NMR “Crystallography” for Uniformly (13C, 15N)-Labeled Oriented Membrane Proteins

In oriented-sample (OS) solid-state NMR of membrane proteins, the angular-dependent dipolar couplings and chemical shifts provide a direct input for structure calculations. However, so far only ¹H–¹⁵N dipolar couplings and ¹⁵N chemical shifts have been routinely assessed in oriented ¹⁵N-labeled samples. The main obstacle for extending this technique to membrane proteins of arbitrary topology has remained in the lack of additional experimental restraints. We have developed a new experimental triple-resonance NMR technique, which was applied to uniformly doubly (¹⁵N, ¹³C)-labeled Pf1 coat protein in magnetically aligned DMPC/DHPC bicelles. The previously inaccessible ¹H_α–¹³C_α dipolar couplings have been measured, which make it possible to determine the torsion angles between the peptide planes without assuming α-helical structure a priori. The fitting of three angular restraints per peptide plane and filtering by Rosetta scoring functions has yielded a consensus α-helical transmembrane structure for Pf1 protein.     

Synthesis and conformational analysis of efrapeptins

The efrapeptin family of peptide antibiotics produced by the fungus Tolypocladium niveum, and the neo‐efrapeptins from the fungus Geotrichum candidumare inhibitors of F₁‐ATPase with promising antitumor, antimalaria, and insecticidal activity. They are rich in C^α‐dialkyl amino acids (Aib, Iva, Acc) and contain one β‐alanine and several pipecolic acid residues. The C‐terminus bears an unusual heterocyclic cationic cap. The efrapeptins C–G and three analogues of efrapeptin C were synthesized using α‐azido carboxylic acids as masked amino acid derivatives. All compounds display inhibitory activity toward F₁‐ATPase. The conformation in solution of the peptides was investigated with electronic CD spectroscopy, FT‐IR spectroscopy, and VCD spectroscopy. All efrapeptins and most efrapeptin analogues were shown to adopt helical conformations in solution. In the case of efrapeptin C, VCD spectra proved that a 3₁₀‐helix prevails. In addition, efrapeptin C was conformationally studied in detail with NMR and molecular modeling. Besides NOE distance restraints, residual dipolar couplings (RDC) observed upon partial alignment with stretched PDMS gels were used for the conformational analysis and confirmed the 3₁₀‐helical conformation.     

Helix Nucleation by the Smallest Known α‐Helix in Water

Cyclic pentapeptides (e.g. Ac‐(cyclo‐1,5)‐[KAXAD]‐NH₂; X=Ala, 1 ; Arg, 2 ) in water adopt one α‐helical turn defined by three hydrogen bonds. NMR structure analysis reveals a slight distortion from α‐helicity at the C‐terminal aspartate caused by torsional restraints imposed by the K(i)–D(i+4) lactam bridge. To investigate this effect on helix nucleation, the more water‐soluble 2 was appended to N‐, C‐, or both termini of a palindromic peptide ARAARAARA (≤5 % helicity), resulting in 67, 92, or 100 % relative α‐helicity, as calculated from CD spectra. From the C‐terminus of peptides, 2 can nucleate at least six α‐helical turns. From the N‐terminus, imperfect alignment of the Asp5 backbone amide in 2 reduces helix nucleation, but is corrected by a second unit of 2 separated by 0–9 residues from the first. These cyclic peptides are extremely versatile helix nucleators that can be placed anywhere in 5–25 residue peptides, which correspond to most helix lengths in protein–protein interactions.     

RDC‐enhanced structure calculation of a β‐heptapeptide in methanol

Residual dipolar couplings (RDCs) are a rich source of structural information that goes beyond the range covered by the nuclear Overhauser effect or scalar coupling constants. They can only be measured in partially oriented samples. RDC studies of peptides in organic solvents have so far been focused on samples in chloroform or DMSO. Here, we show that stretched poly(vinyl acetate) can be used for the partial alignment of a linear β‐peptide with proteinogenic side chains in methanol. ¹D_CH, ¹D_NH, and ²D_HH RDCs were collected with this sample and included as restraints in a simulated annealing calculation. Incorporation of RDCs in the structure calculation process improves the long‐range definition in the backbone of the resulting 3₁₄‐helix and uncovers side‐chain mobility. Experimental side‐chain RDCs of the central leucine and valine residues are in good agreement with predicted values from a local three‐state model. Copyright © 2016 John Wiley & Sons, Ltd.     

Designing Point Mutants to Detect Structural Coupling in a Heterotrimeric G Protein α‐subunit by NMR Spectroscopy†

To better understand the mechanism by which the activating signal is transmitted from the receptor‐interacting regions on the G protein α‐subunit (G_α) to the guanine nucleotide‐binding pocket, we generated and characterized mutant forms of G_α with alterations in switch II (Trp‐207→Phe) and the carboxyl‐terminus (Phe‐350→Ala). Previously reported bacterial expression methods for the high‐level production of a uniformly isotope‐labeled G_tα/G_i1α chimera, ChiT, were successfully used to isolate milligram quantities of ¹⁵N‐labeled mutant protein. NMR analysis showed that while the GDP/Mg²⁺‐bound state of both mutants shared an overall conformation similar to that of the GDP/Mg²⁺‐bound state of ChiT, formation of the “transition/activated” state in the presence of aluminum fluoride (AlF₄^?) revealed distinct differences between the wild‐type and mutant G_α subunits, particularly in the response of the ¹HN, ¹⁵N cross‐peak for the Trp‐254 indole in the Trp‐207→Phe mutant and the ¹HN, ¹⁵N cross‐peak for Ala‐350 in the Phe‐350→Ala mutant. Consistent with the NMR data, the F350→Ala mutant showed an increase in intrinsic fluorescence that was similar to G_tα and ChiT upon formation of the “transition/activated” state in the presence of AlF₄^?, whereas the intrinsic fluorescence of the Trp‐207→Phe mutant decreased. These results show that the substitution of key amino acid positions in G_α can effect structural changes that may compromise receptor interactions and GDP/GTP exchange.     

The Propensity of α‐Aminoisobutyric Acid (=2‐Methylalanine; Aib) to Induce Helical Secondary Structure in an α‐Heptapeptide: A Computational Study

We present a molecular‐dynamics simulation study of an α‐heptapeptide containing an α‐aminoisobutyric acid (=2‐methylalanine; Aib) residue, Val¹‐Ala²‐Leu³‐Aib⁴‐Ile⁵‐Met⁶‐Phe⁷, and a quantum‐mechanical (QM) study of simplified models to investigate the propensity of the Aib residue to induce 3₁₀/α‐helical conformation. For comparison, we have also performed simulations of three analogues of the peptide with the Aib residue being replaced by L ‐Ala, D ‐Ala, and Gly, respectively, which provide information on the subtitution effect at C(α) (two Me groups for Aib, one for L ‐Ala and D ‐Ala, and zero for Gly). Our simulations suggest that, in MeOH, the heptapeptide hardly folds into canonical helical conformations, but appears to populate multiple conformations, i.e., C₇ and 3₁₀‐helical ones, which is in agreement with results from the QM calculations and NMR experiments. The populations of these conformations depend on the polarity of the solvent. Our study confirms that a short peptide, though with the presence of an Aib residue in the middle of the chain, does not have to fold to an α‐helical secondary structure. To generate a helical conformation for a linear peptide, several Aib residues should be present in the peptide, either sequentially or alternatively, to enhance the propensity of Aib‐containing peptides towards the helical conformation. A correction of a few of the published NMR data is reported.     

Proton evolved local field solid-state nuclear magnetic resonance using Hadamard encoding: theory and application to membrane proteins

NMR anisotropic parameters such as dipolar couplings and chemical shifts are central to structure and orientation determination of aligned membrane proteins and liquid crystals. Among the separated local field experiments, the proton evolved local field (PELF) scheme is particularly suitable to measure dynamically averaged dipolar couplings and give information on local molecular motions. However, the PELF experiment requires the acquisition of several 2D datasets at different mixing times to optimize the sensitivity for the complete range of dipolar couplings of the resonances in the spectrum. Here, we propose a new PELF experiment that takes the advantage of the Hadamard encoding (HE) to obtain higher sensitivity for a broad range of dipolar couplings using a single 2D experiment. The HE scheme is obtained by selecting the spin operators with phase switching of hard pulses. This approach enables one to detect four spin operators, simultaneously, which can be processed into two 2D spectra covering a broader range of dipolar couplings. The advantages of the new approach are illustrated for a U-(15)N NAL single crystal and the U-(15)N labeled single-pass membrane protein sarcolipin reconstituted in oriented lipid bicelles. The HE-PELF scheme can be implemented in other multidimensional experiments to speed up the characterization of the structure and dynamics of oriented membrane proteins and liquid crystalline samples.     

Polyoxometalates as a Novel Class of Artificial Proteases: Selective Hydrolysis of Lysozyme under Physiological pH and Temperature Promoted by a Cerium(IV) Keggin‐Type Polyoxometalate

Hen‐egg‐white lysozyme (HEWL) is specifically cleaved at the Trp28–Val29 and Asn44–Arg45 peptide bonds in the presence of a Keggin‐type [Ce(α‐PW₁₁O₃₉)₂]^10? polyoxometalate (POM; 1 ) at pH 7.4 and 37 °C. The reactivity of 1 towards a range of dipeptides was also examined and the calculated reaction rates were comparable to those observed for the hydrolysis of HEWL. Experiments with α‐lactalbumin (α‐LA), a protein that is structurally highly homologous to HEWL but has a different surface potential, showed no evidence of hydrolysis, which indicates the importance of electrostatic interactions between 1 and the protein surface for the hydrolytic reaction to occur. A combination of spectroscopic techniques was used to reveal the molecular interactions between HEWL and 1 that lead to hydrolysis. NMR spectroscopy titration experiments showed that on protein addition the intensity of the ³¹P NMR signal of 1 gradually decreased due to the formation of a large protein/polyoxometalate complex and completely disappeared when the HEWL/ 1 ratio reached 1:2. Circular dichroism (CD) measurements of HEWL indicate that addition of 1 results in a clear decrease in the signal at λ=208 nm, which is attributed to changes in the α‐helical content of the protein. ¹⁵N–¹H heteronuclear single quantum coherence (HSQC) NMR measurements of HEWL in the presence of 1 reveal that the interaction is mainly observed for residues that are located in close proximity to the first site in the α‐helical part of the structure (Trp28–Val29). The less pronounced NMR spectroscopic shifts around the second cleavage site (Asn44–Arg45), which is found in the β‐strand region of the protein, might be caused by weaker metal‐directed binding, compared with strong POM‐directed binding at the first site.     

The “Speedy” Synthesis of Atom‐Specific 15N Imino/Amido‐Labeled RNA

Although numerous reports on the synthesis of atom‐specific ¹⁵N‐labeled nucleosides exist, fast and facile access to the corresponding phosphoramidites for RNA solid‐phase synthesis is still lacking. This situation represents a severe bottleneck for NMR spectroscopic investigations on functional RNAs. Here, we present optimized procedures to speed up the synthesis of ¹⁵N(1) adenosine and ¹⁵N(1) guanosine amidites, which are the much needed counterparts of the more straightforward‐to‐achieve ¹⁵N(3) uridine and ¹⁵N(3) cytidine amidites in order to tap full potential of ¹H/¹⁵N/¹⁵N‐COSY experiments for directly monitoring individual Watson–Crick base pairs in RNA. Demonstrated for two preQ₁ riboswitch systems, we exemplify a versatile concept for individual base‐pair labeling in the analysis of conformationally flexible RNAs when competing structures and conformational dynamics are encountered.     

Interaction of PiB‐Derivative Metal Complexes with Beta‐Amyloid Peptides: Selective Recognition of the Aggregated Forms

Metal complexes are increasingly explored as imaging probes in amyloid peptide related pathologies. We report the first detailed study on the mechanism of interaction between a metal complex and both the monomer and the aggregated form of Aβ_1–40 peptide. We have studied lanthanide(III) chelates of two PiB‐derivative ligands (PiB=Pittsburgh compound B), L¹ and L², differing in the length of the spacer between the metal‐complexing DO3A macrocycle (DO3A= 1,4,7,10‐tetraazacyclododecane‐1,4,7‐triacetic acid) and the peptide‐recognition PiB moiety. Surface plasmon resonance (SPR) and saturation transfer difference (STD) NMR spectroscopy revealed that they both bind to aggregated Aβ_1–40 (K_D=67–160 μM ), primarily through the benzothiazole unit. HSQC NMR spectroscopy on the ¹⁵N‐labeled, monomer Aβ_1–40 peptide indicates nonsignificant interaction with monomeric Aβ. Time‐dependent circular dichroism (CD), dynamic light scattering (DLS), and TEM investigations of the secondary structure and of the aggregation of Aβ_1–40 in the presence of increasing amounts of the metal complexes provide coherent data showing that, despite their structural similarity, the two complexes affect Aβ fibril formation distinctly. Whereas GdL¹, at higher concentrations, stabilizes β‐sheets, GdL² prevents aggregation by promoting α‐helical structures. These results give insight into the behavior of amyloid‐targeted metal complexes in general and contribute to a more rational design of metal‐based diagnostic and therapeutic agents for amyloid‐ associated pathologies.     

Investigation of the Structure and Dynamics of the Capsid–Spacer Peptide 1–Nucleocapsid Fragment of the HIV‐1 Gag Polyprotein by Solution NMR Spectroscopy

Structural studies of HIV‐1 Gag, the primary structural polyprotein involved in retroviral assembly, have been challenging, owing to its flexibility and conformational heterogeneity. Using residual dipolar couplings, we show that the four structural units of the capsid (CA)–spacer peptide 1 (SP1)–nucleocapsid (NC) fragment of HIV‐1 Gag (namely, the N‐ and C‐terminal domains of capsid, and the N‐ and C‐terminal Zn knuckles of nucleocapsid) have the same structures as their individually isolated counterparts, and tumble semi‐independently of one another in the absence of nucleic acids. Nucleic acids bind exclusively to the nucleocapsid domain and fix the orientation of the two Zn knuckles relative to one another so that the nucleocapsid domain/nucleic acid complex behaves as a single structural unit. The low ¹⁵N–{¹H} heteronuclear NOE values (≤0.4), the close to zero values for the residual dipolar couplings of the backbone amides, and minimal deviations from random‐coil chemical shifts for the C‐terminal tail of capsid and SP1, both in the absence and presence of nucleic acids, indicate that these regions are intrinsically disordered in the context of CA–SP1–NC.     

α‐Aminoxy Oligopeptides: Synthesis,Secondary Structure,and Cytotoxicity of a New Class of Anticancer Foldamers

α‐Aminoxy peptides are peptidomimetic foldamers with high proteolytic and conformational stability. To gain an improved synthetic access to α‐aminoxy oligopeptides we used a straightforward combination of solution‐ and solid‐phase‐supported methods and obtained oligomers that showed a remarkable anticancer activity against a panel of cancer cell lines. We solved the first X‐ray crystal structure of an α‐aminoxy peptide with multiple turns around the helical axis. The crystal structure revealed a right‐handed 2₈‐helical conformation with precisely two residues per turn and a helical pitch of 5.8 Å. By 2D ROESY experiments, molecular dynamics simulations, and CD spectroscopy we were able to identify the 2₈‐helix as the predominant conformation in organic solvents. In aqueous solution, the α‐aminoxy peptides exist in the 2₈‐helical conformation at acidic pH, but exhibit remarkable changes in the secondary structure with increasing pH. The most cytotoxic α‐aminoxy peptides have an increased propensity to take up a 2₈‐helical conformation in the presence of a model membrane. This indicates a correlation between the 2₈‐helical conformation and the membranolytic activity observed in mode of action studies, thereby providing novel insights in the folding properties and the biological activity of α‐aminoxy peptides.     

Conformational Studies on Peptides of α‐Aminoxy Acids with Functionalized Side‐Chains

Peptides of homochiral α‐aminoxy acids of nonpolar side chains can form a 1.8₈‐helix. In this paper, we report the conformational studies of α‐aminoxy peptides 1 , 2 , 3 , which have functionalized side chains, in both nonpolar and polar solvents. ¹H NMR, XRD, and FTIR absorption studies confirm the presence of the eight‐membered‐ring intramolecular hydrogen bonds (the N‐O turns) in nonpolar solvents as well as in methanol. CD studies of peptides 1 , 2 , 3 in different solvents indicate that a substantial degree of helical content is retained in methanol and acidic aqueous buffers. The introduction of functionalized side chains in α‐aminoxy peptides provides opportunities for designing biologically active peptides.     

Exclusively Heteronuclear NMR Experiments to Obtain Structural and Dynamic Information on Proteins

Provided that ¹³C‐detected NMR experiments are either preferable or complementary to ¹H detection, we report here tools to determine C^α? C′, C′? N, and C^α? H^α residual dipolar couplings on the basis of the CON experiment. The coupling constants determined on ubiquitin are consistent with the subset measured with the ¹H‐detected HNCO sequences. Since the utilization of residual dipolar couplings may depend on the mobility of the involved nuclei, we also provide tools to measure longitudinal and transverse relaxation rates of N and C′. This new set of experiments is a further development of a whole strategy based on ¹³C direct‐detection NMR spectroscopy for the study of biological macromolecules.     

POSS end‐linked peptide‐functionalized poly(ɛ‐caprolactone)s and their inclusion complexes with α‐cyclodextrin

In this report, we have synthesized organic/inorganic hybrid peptide–poly(?‐caprolactone) (PCL) conjugates via ring opening polymerization (ROP) of ?‐caprolactone (CL) in the presence of two sequence defined peptide initiators, namely POSS‐Leu‐Aib‐Leu‐NH₂ (POSS: polyhedral oligomeric silsesquioxane; Leu: Leucine; Aib: α‐aminoisobutyric acid) and OMe‐Leu‐Aib‐Leu‐NH₂. Covalent attachment of peptide segments with the PCLs were examined by ¹H and ²⁹Si NMR spectroscopy, matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) and FTIR spectroscopy. Supramolecular inclusion complexations of synthesized peptide‐PCL conjugates with α‐cyclodextrin (α‐CyD) were studied to understand the effect of POSS/OMe‐peptide moieties at the PCL chain ends. Inclusion complexation of peptide‐PCL conjugates with α‐CyD produced linear polypseudorotaxane, confirmed by ¹H NMR, FTIR, powder X‐ray diffraction (PXRD), polarized optical microscopy (POM) and differential scanning calorimetry (DSC). Extent of α‐CyD threading onto the hybrid peptide‐PCL conjugated polymers is less than that of α‐CyD threaded onto the linear PCL. Thus, PCL chains were not fully covered by the host α‐CyD molecules due to the bulky POSS/OMe‐peptide moieties connected with the one edge of the PCL chains. PXRD experiment reveals channel like structures by the synthesized inclusion complexes (ICs). Spherulitic morphologies of POSS/OMe‐peptide‐PCL conjugates were fully destroyed after inclusion complexation with α‐CyD and tiny nanoobjects were produced. © 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016 , 54, 3643–3651.     

Phosphorus NMR Characterisation of P–N Bond Rotamers of a α‐P4S3 Amide with a Conformationally Constrained Chiral Substituent

Reactions of bicyclic α‐P₄S₃I₂ with Hpthiq gave solutions containing α‐P₄S₃(pthiq)I and α‐P₄S₃(pthiq)₂, where Hpthiq is the conformationally constrained chiral secondary amine 1‐phenyl‐1,2,3,4‐tetrahydroisoquinoline. The expected diastereomers have been characterised by complete analysis of their ³¹P{¹H} NMR spectra. Hindered P–N bond rotation in the amide iodide α‐P₄S₃(pthiq)I caused greater broadening of peaks in the room‐temperature spectrum of one diastereomer than in that of the other. At 183 K, spectra of two P–N bond rotamers for each diastereomer were observed and analysed. The minor rotamers showed strong evidence for steric crowding, having large diastereomeric differences in ¹J(P–P) and ²J(P–S–P) couplings (49 Hz, 16 % of value, and 4.4 Hz, 19 % of value, respectively).     

A rare occurrence of a β‐turn in an amyloid βA4 peptide

The fragment β(25–35) of the amyloid β‐peptide, like its parent βA4, has shown neurotrophic and late neurotoxic activities in cultured cells. The 3D structure of this important peptide was examined by ¹H and ¹³C 2D‐NMR and MD simulations in DMSO‐d₆ and water. The NMR parameters of chemical shift, ³J(N,Hα) coupling constants, temperature coefficients of NH chemical shifts and the pattern of intra and inter‐residue NOEs were used to deduce the structures. In DMSO‐d₆, the peptide was found to take up a type I β‐turn around the C‐terminal residues Ile⁸–Gly⁹–Leu¹⁰–Met¹¹, whereas in water at pH 5.5, it adopts a random coil conformation. This is only the second report of a β‐turn in the β‐amyloid class of peptides. The solution structures generated using restrained molecular dynamics were refined by MARDIGRAS to an R factor of 0.33 in the case of DMSO‐d₆ and to 0.56 for water. Copyright © 2002 John Wiley & Sons, Ltd.     

Aggregation of amyloid Aβ(1–40) peptide in perdeuterated 2,2,2‐trifluoroethanol caused by ultrasound sonication

Ultrasound sonication of protein and peptide solutions is routinely used in biochemical, biophysical, pharmaceutical and medical sciences to facilitate and accelerate dissolution of macromolecules in both aqueous and organic solvents. However, the impact of ultrasound waves on folding/unfolding of treated proteins, in particular, on aggregation kinetics of amyloidogenic peptides and proteins is not understood. In this work, effects of ultrasound sonication on the misfolding and aggregation behavior of the Alzheimer's Aβ_(1–40)‐peptide is studied by pulsed‐field gradient (PFG) spin–echo diffusion NMR and UV circular dichroism (CD) spectroscopy. Upon simple dissolution of Aβ_(1–40) in perdeuterated trifluoroethanol, CF₃‐CD₂‐OD (TFE‐d₃), the peptide is present in the solution as a stable monomer adopting α‐helical secondary structural motifs. The self‐diffusion coefficient of Aβ_(1–40) monomers in TFE‐d₃ was measured as 1.35 × 10^?10 m² s^?1, reflecting its monomeric character. However, upon ultrasonic sonication for less than 5 min, considerable populations of Aβ molecules (ca 40%) form large aggregates as reflected in diffusion coefficients smaller than 4.0 × 10^?13 m² s^?1. Sonication for longer times (up to 40 min in total) effectively reduces the fraction of these aggregates in ¹H PFG NMR spectra to ca 25%. Additionally, absorption below 230 nm increased significantly upon sonication treatment, an observation, which also clearly confirms the ongoing aggregation process of Aβ_(1–40) in TFE‐d₃. Surprisingly, upon ultrasound sonication only small changes in the peptide secondary structure were detected by CD: the peptide molecules mainly adopt α‐helical motifs in both monomers and aggregates formed upon sonication. Copyright © 2010 John Wiley & Sons, Ltd.     

Dipolar waves as NMR maps of protein structure

The anisotropy of nuclear spin interactions results in a unique mapping of structure to the resonance frequencies and split tings observed in NMR spectra, however, the determination of molecular structure from experimentally measured spectral parameters is complicated by angular ambiguities resulting from the symmetry properties of dipole-dipole and chemical shift interactions. This issue can be addressed through the periodicity inherent in secondary structure elements, which can be used as an index of topology. Distinctive wheel-like patterns are observed in two-dimensional 1H-15N heteronuclear dipolar/15N chemical shift PISEMA (polarization inversion spin-exchange at the magic angle) spectra of helical membrane proteins in highly aligned lipid bilayer samples. One-dimensional dipolar waves are an extension of two-dimensional PISA (polarity index slant angle) wheels to map protein structure in NMR spectra of both highly and weakly aligned samples. Dipolar waves describe the periodic wavelike variations of the magnitudes of the static heteronuclear dipolar couplings as a function of residue number in the absence of chemical shift effects. Weakly aligned samples of proteins display these same effects, primarily as residual dipolar couplings (RDCs), in solution NMR spectra. The corresponding properties of the RDCs in solution NMR spectra of weakly aligned helices represent a convergence of solid-state and solution NMR approaches to structure determination.     

CABS‐NMR—De novo tool for rapid global fold determination from chemical shifts,residual dipolar couplings and sparse methyl‐methyl noes

Recent development of nuclear magnetic resonance (NMR) techniques provided new types of structural restraints that can be successfully used in fast and low‐cost global protein fold determination. Here, we present CABS‐NMR, an efficient protein modeling tool, which takes advantage of such structural restraints. The restraints are converted from original NMR data to fit the coarse grained protein representation of the C‐Alpha‐Beta‐Side‐group (CABS) algorithm. CABS is a Monte Carlo search algorithm that uses a knowledge‐based force field. Its versatile structure enables a variety of protein‐modeling protocols, including purely de novo folding, folding guided by restraints derived from template structures or, structure assembly based on experimental data. In particular, CABS‐NMR uses the distance and angular restraints set derived from various NMR experiments. This new modeling technique was successfully tested in structure determination of 10 globular proteins of size up to 216 residues, for which sparse NMR data were available. Additional detailed analysis was performed for a S100A1 protein. Namely, we successfully predicted Nuclear Overhauser Effect signals on the basis of low‐energy structures obtained from chemical shifts by CABS‐NMR. It has been observed that utility of chemical shifts and other types of experimental data (i.e. residual dipolar couplings and methyl‐methyl Nuclear Overhauser Effect signals) in the presented modeling pipeline depends mainly on size of a protein and complexity of its topology. In this work, we have provided tools for either post‐experiment processing of various kinds of NMR data or fast and low‐cost structural analysis in the still challenging field of new fold predictions. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2011