integrated structural approaches.

The Journal of Chemical Physics, 2019

Chemistry – A European Journal, 2019

Biochimica et biophysica acta. General subjects, 2018

Natural products offer a wide range of biological activities, but they are not easily integrated ... more Natural products offer a wide range of biological activities, but they are not easily integrated in the drug discovery pipeline, because of their inherent scaffold intricacy and the associated complexity in their synthetic chemistry. Enzymes may be used to perform regioselective and stereoselective incorporation of functional groups in the natural product core, avoiding harsh reaction conditions, several protection/deprotection and purification steps. Herein, we developed a three step protocol carried out inside an NMR-tube. 1st-step: STD-NMR was used to predict the: i) capacity of natural products as enzyme substrates and ii) possible regioselectivity of the biotransformations. 2nd-step: The real-time formation of multiple-biotransformation products in the NMR-tube bioreactor was monitored in-situ. 3rd-step: STD-NMR was applied in the mixture of the biotransformed products to screen ligands for protein targets. Herein, we developed a simple and time-effective process, the "NMR...

Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry, 2018

Paramagnetic NMR data can be profitably incorporated in structural refinement protocols of metall... more Paramagnetic NMR data can be profitably incorporated in structural refinement protocols of metalloproteins or metal-substituted proteins, mostly as distance or angle restraints. However, they could in principle provide much more information, because the magnetic susceptibility of a paramagnetic metal ion is largely determined by its coordination sphere. This information can in turn be used to evaluate changes occurring in the coordination sphere of the metal when ligands (e.g.: inhibitors) are bound to the protein. This gives an experimental handle on the molecular structure in the vicinity of the metal which falls in the so-called blind sphere. The magnetic susceptibility anisotropy tensors of cobalt(II) and nickel(II) ions bound to human carbonic anhydrase II in free and inhibited forms have been determined. The change of the magnetic susceptibility anisotropy is directly linked to the binding mode of different ligands in the active site of the enzyme. Indication about the metal c...

Journal of medicinal chemistry, Jan 3, 2018

Biophysical parameters can accelerate drug development; e.g., rigid ligands may reduce entropic p... more Biophysical parameters can accelerate drug development; e.g., rigid ligands may reduce entropic penalty and improve binding affinity. We studied systematically the impact of ligand rigidification on thermodynamics using a series of fasudil derivatives inhibiting protein kinase A by crystallography, isothermal titration calorimetry, nuclear magnetic resonance, and molecular dynamics simulations. The ligands varied in their internal degrees of freedom but conserve the number of heteroatoms. Counterintuitively, the most flexible ligand displays the entropically most favored binding. As experiment shows, this cannot be explained by higher residual flexibility of ligand, protein, or formed complex nor by a deviating or increased release of water molecules upon complex formation. NMR and crystal structures show no differences in flexibility and water release, although strong ligand-induced adaptations are observed. Instead, the flexible ligand entraps more efficiently water molecules in s...

Inorganic chemistry, Jan 21, 2018

A fundamental challenge in the design of bioresponsive (or bioactivated) Gd-based magnetic resona... more A fundamental challenge in the design of bioresponsive (or bioactivated) Gd-based magnetic resonance (MR) imaging probes is the considerable background signal present in the "preactivated" state that arises from outer-sphere relaxation processes. When sufficient concentrations of a bioresponsive agent are present (i.e., a detectable signal in the image), the inner- and outer-sphere contributions to r may be misinterpreted to conclude that the agent has been activated, when it has not. Of the several parameters that determine the observed MR signal of an agent, only the electron relaxation time ( T) impacts both the inner- and outer-sphere relaxation. Therefore, strategies to minimize this background signal must be developed to create a near zero-background (or truly "off" state) of the agent. Here, we demonstrate that intramolecular magnetic exchange coupling when Gd is coupled to a paramagnetic transition metal provides a means to overcome the contribution of se...

Journal of Magnetic Resonance, 2017

Proceedings of the National Academy of Sciences of the United States of America, Mar 7, 2017

Well-defined, stereospecific states in protein complexes are often in exchange with an ensemble o... more Well-defined, stereospecific states in protein complexes are often in exchange with an ensemble of more dynamic orientations: the encounter states. The structure of the stereospecific complex between cytochrome P450cam and putidaredoxin was solved recently by X-ray diffraction as well as paramagnetic NMR spectroscopy. Other than the stereospecific complex, the NMR data clearly show the presence of additional states in the complex in solution. In these encounter states, populated for a small percentage of the time, putidaredoxin assumes multiple orientations and samples a large part of the surface of cytochrome P450cam. To characterize the nature of the encounter states, an extensive paramagnetic NMR dataset has been analyzed using the Maximum Occurrence of Regions methodology. The analysis reveals the location and maximal spatial extent of the additional states needed to fully explain the NMR data. Under the assumption of sparsity of the size of the conformational ensemble, several ...

Solution NMR of Paramagnetic Molecules, 2017

Hyperfine shifts, residual dipolar couplings and paramagnetic enhancements of the nuclear relaxat... more Hyperfine shifts, residual dipolar couplings and paramagnetic enhancements of the nuclear relaxation rates, experimentally available when the system contains a paramagnetic site, provide a wealth of information on the structure and mobility of these systems. These paramagnetic restraints are increasingly used in structural biology studies, up to the point that diamagnetic complexes are conveniently made paramagnetic through the use of paramagnetic binding tags. In this chapter, we will discuss the advantages provided by these restraints and present some guidelines for their use.

Solution NMR of Paramagnetic Molecules, 2017

Chemical exchange is capable of affecting the NMR parameters both in terms of shifts and relaxati... more Chemical exchange is capable of affecting the NMR parameters both in terms of shifts and relaxation. The matter is complex and the various detailed treatments may be laborious. Here the exchange of a nucleus between two sites is presented both qualitatively and quantitatively. The exchange time can be measured through saturation transfer experiments. Some thermodynamic parameters are obtained from the NMR parameters by varying the concentration of the species at equilibrium. In this chapter, the relaxation due to outer-sphere interactions is introduced as well as the chemical shifts due to bulk paramagnetic effects, which are used to measure the paramagnetism of molecules in solution.

Progress in Nuclear Magnetic Resonance Spectroscopy, 2016

Journal of biomolecular NMR, 2015

Long-range NMR data, namely residual dipolar couplings (RDCs) from external alignment and paramag... more Long-range NMR data, namely residual dipolar couplings (RDCs) from external alignment and paramagnetic data, are becoming increasingly popular for the characterization of conformational heterogeneity of multidomain biomacromolecules and protein complexes. The question addressed here is how much information is contained in these averaged data. We have analyzed and compared the information content of conformationally averaged RDCs caused by steric alignment and of both RDCs and pseudocontact shifts caused by paramagnetic alignment, and found that, despite the substantial differences, they contain a similar amount of information. Furthermore, using several synthetic tests we find that both sets of data are equally good towards recovering the major state(s) in conformational distributions.

Journal of magnetic resonance (San Diego, Calif. : 1997), 2015

Resolution and sensitivity in solid state NMR (SSNMR) can rival the results achieved by solution ... more Resolution and sensitivity in solid state NMR (SSNMR) can rival the results achieved by solution NMR, and even outperform them in the case of large systems. However, several factors affect the spectral quality in SSNMR samples, and not all systems turn out to be equally amenable for this methodology. In this review we attempt at analyzing the causes of this variable behavior and at providing hints to increase the chances of experimental success.

Journal of Biomolecular NMR, 2014

NMR experiments on proteins in simultaneous equilibria with multiple binding partners can provide... more NMR experiments on proteins in simultaneous equilibria with multiple binding partners can provide a tool to understand complex biological interaction networks. Competition among proteins for binding to signaling hubs is often at the basis of the information transmission across signaling networks in every organism. Changes in affinity towards one or more partners, as well as changes of the relative concentration of the competing partners, can determine pathways alterations that lead to pathological consequences. Overall, the knowledge of the interaction hierarchy of the multiple partners to a single signaling hub can lead to new therapeutic strategies. Smith and Ikura (Nat Chem Biol 10:223–230, 2014) have recently proposed pairwise competition NMR experiments to determine the binding hierarchy in network interactions. We have taken the moves from their approach to show how from pairwise competition NMR experiments the ratios between the equilibrium constants for multiple binding partners can be determined, and thus, given their concentration in solution, the concentrations of all the possible complexes can be obtained.

Proceedings of the National Academy of Sciences, 2004

The conformational space sampled by the two-domain protein calmodulin has been explored by an app... more The conformational space sampled by the two-domain protein calmodulin has been explored by an approach based on four sets of NMR observables obtained on Tb 3+ - and Tm 3+ -substituted proteins. The observables are the pseudocontact shifts and residual dipolar couplings of the C-terminal domain when lanthanide substitution is at the N-terminal domain. Each set of observables provides independent information on the conformations experienced by the molecule. It is found that not all sterically allowed conformations are equally populated. Taking the N-terminal domain as the reference, the C-terminal domain preferentially resides in a region of space inscribed in a wide elliptical cone. The axis of the cone is tilted by ≈30° with respect to the direction of the N-terminal part of the interdomain helix, which is known to have a flexible central part in solution. The C-terminal domain also undergoes rotation about the axis defined by the C-terminal part of the interdomain helix. Neither th...

NMR in Biomedicine, 2021

Several fruit juices are used as oral contrast agents to improve the quality of images in magneti... more Several fruit juices are used as oral contrast agents to improve the quality of images in magnetic resonance cholangiopancreatography. They are often preferred to conventional synthetic contrast agents because of their very low cost, natural origin, intrinsic safety, and comparable image qualities. Pineapple and blueberry juices are the most employed in clinical practice due to their higher content of manganese(II) ions. The interest of pharmaceutical companies in these products is testified by the appearance in the market of fruit juice derivatives with improved contrast efficacy. Here, we investigate the origin of the contrast of blueberry juice, analyze the parameters that can effect it, and elucidate the differences with pineapple juice and manganese(II) solutions. It appears that, although manganese(II) is the paramagnetic ion responsible for the contrast, it is the interaction of manganese(II) with other juice components that modulates the efficiency of the juice as a magnetic resonance contrast agent. On these grounds, we conclude that blueberry juice concentrated to the same manganese concentration of pineapple juice would prove a more efficient contrast agent than pineapple juice.

Magnetochemistry, 2021

Nuclear Magnetic Resonance is particularly sensitive to the electronic structure of matter and is... more Nuclear Magnetic Resonance is particularly sensitive to the electronic structure of matter and is thus a powerful tool to characterize in-depth the magnetic properties of a system. NMR is indeed increasingly recognized as an ideal tool to add precious structural information for the development of Single Ion Magnets, small complexes that are recently gaining much popularity due to their quantum computing and spintronics applications. In this review, we recall the theoretical principles of paramagnetic NMR, with particular attention to lanthanoids, and we give an overview of the recent advances in this field.

Emerging Topics in Life Sciences, 2018

NMR (nuclear magnetic resonance) investigation through the exploitation of paramagnetic effects i... more NMR (nuclear magnetic resonance) investigation through the exploitation of paramagnetic effects is passing from an approach limited to few specialists in the field to a generally applicable method that must be considered, especially for the characterization of systems hardly affordable with other techniques. This is mostly due to the fact that paramagnetic data are long range in nature, thus providing information for the structural and dynamic characterization of complex biomolecular architectures in their native environment. On the other hand, this information usually needs to be complemented by data from other sources. Integration of paramagnetic NMR with other techniques, and the development of protocols for a joint analysis of all available data, is fundamental for achieving a comprehensive characterization of complex biological systems. We describe here a few examples of the new possibilities offered by paramagnetic data used in integrated structural approaches.

The Journal of Chemical Physics, 2019

Chemistry – A European Journal, 2019

Biochimica et biophysica acta. General subjects, 2018

Natural products offer a wide range of biological activities, but they are not easily integrated ... more Natural products offer a wide range of biological activities, but they are not easily integrated in the drug discovery pipeline, because of their inherent scaffold intricacy and the associated complexity in their synthetic chemistry. Enzymes may be used to perform regioselective and stereoselective incorporation of functional groups in the natural product core, avoiding harsh reaction conditions, several protection/deprotection and purification steps. Herein, we developed a three step protocol carried out inside an NMR-tube. 1st-step: STD-NMR was used to predict the: i) capacity of natural products as enzyme substrates and ii) possible regioselectivity of the biotransformations. 2nd-step: The real-time formation of multiple-biotransformation products in the NMR-tube bioreactor was monitored in-situ. 3rd-step: STD-NMR was applied in the mixture of the biotransformed products to screen ligands for protein targets. Herein, we developed a simple and time-effective process, the "NMR...

Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry, 2018

Paramagnetic NMR data can be profitably incorporated in structural refinement protocols of metall... more Paramagnetic NMR data can be profitably incorporated in structural refinement protocols of metalloproteins or metal-substituted proteins, mostly as distance or angle restraints. However, they could in principle provide much more information, because the magnetic susceptibility of a paramagnetic metal ion is largely determined by its coordination sphere. This information can in turn be used to evaluate changes occurring in the coordination sphere of the metal when ligands (e.g.: inhibitors) are bound to the protein. This gives an experimental handle on the molecular structure in the vicinity of the metal which falls in the so-called blind sphere. The magnetic susceptibility anisotropy tensors of cobalt(II) and nickel(II) ions bound to human carbonic anhydrase II in free and inhibited forms have been determined. The change of the magnetic susceptibility anisotropy is directly linked to the binding mode of different ligands in the active site of the enzyme. Indication about the metal c...

Journal of medicinal chemistry, Jan 3, 2018

Biophysical parameters can accelerate drug development; e.g., rigid ligands may reduce entropic p... more Biophysical parameters can accelerate drug development; e.g., rigid ligands may reduce entropic penalty and improve binding affinity. We studied systematically the impact of ligand rigidification on thermodynamics using a series of fasudil derivatives inhibiting protein kinase A by crystallography, isothermal titration calorimetry, nuclear magnetic resonance, and molecular dynamics simulations. The ligands varied in their internal degrees of freedom but conserve the number of heteroatoms. Counterintuitively, the most flexible ligand displays the entropically most favored binding. As experiment shows, this cannot be explained by higher residual flexibility of ligand, protein, or formed complex nor by a deviating or increased release of water molecules upon complex formation. NMR and crystal structures show no differences in flexibility and water release, although strong ligand-induced adaptations are observed. Instead, the flexible ligand entraps more efficiently water molecules in s...

Inorganic chemistry, Jan 21, 2018

A fundamental challenge in the design of bioresponsive (or bioactivated) Gd-based magnetic resona... more A fundamental challenge in the design of bioresponsive (or bioactivated) Gd-based magnetic resonance (MR) imaging probes is the considerable background signal present in the "preactivated" state that arises from outer-sphere relaxation processes. When sufficient concentrations of a bioresponsive agent are present (i.e., a detectable signal in the image), the inner- and outer-sphere contributions to r may be misinterpreted to conclude that the agent has been activated, when it has not. Of the several parameters that determine the observed MR signal of an agent, only the electron relaxation time ( T) impacts both the inner- and outer-sphere relaxation. Therefore, strategies to minimize this background signal must be developed to create a near zero-background (or truly "off" state) of the agent. Here, we demonstrate that intramolecular magnetic exchange coupling when Gd is coupled to a paramagnetic transition metal provides a means to overcome the contribution of se...

Journal of Magnetic Resonance, 2017

Proceedings of the National Academy of Sciences of the United States of America, Mar 7, 2017

Well-defined, stereospecific states in protein complexes are often in exchange with an ensemble o... more Well-defined, stereospecific states in protein complexes are often in exchange with an ensemble of more dynamic orientations: the encounter states. The structure of the stereospecific complex between cytochrome P450cam and putidaredoxin was solved recently by X-ray diffraction as well as paramagnetic NMR spectroscopy. Other than the stereospecific complex, the NMR data clearly show the presence of additional states in the complex in solution. In these encounter states, populated for a small percentage of the time, putidaredoxin assumes multiple orientations and samples a large part of the surface of cytochrome P450cam. To characterize the nature of the encounter states, an extensive paramagnetic NMR dataset has been analyzed using the Maximum Occurrence of Regions methodology. The analysis reveals the location and maximal spatial extent of the additional states needed to fully explain the NMR data. Under the assumption of sparsity of the size of the conformational ensemble, several ...

Solution NMR of Paramagnetic Molecules, 2017

Hyperfine shifts, residual dipolar couplings and paramagnetic enhancements of the nuclear relaxat... more Hyperfine shifts, residual dipolar couplings and paramagnetic enhancements of the nuclear relaxation rates, experimentally available when the system contains a paramagnetic site, provide a wealth of information on the structure and mobility of these systems. These paramagnetic restraints are increasingly used in structural biology studies, up to the point that diamagnetic complexes are conveniently made paramagnetic through the use of paramagnetic binding tags. In this chapter, we will discuss the advantages provided by these restraints and present some guidelines for their use.

Solution NMR of Paramagnetic Molecules, 2017

Chemical exchange is capable of affecting the NMR parameters both in terms of shifts and relaxati... more Chemical exchange is capable of affecting the NMR parameters both in terms of shifts and relaxation. The matter is complex and the various detailed treatments may be laborious. Here the exchange of a nucleus between two sites is presented both qualitatively and quantitatively. The exchange time can be measured through saturation transfer experiments. Some thermodynamic parameters are obtained from the NMR parameters by varying the concentration of the species at equilibrium. In this chapter, the relaxation due to outer-sphere interactions is introduced as well as the chemical shifts due to bulk paramagnetic effects, which are used to measure the paramagnetism of molecules in solution.

Progress in Nuclear Magnetic Resonance Spectroscopy, 2016

Journal of biomolecular NMR, 2015

Long-range NMR data, namely residual dipolar couplings (RDCs) from external alignment and paramag... more Long-range NMR data, namely residual dipolar couplings (RDCs) from external alignment and paramagnetic data, are becoming increasingly popular for the characterization of conformational heterogeneity of multidomain biomacromolecules and protein complexes. The question addressed here is how much information is contained in these averaged data. We have analyzed and compared the information content of conformationally averaged RDCs caused by steric alignment and of both RDCs and pseudocontact shifts caused by paramagnetic alignment, and found that, despite the substantial differences, they contain a similar amount of information. Furthermore, using several synthetic tests we find that both sets of data are equally good towards recovering the major state(s) in conformational distributions.

Journal of magnetic resonance (San Diego, Calif. : 1997), 2015

Resolution and sensitivity in solid state NMR (SSNMR) can rival the results achieved by solution ... more Resolution and sensitivity in solid state NMR (SSNMR) can rival the results achieved by solution NMR, and even outperform them in the case of large systems. However, several factors affect the spectral quality in SSNMR samples, and not all systems turn out to be equally amenable for this methodology. In this review we attempt at analyzing the causes of this variable behavior and at providing hints to increase the chances of experimental success.

Journal of Biomolecular NMR, 2014

NMR experiments on proteins in simultaneous equilibria with multiple binding partners can provide... more NMR experiments on proteins in simultaneous equilibria with multiple binding partners can provide a tool to understand complex biological interaction networks. Competition among proteins for binding to signaling hubs is often at the basis of the information transmission across signaling networks in every organism. Changes in affinity towards one or more partners, as well as changes of the relative concentration of the competing partners, can determine pathways alterations that lead to pathological consequences. Overall, the knowledge of the interaction hierarchy of the multiple partners to a single signaling hub can lead to new therapeutic strategies. Smith and Ikura (Nat Chem Biol 10:223–230, 2014) have recently proposed pairwise competition NMR experiments to determine the binding hierarchy in network interactions. We have taken the moves from their approach to show how from pairwise competition NMR experiments the ratios between the equilibrium constants for multiple binding partners can be determined, and thus, given their concentration in solution, the concentrations of all the possible complexes can be obtained.

Proceedings of the National Academy of Sciences, 2004

The conformational space sampled by the two-domain protein calmodulin has been explored by an app... more The conformational space sampled by the two-domain protein calmodulin has been explored by an approach based on four sets of NMR observables obtained on Tb 3+ - and Tm 3+ -substituted proteins. The observables are the pseudocontact shifts and residual dipolar couplings of the C-terminal domain when lanthanide substitution is at the N-terminal domain. Each set of observables provides independent information on the conformations experienced by the molecule. It is found that not all sterically allowed conformations are equally populated. Taking the N-terminal domain as the reference, the C-terminal domain preferentially resides in a region of space inscribed in a wide elliptical cone. The axis of the cone is tilted by ≈30° with respect to the direction of the N-terminal part of the interdomain helix, which is known to have a flexible central part in solution. The C-terminal domain also undergoes rotation about the axis defined by the C-terminal part of the interdomain helix. Neither th...