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The increased interest in using monoclonal antibodies (mAbs) as a platform for biopharmaceuticals has led to the need for new analytical techniques that can precisely assess physicochemical properties of these large and very complex drugs for the purpose of correctly identifying quality attributes (QA). One QA, higher order structure (HOS), is unique to biopharmaceuticals and essential for establishing consistency in biopharmaceutical manufacturing, detecting process-related variations from manufacturing changes and establishing comparability between biologic products. To address this measurement challenge, two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) methods were introduced that allow for the precise atomic-level comparison of the HOS between two proteins, including mAbs. Here, an inter-laboratory comparison involving 26 industrial, government and academic laboratories worldwide was performed as a benchmark using the NISTmAb, from the National Institute of Standards and Technology (NIST), to facilitate the translation of the 2D-NMR method into routine use for biopharmaceutical product development. Two-dimensional 1H,15N and 1H,13C NMR spectra were acquired with harmonized experimental protocols on the unlabeled Fab domain and a uniformly enriched-15N, 20%-13C-enriched system suitability sample derived from the NISTmAb. Chemometric analyses from over 400 spectral maps acquired on 39 different NMR spectrometers ranging from 500 MHz to 900 MHz demonstrate spectral fingerprints that are fit-for-purpose for the assessment of HOS. The 2D-NMR method is shown to provide the measurement reliability needed to move the technique from an emerging technology to a harmonized, routine measurement that can be generally applied with great confidence to high precision assessments of the HOS of mAb-based biotherapeutics., mAbs, 11 (1), ISSN:1942-0862, ISSN:1942-0870

Prion diseases are a group of fatal neurodegenerative disorders that can be of sporadic, genetic or acquired origin. The key molecular event in prion diseases is the conformational conversion of the physiological cellular prion protein, PrPC, into a disease- associated form, prion or PrPSc (scrapie PrP). Understanding of the earliest stages of the conformational changes leading to spontaneous generation of prions in genetic prion diseases, which are linked with mutations in the human prion protein gene, may benefit from structural characterization of various human (Hu) PrP variants. We determined NMR structures of the truncated recombinant HuPrPs (residues 90-231) with pathological Q212P [1] and V210I [2] mutations associated with Gerstmann- Sträussler- Scheinker (GSS) syndrome and familial Creutzfeldt- Jakob disease (CJD), respectively, and of HuPrP with naturally occurring E219K polymorphism [3] considered to act protectively against sporadic CJD. Comparison of 3D structures of HuPrP variants with the WT HuPrP revealed that mutations do not affect global architecture of the protein. However, 3D structures of HuPrPs with pathological Q212P and V210I mutations highlighted several common structural perturbations. These include disruption of hydrophobic contacts at the α2-α3 inter-helical interface, loosening of tertiary contacts between the β2-α2 loop and the C-terminus of helix α3 and increased exposure of hydrophobic residues to solvent. In addition, we determined NMR structure of HuPrP(V210I) under physiological pH conditions which was found to exhibit higher structural stability when compared to the structure of the same protein at pH 5.5 [4].

The cellular form of the prion protein (PrPC) is a highly conserved glycoprotein mostly expressed in the central and peripheral nervous systems by different cell types in mammals. A misfolded, pathogenic isoform, denoted as prion, is related to a class of neurodegenerative diseases known as transmissible spongiform encephalopathy. PrPC function has not been unequivocally clarified and it is rather defined as a pleiotropic protein likely acting as a dynamic cell surface scaffolding protein for the assembly of different signalling modules. Among the variety of PrPC protein interactors, the neuronal cell adhesion molecule (NCAM) has been studied in vivo, but the structural basis of this functional interaction is still a matter of debate. Here we focused on the structural determinants responsible for human PrPC (HuPrP) and NCAM interaction using Stimulated Emission Depletion (STED) nanoscopy, surface plasma resonance (SPR) and NMR spectroscopy approaches. PrPC co-localizes with NCAM in mouse hyppocampal neurons and this interaction is mainly mediated by the intrinsically disordered PrPC N-terminal tail, which binds with high affinity to NCAM Fibronectin type-3 domain. NMR structural investigations revealed surface interacting epitopes governing the interaction between HuPrP N-terminus and the second module of NCAM Fibronectin type-3 domain. Our data provided molecular details about the interaction between HuPrP and NCAM Fibronectin domain, and revealed a new role of PrPC N-terminus as a dynamic and functional element responsible for protein-protein interaction.

The activity of several cytosolic proteins critically depends on the concentration of calcium ions. One important intracellular calcium-sensing protein is alpha-actinin-1, the major actin crosslinking protein in focal adhesions and stress fibers. The actin crosslinking activity of alpha-actinin-1 has been proposed to be negatively regulated by calcium, but the underlying molecular mechanisms are poorly understood. To address this, we determined the first high-resolution NMR structure of its functional calmodulin-like domain (CaMD) in calcium-bound and calcium-free form. These structures reveal that in the absence of calcium, CaMD displays a conformationally flexible ensemble that undergoes a structural change upon calcium binding, leading to limited rotation of the N- and C-terminal lobes around the connecting linker and consequent stabilization of the calcium-loaded structure. Mutagenesis experiments, coupled with mass-spectrometry and isothermal calorimetry data designed to validate the calcium binding stoichiometry and binding site, showed that human non-muscle alpha-actinin-1 binds a single calcium ion within the N-terminal lobe. Finally, based on our structural data and analogy with other alpha-actinins, we provide a structural model of regulation of the actin crosslinking activity of alpha-actinin-1 where calcium induced structural stabilisation causes fastening of the juxtaposed actin binding domain, leading to impaired capacity to crosslink actin.

Prion diseases, also known as transmissible spongiform encephalopathies (TSE), are a group of neuropathies characterized by a fatal spongiform neurodegeneration of the brain, facilitated by conformational changes in the prion protein (PrP) observed in humans and other animals. However, the initiation and propagation mechanisms by which PrP convert from normal cellular form (PrPC) to the protease resistant PrPSc form is still not understood in detail. One of the important factors of this phenomenon are processes of molecular dynamics which facilitate and control the conversion of PrPC to PrPSc forms. In the presented study, the backbone dynamics of the human pathogenic mutant (Q212P), protective mutant (Q218K), and two wild-type constructs PrP(90-231) and PrP(23-231) were analyzed at pH 5.5. The high resolution 3D structures of all mutants were solved previously in our group with the exception of the PrP(23-231) protein. There were several studies focused on analysis of 15N relaxation data performed on mouse PrP protein demonstrated similarity of dynamic performed on the mouse wild-type and several PrP mutants. Our studies focused on human pathological and protective PrP mutants. Experimental data (15N R1, R2 relaxation rates, and 1H-15N NOE) were acquired under two magnetic fields (16.4 T and 18.8 T) and analyzed with both Spectral Density Mapping and ModelFree formalisms. Moreover, results evaluated for the PrP(90-231) construct are analyzed together with whole length PrP(23-231) protein, which provide possibilities to explore tiny changes in dynamic processes caused by a long, unstructured N-terminal domain. Our studies reveal the possibility to explore the effects of molecular dynamics in the initial steps of the transformation of cellular PrPC into the pathogenic PrPSc form.

Prion diseases are a rare group of fatal neurodegenerative disorders affecting humans and animals. The key event in prion diseases is the conformational conversion of the normal, cellular prion protein, PrPC, into a disease-related form known as prion or PrPSc. Approximately 10-15% of prion diseases in humans are caused by mutations in the PrP gene (PRNP). Structural characterization of different human (Hu) PrP variants may be helpful for better understanding of the earliest stages of the conformational changes leading to spontaneous generation of prions in inherited forms of prion diseases. In our recent NMR studies we examined the effects of several disease-related mutations on HuPrP structure. We determined solution-state NMR structures of the truncated recombinant HuPrPs (residues 90-231) carrying pathological Q212P and V210I mutations linked to Gerstmann-Straussler-Schenker (GSS) syndrome and familial Creutzfeldt-Jakob disease (CJD), respectively, and of HuPrP with naturally occurring E219K polymorphism considered to protect against sporadic CJD (sCJD). NMR structures of HuPrP(Q212P), HuPrP(V210I) and HuPrP(E219K) proteins share similar global architecture with the WT HuPrP comprising unstructured N-terminal tail and a globular C-terminal domain. Detailed analysis revealed that pathological Q212P and V210I mutations introduce subtle local structural changes with respect to the WT HuPrP. These include perturbation of hydrophobic contacts at the α2-α3 inter-helical interface, altered conformation of the β2-α2 loop along with loosening of tertiary contacts between the loop and the C-terminal end of helix α3 leading to higher exposure of hydrophobic residues to solvent. Notably, the structure of HuPrP(E219K) does not point to interruption of hydrophobic interactions in the β2-α2 loop region. Our structural findings on disease-related HuPrP mutants offer new clues about the earliest events of the pathogenic conversion process. In addition, we examined the effect of pH on the structure of HuPrP(V210I). Comparison of NMR structures of HuPrP(V210I) at pH 5.5 and 7.2 revealed that the tertiary contacts are less perturbed at neutral pH conditions suggesting that spontaneous formation of prions probably occurs under acidic pH conditions in endosomal compartments.

Prion diseases are fatal neurodegenerative disorders which can be of sporadic, genetic and infectious origin. In genetic forms of prion diseases, misfolding of cellular prion protein, PrPC, into its pathological form, PrPSc, is caused by mutations in the human prion protein gene. Understanding of the earliest stages of the conformational changes leading to spontaneous generation of prions in genetic forms of prion diseases may benefit from detailed structural characterization of various human (Hu) PrP variants. NMR spectroscopy is a powerful tool for providing information on three-dimensional (3D) structural features at the atomic level. Therefore, we undertook solution-state NMR studies of HuPrPs with pathological Q212P and V210I mutations linked to Gerstmann-Sträussler-Scheinker (GSS) syndrome and genetic Creutzfeldt–Jakob disease (CJD), respectively and of HuPrP carrying naturally occurring E219K polymorphism considered to protect against sporadic CJD (sCJD). Nearly complete backbone and side-chain assignments along with a high number of NOE distance restraints enabled high-resolution structure determination of HuPrP variants. None of the mutation affects the overall fold of HuPrP which highly resembles that of the WT protein consisting of unstructured N-terminal tail and a globular C-terminal domain. However, pathological Q212P and V210I mutations introduce several local structural differences in comparison to the WT HuPrP. Variations are mostly clustered at the α2-α3 interhelical interface and in the β2-α2 loop region leading to higher exposure of hydrophobic residues to solvent what may facilitate intermolecular interactions involved in spontaneous generation of prions. Notably, hydrophobic interactions in the β2-α2 loop region are not interrupted in HuPrP with protective E219K polymorphism. The NMR structures of HuPrP variants provide new insights into the possible key structural determinants underlying conversion of PrPC to PrPSc. In addition, we analyzed the effect of pH on the structural features of HuPrP(V210I). Comparison of NMR structures of HuPrP(V210I) at pH 5.5 and 7.2 revealed that the tertiary contacts are less perturbed at neutral pH conditions suggesting that spontaneous formation of prions may occur under acidic pH conditions in endosomal compartments.

In human genetic forms of prion diseases, mutations in the globular C-terminal domain of PrPC are hypothesized to favor spontaneous generation of PrPSc in specific brain regions leading to neuronal cell degeneration and death. We hypothesize that high-resolution solution-state NMR structures on HuPrPs with pathological Q212P and V210I mutations linked to GSS and fCJD respectively, and HuPrP carrying naturally occurring E219K polymorphism considered to protect against sCJD might provide insights into the early stages of prion diseases. Nuclear Magnetic Resonance spectroscopy offers unique potential amongst the experimental methods to obtain topological assignment and detailed atomic-level structure information. The sequence-specific 1H, 13C and 15N backbone assignment was achieved to a very high degree, and it enabled protein structure determination of mutant PrP protein(s). Finally, 120 out of 141 (excluding C-terminus His tag) residues were successfully assigned, which corresponds to 85% of the HuPrP(90-231) sequence. The unassigned residues are located mostly around Phe175 and constitute a very flexible and hydrophilic fragment of HuPrP(90-231) structure. For those residues, the signals from NH protons are not observed in 1H-15N HSQC spectrum probably due to fast exchange with water. Moreover, this part of HuPrP(90-231) structure is involved in a pronounced chemical exchange process at the intermediate NMR timescale, which results in an additional increase of resonance linewidths. The NMR structure of the recombinant human PrP, HuPrP(90-231, M129, Q212P), believed to cause inherited GSS, provides new insights into the conformational preorganization and early stages of spontaneous conversion of PrPC into PrPSc. The secondary structure of the Q212P mutant consists of a flexible disordered tail (residues 90-124) and a globular domain (residues 125-231). The substitution of a proline at position 212 introduces unique structural differences in comparison to the known structures of either wild-type or other mammalian PrP, which are likely to have implications for understanding why and how changes in the native fold of endogenous PrPC lead to neurodegeneration or its degradation. Subtle local structural changes include an altered conformation of the β2-α2 loop along with loosening of tertiary contacts between the loop and the C-terminal end of helix α3, higher exposure of hydrophobic residues to solvent and perturbation of hydrophobic contacts at the α2-α3 inter-helical interface. In contrast to pathological mutants, hydrophobic interactions between residues from the β2-α2 loop and helix α3 are not interrupted in HuPrP with E219K polymorphismP

NMR spectroscopy allows to obtain topological assignment and detailed atomic-level structure information. In human genetic forms of prion diseases, mutations in the C-terminal globular domain of PrPC (HuPrP) are hypothesized to favor spontaneous generation of PrPSc in the brain leading to neurodegeneration. High-resolution solution-state NMR structures on HuPrPs with Q212P and V210I mutations (linked to GSS and fCJD respectively) and HuPrP carrying the E219K polymorphism considered to protect against sCJD might provide insights into the early stages of prion conversion. Sequence-specific backbone and side-chain assignments were achieved to a very high degree enabling structure determination of mutant PrPs. Hundred twenty out of 141 residues were assigned, corresponding to 85% of the HuPrP sequence. Unassigned residues are mostly located around Phe175 and constitute a flexible segment of HuPrP structure. For those residues, the signals from NH protons are not observed in 1H, 15N-HSQC spectrum due to fast exchange with water. This part is involved in a pronounced chemical exchange process at the intermediate NMR timescale, resulting in an additional increase of resonance linewidths. The NMR structure of HuPrP carrying the Q212P mutation provides new insights into the early stages of spontaneous PrPC conversion into PrPSc. The secondary structure of the Q212P mutant consists of a flexible disordered tail (residues 90-124) and a globular domain (residues 125-231). The substitution of a proline at position 212 introduces unique structural differences in comparison to the known mammalian PrP structures, which are likely to have implications for understanding why and how changes in the native PrPC fold lead to neurodegeneration. Subtle local structural changes include an altered conformation of the β2-α2 loop along with loosening of tertiary contacts between the loop and the C-terminal end of helix α3, higher exposure of hydrophobic residues to solvent and perturbation of hydrophobic contacts at the α2-α3 inter-helical interface. Similar findings were found in the V210I mutant. However, hydrophobic interactions between residues from the β2-α2 loop and helix α3 are not interrupted in HuPrP with E219K polymorphism. In conclusion, our findings on disease-associated mutants and on E219K polymorphism provide new clues on the possible early events of HuPrP misfolding. The structural disorder of the β2-α2 loop, together with the increased distance between this loop and helix α3, observed in different mutants, lead to exposure of hydrophobic residues to solvent, thus facilitating intermolecular interactions with other PrP or with yet unknown cellular factors and abnormal accumulation inside the cells.

The post-translational conversion of the ubiquitously expressed cellular form of the prion protein, PrPC, into its misfolded and pathogenic isoform, known as prion or PrPSc, plays a key role in prion diseases. These maladies are denoted transmissible spongiform encephalopathies (TSEs) and affect both humans and animals. A prerequisite for understanding TSEs is unraveling the molecular mechanism leading to the conversion process whereby most α-helical motifs are replaced by β-sheet secondary structures. Importantly, most point mutations linked to inherited prion diseases are clustered in the C-terminal domain region of PrPC and cause spontaneous conversion to PrPSc. Structural studies with PrP variants promise new clues regarding the proposed conversion mechanism and may help identify “hot spots” in PrPC involved in the pathogenic conversion. These investigations may also shed light on the early structural rearrangements occurring in some PrPC epitopes thought to be involved in modulating prion susceptibility. Here we present a detailed overview of our solution-state NMR studies on human prion protein carrying different pathological point mutations and the implications that such findings may have for the future of prion research.

The development of transmissible spongiphorm encephalopathies (TSE) is associated with the conversion of the cellular prion protein (PrPC) into the misfolded, pathogenic isoform (PrPSc). In human genetic forms of these diseases, mutations in the globular C-terminal domain of PrPC are hypothesized to favor spontaneous generation of PrPSc in specific brain regions, leading to neuronal cell degeneration and death. Approximately 10–15% of TSEs are associated with the mutations. Our recent NMR studies were focused on structural characterization of different truncated recombinant human (Hu) PrPs carrying the pathological Q212P (90-231, M129), V210I (90-231, M129) mutations and protective E219K (90-231, M129) polymorphism. While Q212P mutation is linked to GSS, the V210I mutation is linked to genetic CJD. The naturally occurring E219K polymorphism in the HuPrP is considered to protect against sCJD. We have demonstrated that the determined structures of variants consist of unstructured N-terminal part (residues 90-124) and well-defined C-terminal domain (residues 125- 228). Analysis and comparison with the structure of the WT Hu-PrP revealed that although structures share a similar global fold, mutations introduce some local structural differences. The determined NMR structures offer new clues on the earliest events of the pathogenic conversion process and could be used for the development of antiprion drugs.

Prion diseases are fatal neurodegenerative disorders caused by an aberrant accumulation of the infectious scrapie isoform (PrPSc). Understanding of the mechanisms by which cellular prion protein (PrPC) misfolds and leads to disease can benefit from detailed analysis of 3D structures. Our recent NMR studies have focused on structural characterization of different human PrPC variants that are linked to genetic prion diseases. We have demonstrated that structures of Q212P (Ilc et al., 2010), V210I (Biljan et al., 2011) and E219K (Biljan et al., 2012a) mutants exhibit unique structural features that are caused by a single amino acid substitution. Interestingly, the naturally occurring E219K polymorphism in the human prion protein is considered to protect against sporadic Creutzfeldt–Jakob disease. In all studies, 13C, 15N-double labeled mutants were prepared and exhibited favorable cross-peak dispersion in 1H-15N HSQC spectra thus enabling complete spectral assignment and acquisition of a large number of NOE based distance restraints. In general, the structure of the mutant proteins consist of a disordered N-terminal tail (residues 90–124) and a well-structured C-terminal segment (residues 125–231) containing three α-helices and two short antiparallel β-strands. More recently, we have characterized HuPrP(90-231) carrying V210I mutation under two different conditions with pH 5.5 and 7.2 (Biljan et al., 2012b). While nearly complete assignment was obtained for V210I mutant at pH 5.5, amide protons are involved in fast exchange with solvent at pH 7.2 which renders observation of some amino acids residues from unstructured N-terminal region. Significant pH-related local structural differences were observed in the α2−α3 interhelical region, at the interface of the β1−α1 loop, in helices α1 and α3, and in the β2−α2 loop region. The detailed analysis of interactions suggests that spontaneous misfolding of PrPC may occur under acidic-pH conditions in endosomal compartments.

Prion diseases or Transmissible Spongiform Encephalopathies (TSE) are a group of fatal neurodegenerative illnesses affecting humans and animals. They are classified into sporadic, genetic and infectious forms. Genetic prion diseases are caused by mutations in the human prion protein gene and include Gerstmann-Straussler-Scheinker (GSS) syndrome, Fatal Familial Insomnia and genetic Creutzfeldt-Jakob disease (CJD). Approximately 10-15% of all TSE cases in humans are associated with mutations. The development of TSEs is associated with the conversion of the cellular prion protein (PrPC) into a misfolded, pathogenic isoform (PrPSc). Our recent NMR studies were focused on structural characterization of different truncated recombinant human (Hu) PrPs carrying the pathological Q212P (90-231, M129) and V210I(90-231, M129) mutations, and protective E219K (90-231, M129) polymorphism. While Q212P mutation is linked to GSS, the V210I mutation is linked to genetic CJD. The naturally occurring E219K polymorphism in the HuPrP is considered to protect against sCJD. We have demonstrated that the determined structures of variants consist of unstructured N-terminal part (residues 90-124) and well defined C-terminal domain (residues 125-228). Analysis and comparison with the structure of the WT HuPrP revealed that although structures share similar global fold, mutations introduces several local structural differences. The observed differences are mostly clustered at the alpha2-alpha3 inter-helical interface and in the beta2-alpha2 loop region. The determined NMR structures offer new insights on the earliest events of the pathogenic conversion process and could be used for the development of antiprion drugs. More recently we have determined solution state structures of V210I (90-231, M129) pathogenic mutation at two different conditions with pH 5.5 and 7.2. The detailed comparison of three-dimensional structures of HuPrP(V210I) at two different pH values revealed that interactions among secondary structure elements have a higher degree of structural ordering under neutral pH conditions, thus implying that spontaneous misfolding of PrPC may occur under acidic-pH conditions in endosomal compartments.

Prion diseases or transmissible spongiform encephalopathies are fatal neurodegenerative disorders associated with the conformational conversion of the cellular prion protein, PrPC, into a pathological form known as prion of PrPSc. They can be classified into sporadic, inherited and infectious forms. Spontaneous generation of PrPSc in inherited forms of prion diseases is caused by mutations in the human prion protein gene. A major goal in prion biology is unravelling the molecular mechanism by which PrPC misfolds and leads to development of diseases. Structural characterization of various human PrP (HuPrP) variants may be helpful for better understanding of the earliest stages of the conformational changes leading to spontaneous generation of prions. Our recent NMR studies were focused on structural characterization of different truncated recombinant human PrPs carrying the pathological Q212P (90-231, M129), V210I (90-231, M129) mutations and protective E219K (90-231, M129) polymorphism. While Q212P mutation is linked to GSS the V210I mutation is linked to genetic CJD. The naturally occurring E219K polymorphism in the HuPrP is considered to be protective against sCJD. We have demonstrated that the determined structures of variants consist of unstructured N-terminal part (residues 90-124) and well-defined C-terminal domain (residues 125-228). Analysis and comparison of determined structures with respect to the structure of the WT Hu-PrP revealed that although structures share similar global fold, mutations introduce local structural differences. The determined NMR structures offer new clues on the earliest events of the pathogenic conversion process, which is particularly interesting for the development of antiprion drugs.

Prion diseases or Transmissible Spongiform Encephalopathies (TSEs) are a group of fatal neurodegenerative illnesses affecting humans and animals. They are classified into sporadic, genetic and infectious forms. Genetic prion diseases are caused by mutations in the human prion protein gene and include Gerstmann-Sträussler-Scheinker (GSS) syndrome, Fatal Familial Insomnia and genetic Creutzfeldt–Jakob disease (CJD). Approximately 10-15% of all TSE cases in humans are associated with mutations. The development of TSEs is associated with the conversion of the cellular prion protein (PrPC) into a misfolded, pathogenic isoform (PrPSc). In our recent works, we have determined the NMR solution-state structures of the truncated recombinant human (Hu) PrPs carrying the pathological Q212P (90-231, M129) and V210I (90-231, M129) mutations. While Q212P mutation is linked to GSS the V210I mutation is linked to genetic CJD. The determined structures of both mutants consist of unstructured N-terminal part (residues 90-124) and well-defined C-terminal domain (residues 125-228). Analysis and comparison with the structure of the WT HuPrP revealed that although structures share similar global fold, mutations introduces some local structural differences. The observed variations are mostly clustered at the α2-α3 inter-helical interface and in the β2-α2 loop region. NMR structures offer new clues on the earliest events of the pathogenic conversion process and could be used for the development of antiprion drugs.

Transmissible spongiform encephalopathies (TSEs) are a rare group of rapidly progressive, invariably fatal neurodegenerative diseases affecting humans and animals. A hallmark of TSEs is the conformational conversion of the physiological cellular prion protein, PrPC, into a disease-associated form known as prion or PrPSc. A major focus in prion biology is unraveling the molecular mechanism leading to the structural conversion of PrPC to its pathological form PrPSc. In our recent studies, we tried to understand the early events of the conformational changes leading to PrPSc using as investigative tools point mutations clustered in the open reading frame of the human PrP gene and linked to genetic forms of human prion diseases. We have determined high- resolution solution-state NMR structures of the truncated recombinant human (Hu) PrP (residues 90-231) with pathological Q212P and V210I mutations linked to Gerstmann-Sträussler- Scheinker (GSS) syndrome and genetic Creutzfeldt–Jakob disease (CJD), respectively. In addition, we examined the NMR structure of the recombinant HuPrP (residues 90-231) carrying naturally occurring E219K polymorphism considered to protect against sporadic CJD (sCJD). 3D structures of HuPrP(Q212P), HuPrP(V210I) and HuPrP(E219K) proteins share similar global architecture with the WT HuPrP consisting of highly flexible N-terminal tail and a well-structured C-terminal domain, the latter containing three alpha-helices and a short antiparallel beta-sheet. Detailed inspection revealed that pathological Q212P and V210I mutations introduce some local structural variations with respect to the WT HuPrP which are mostly clustered at the alpha2-alpha3 interhelical interface and in the beta2-alpha2 loop region. Alteration of conformation of the beta2-alpha2 loop region and subsequent higher exposure of hydrophobic residues to solvent may facilitate intermolecular interactions involved in spontaneous generation of PrPSc in genetic TSEs. On contrary to the HuPrP(Q212P) and HuPrP(V210I), the structure of HuPrP(E219K) does not point to interruption of aromatic and hydrophobic interactions in the beta2-alpha2 loop region. Noteworthy, it seems that the beta2-alpha2 loop in HuPrP(E219K) is somewhat better defined in comparison to the same region in the WT protein and proteins with pathological Q212P and V210I mutations. In addition to such subtle structural rearrangements localized within the epitopes critical for prion conversion, replacement of Glu219 by Lys induces significant alteration of surface charge distribution with respect to the WT protein what may further account for protective influence of E219K polymorphism against sCJD.

Prion diseases or transmissible spongiform encephalopathies (TSEs) are a group of rare neuropathies characterized by a spongiform neurodegeneration of the central nervous system caused by prions. These disorders include Creutzfeldt–Jakob disease (CJD), Gerstmann-Sträussler-Scheinker (GSS) syndrome, Fatal Familial Insomnia and kuru in humans, bovine spongiform encephalopathy in cattle, scrapie in sheep and goats, and chronic wasting disease in elk, deer and moose. Prions are thought to consist solely of a misfolded isoform (PrPSc) of the normal, host-encoded cellular protein (PrPC), whose function is still unknown. Human (Hu) PrPC is a 209 residues long glycoprotein, tethered to the outer leaflet of the plasma membrane by a glycosylphosphatidylinositol (GPI) anchoring and its primary structure is highly conserved among mammals. According to the „protein-only hypothesis‟, during the course of prion diseases, PrPC is converted into the abnormal form by a conversion process whereby most α-helix motives are replaced by β-sheet secondary structures. One of the strongest arguments supporting the „protein-only hypothesis‟ is the link between inherited prion diseases and mutations in the PRNP gene. In our recent studies, we have determined the NMR solution-state structures of the truncated recombinant human (Hu) PrPs carrying the pathological Q212P (90-231, M129)1 and V210I (90-231, M129)2 mutations linked to GSS and genetic CJD, respectively. In order to determine high-resolution structures triple resonance (1H, 13C and 15N) NMR experiments were performed using 800 MHz NMR spectrometer. The determined structures of both mutants consist of unstructured N-terminal part (residues 90-124) and well-defined C-terminal domain (residues 125-228). The C-terminal part contains three -helices (residues 144-156, 173-193 and 200-230) and a short, antiparallel -sheet (residues 129-130 and 162-163). Detailed analysis and comparison with the structure of the WT Hu-PrP revealed that although structures share similar global fold, mutations introduces some local structural differences. The observed variations are mostly clustered at the α2-α3 inter-helical interface and in the β2-α2 loop region. The alteration of conformation of the β2–α2 loop region and the subsequent changes in hydrophobic cluster may facilitate intermolecular interactions between PrPs. The high-resolution NMR structures offer new clues on the earliest events of the pathogenic conversion process and could be used for the development of antiprion drugs.

Prion diseases belong to a group of fatal neurodegenerative disorders caused by the conversion of the normal cellular prion protein (PrPC) into its pathogenic form (PrPSc). According to the "protein-only hypothesis" PrPSc is the sole component of the infectious agents. One of the key arguments supporting this hypothesis is the link between inherited prion diseases and mutations in the gene coding for human PrP. Several pathogenic mutations leading to familial prion diseases have been identified in the prion protein gene (PRNP) open reading frame. However, it is still largely unknown how these mutations affect the PrPC→PrPSc conversion. Structural studies on PrP variants carrying familial mutations may provide new clues about the molecular mechanism at early stages of the disease. In the current study we have determined a high-resolution 3D structure of the truncated recombinant HuPrP(90-231) containing the pathological Q212P mutation that is associated with a Gerstmann-Sträussler-Scheinker (GSS) syndrome. In comparison to the other known PrP structures, structure of Q212P mutant shows some unique structural features. The most remarkable differences involve the C-terminal end of the protein and the β2-α2 loop. Spontaneous generation of PrPSc in inherited prion diseases might be due to the disruptions of the hydrophobic core consisting of β2-α2 loop and α3 helix.

Prion diseases, or transmissible spongiform encephalopathies (TSEs), are fatal neurodegenerative disorders associated with the conversion of a normal (cellular) prion protein, PrPC, into a misfolded isoform, PrPSc. According to the "protein-only hypothesis" PrPSc itself is the infectious TSE pathogen. One of the strongest arguments supporting this hypothesis is the link between inherited prion diseases and mutations in the gene coding for human PrP. Several point mutations leading to familial prion diseases have been identified in PRNP gene but largely still unknown is how these mutations affect the transition of PrPC to PrPSc. Structural studies on PrP variants carrying familial mutations, more prone to spontaneous conversion into the infectious form, may be an important step toward understanding the molecular mechanism at early stages of the disease. In the current study we have determined a high-resolution 3D structure of the truncated recombinant HuPrP(90-231) with the pathological Q212P mutation that is associated with a Gerstmann-Sträussler-Scheinker (GSS) syndrome. The structure of Q212P mutant reveals unique structural features in comparison to the known wild-type PrP structures. The most remarkable differences involve the C-terminal end of the protein and the β2-α2 loop region. The spontaneous generation of PrPSc in familial cases might be due to the disruptions of the hydrophobic core consisting of β2-α2 loop and α3 helix.

The work describes NMR and functional investigations of the interaction between the Prion Protein and the Fibronectin type III domain of the Neuronal Cell Adhesion Molecule.

We have prepared and structurally characterized the first examples of doubly ortho-palladated azobenzenes. The structures of complexes were determined by elementary analysis, 1H and 13C NMR spectroscopies, and X-ray diffraction. The products were additionally characterized by UV-vis, IR and fluorimetric spectroscopies. The experimental investigation was complemented by DFT (BP86/SDD/6-31G(d, p)) quantum-chemical calculations in order to provide rational explanation of the structures by comparison of their energies with other conceivable isomers. Two metalated phenyl rings and two easily replaceable solvent ligands make these complexes excellent building blocks for designing a wide variety of metallomesogenes, organometallic polymers and even more elaborate supramolecular systems.

Already predicted in 1946 by Bloch[1] nuclear spin noise, stemming from incomplete cancellation of transverse magnetization components, could not be detected before 1985, when detection of NQR noise was first reported[2]. With state-of-the-art high-resolution NMR spectrometers, in particular when using cryogenically cooled probes, spin noise observation is fairly straightforward today. It has been used previously to generate images of proton spin density without radio frequency excitation[3]. Our current research focuses on the fundamentals of spin noise in NMR spectroscopy and systematic assessment of experimental manifestation of spin noise phenomena. Quantification of spin noise amplitudes and line shapes are complicated due the effects of radiation damping. Only if radiation damping is quenched, e.g. by a static field gradient, linear dependence between the power spectral amplitude and the number of spins can be observed as was the case in our earlier study[3]. Of particular interest is the dependence of spin noise line shape on the tuning of the receiving resonance circuit. The large offset of -570 kHz between the tuning optimum and the “ spin noise dip tuning” , deviates significantly from what is intuitively expected and in important aspects also from what is predicted by a formalism based on Nyquist noise introduced by Ernst and McCoy[4]. This tuning dependence may be used to optimise tuning and as a guide for the design of resonance circuits in magnetic resonance probes. References [1] Bloch, F. (1946) Phys. Rev. 70, 460-475 [2] Sleator, T., Hahn, E.L., Hilbert, C. and Clarke, J. (1985) Phys. Rev. Lett. 55, 1742-1746 [3] Müller, N. and Jerschow, A. (2006) Proc. Nat. Acad. Sci. (USA) 103, 6790-6792 [4] McCoy, M.A. and Ernst, R.R. (1989) Chem. Phys. Lett. 139, 587-593 Acknowledgement This research is supported by the Austrian Science Funds FWF, project P19635-N17 (to N.M.), the ÖAD (WTZ AT-HR, to N.M.), a grant to A.J. by the US NSF (CHE-0550054), and by the Croatian Ministry of Science, Education and Sports (project 098-0982929-2917, to V.S.).