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Author(s): Ivan Anishchenko [sup.1] [sup.2] , Samuel J. Pellock [sup.1] [sup.2] , Tamuka M. Chidyausiku [sup.1] [sup.2] , Theresa A. Ramelot [sup.3] [sup.4] , Sergey Ovchinnikov [sup.5] , Jingzhou Hao [...], There has been considerable recent progress in protein structure prediction using deep neural networks to predict inter-residue distances from amino acid sequences.sup.1-3. Here we investigate whether the information captured by such networks is sufficiently rich to generate new folded proteins with sequences unrelated to those of the naturally occurring proteins used in training the models. We generate random amino acid sequences, and input them into the trRosetta structure prediction network to predict starting residue-residue distance maps, which, as expected, are quite featureless. We then carry out Monte Carlo sampling in amino acid sequence space, optimizing the contrast (Kullback-Leibler divergence) between the inter-residue distance distributions predicted by the network and background distributions averaged over all proteins. Optimization from different random starting points resulted in novel proteins spanning a wide range of sequences and predicted structures. We obtained synthetic genes encoding 129 of the network-'hallucinated' sequences, and expressed and purified the proteins in Escherichia coli; 27 of the proteins yielded monodisperse species with circular dichroism spectra consistent with the hallucinated structures. We determined the three-dimensional structures of three of the hallucinated proteins, two by X-ray crystallography and one by NMR, and these closely matched the hallucinated models. Thus, deep networks trained to predict native protein structures from their sequences can be inverted to design new proteins, and such networks and methods should contribute alongside traditional physics-based models to the de novo design of proteins with new functions. The trRosetta neural network was used to iteratively optimise model proteins from random 100-amino-acid sequences, resulting in 'hallucinated' proteins, which when expressed in bacteria closely resembled the model structures.

Degenerate encoding of 20 amino acids by 61 triplet nucleotide codons enables the same protein sequence to be synthesized by a vast number of synonymous mRNAs. While variations between synonymous [...], Degeneracy in the genetic code, which enables a single protein to be encoded by a multitude of synonymous gene sequences, has an important role in regulating protein expression, but substantial uncertainty exists concerning the details of this phenomenon. Here we analyse the sequence features influencing protein expression levels in 6,348 experiments using bacteriophage T7 polymerase to synthesize messenger RNA in Escherichia coli. Logistic regression yields a new codon-influence metric that correlates only weakly with genomic codon-usage frequency, but strongly with global physiological protein concentrations and also mRNA concentrations and lifetimes in vivo. Overall, the codon content influences protein expression more strongly than mRNA-folding parameters, although the latter dominate in the initial ~16 codons. Genes redesigned based on our analyses are transcribed with unaltered efficiency but translated with higher efficiency in vitro. The less efficiently translated native sequences show greatly reduced mRNA levels in vivo. Our results suggest that codon content modulates a kinetic competition between protein elongation and mRNA degradation that is a central feature of the physiology and also possibly the regulation of translation in E. coli.

For proteins to fold, the interactions favouring the native state must collectively outweigh the non-native interactions, resulting in funnel-shaped energy landscapes (1,3). However, it is not obvious how the ubiquitous [...], Unlike random heteropolymers, natural proteins fold into unique ordered structures. Understanding how these are encoded in amino-acid sequences is complicated by energetically unfavourable non-ideal features--for example kinked α-helices, bulged β-strands, strained loops and buried polar groups--that arise in proteins from evolutionary selection for biological function or from neutral drift. Here we describe an approach to designing ideal protein structures stabilized by completely consistent local and non-local interactions. The approach is based on a set of rules relating secondary structure patterns to protein tertiary motifs, which make possible the design of funnel-shaped protein folding energy landscapes leading into the target folded state. Guided by these rules, we designed sequences predicted to fold into ideal protein structures consisting of α-helices, β-strands and minimal loops. Designs for five different topologies were found to be monomeric and very stable and to adopt structures in solution nearly identical to the computational models. These results illuminate how the folding funnels of natural proteins arise and provide the foundation for engineering a new generation of functional proteins free from natural evolution.

A recently determined set of 20 NMR-derived conformations of a 48-residue all-[alpha]-helical protein, (PDB ID code 2JVD), is validated here by comparing the observed [sup.13][C.sup.[alpha]] chemical shifts with those computed at the density functional level of theory. In addition, a recently introduced physics-based method, aimed at determining protein structures by using NOE-derived distance constraints together with observed and computed [sup.13][C.sup.[alpha]] chemical shifts, was applied to determine a new set of 10 conformations, (Set-bt), as a blind test for the same protein. A cross-validation of these two sets of conformations in terms of the agreement between computed and observed [sup.13][C.sup.[alpha]] chemical shifts, several stereochemical quality factors, and some NMR quality assessment scores reveals the good quality of both sets of structures. We also carried out an analysis of the agreement between the observed and computed [sup.13][C.sup.[alpha]] chemical shifts for a slightly longer construct of the protein solved by x-ray crystallography at 2.0-[Angstrom] resolution (PDB ID code 3BHP) with an identical amino acid residue sequence to the 2JVD structure for the first 46 residues. Our results reveal that both of the NMR-derived sets, namely 2JVD and Set-bt, are somewhat better representations of the observed [sup.13][C.sup.[alpha]] chemical shifts in solution than the 3BHP crystal structure. In addition, the [sup.13][C.sup.[alpha]]-based validation analysis appears to be more sensitive to subtle structural differences across the three sets of structures than any other NMR quality-assessment scores used here, and, although it is computationally intensive, this analysis has potential value as a standard procedure to determine, refine, and validate protein structures.

Influenza A viruses are responsible for seasonal epidemics and high mortality pandemics. A major function of the viral NS1A protein, a virulence factor, is the inhibition of the production of IFN-[beta] mRNA and other antiviral mRNAs. The NS1A protein of the human influenza A/Udorn/72 (Ud) virus inhibits the production of these antiviral mRNAs by binding the cellular 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), which is required for the 3' end processing of all cellular pre-mRNAs. Here we report the 1.95-[Angstrom] resolution X-ray crystal structure of the complex formed between the second and third zinc finger domain (F2F3) of CPSF30 and the C-terminal domain of the Ud NS1A protein. The complex is a tetramer, in which each of two F2F3 molecules wraps around two NS1A effector domains that interact with each other head-to-head. This structure identifies a CPSF30 binding pocket on NS1A comprised of amino acid residues that are highly conserved among human influenza A viruses. Single amino acid changes within this binding pocket eliminate CPSF30 binding, and a recombinant Ud virus expressing an NS1A protein with such a substitution is attenuated and does not inhibit IFN-[beta] pre-mRNA processing. This binding pocket is a potential target for antiviral drug development. The crystal structure also reveals that two amino acids outside of this pocket, F103 and M106, which are highly conserved (>99%) among influenza A viruses isolated from humans, participate in key hydrophobic interactions with F2F3 that stabilize the complex. antiviral drug discovery | bird flu | vaccine engineering | virology | X-ray crystallography

Protein NMR chemical shifts are highly sensitive to local structure. A robust protocol is described that exploits this relation for de novo protein structure generation, using as input experimental parameters the [sup.13][C.sup.[alpha]], [sup.13][C.sup.[beta]], [sup.13][C.sup.], [sup.15]N, [sup.1][H.sup.[alpha]] and [sup.1][H.sup.N] NMR chemical shifts. These shifts are generally available at the early stage of the traditional NMR structure determination process, before the collection and analysis of structural restraints. The chemical shift based structure determination protocol uses an empirically optimized procedure to select protein fragments from the Protein Data Bank, in conjunction with the standard ROSETTA Monte Carlo assembly and relaxation methods. Evaluation of 16 proteins, varying in size from 56 to 129 residues, yielded full-atom models that have 0.7-1.8 [Angstrom] root mean square deviations for the backbone atoms relative to the experimentally determined x-ray or NMR structures. The strategy also has been successfully applied in a blind manner to nine protein targets with molecular masses up to 15.4 kDa, whose conventional NMR structure determination was conducted in parallel by the Northeast Structural Genomics Consortium. This protocol potentially provides a new direction for high-throughput NMR structure determination. molecular fragment replacement | protein structure prediction | ROSETTA | structural genomics

Pathways are integral to systems biology. Their classical representation has proven useful but is inconsistent in the meaning assigned to each arrow (or edge) and inadvertently implies the isolation of one pathway from another. Conversely, modern high-throughput (HTP) experiments offer standardized networks that facilitate topological calculations. Combining these perspectives, classical pathways can be embedded within large-scale networks and thus demonstrate the crosstalk between them. As more diverse types of HTP data become available, both perspectives can be effectively merged, embedding pathways simultaneously in multiple networks. However, the original problem still remains-the current edge representation is inadequate to accurately convey all the information in pathways. Therefore, we suggest that a standardized and well-defined edge ontology is necessary and propose a prototype as a starting point for reaching this goal.

Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) constitute an important, yet relatively poorly understood, family of heme-containing enzymes. Here, we report extensive structural and biochemical studies of the Xanthomonas campestris TDO and a related protein SO4414 from Shewanella oneidensis, including the structure at 1.6-[Angstrom] resolution of the catalytically active, ferrous form of TDO in a binary complex with the substrate L-Trp. The carboxylate and ammonium moieties of tryptophan are recognized by electrostatic and hydrogen-bonding interactions with the enzyme and a propionate group of the heme, thus defining the L-stereospecificity. A second, possibly allosteric, L-Trp-binding site is present at the tetramer interface. The sixth coordination site of the heme-iron is vacant, providing a dioxygenbinding site that would also involve interactions with the ammonium moiety of L-Trp and the amide nitrogen of a glycine residue. The indole ring is positioned correctly for oxygenation at the C2 and C3 atoms. The active site is fully formed only in the binary complex, and biochemical experiments confirm this induced-fit behavior of the enzyme. The active site is completely devoid of water during catalysis, which is supported by our electrochemical studies showing significant stabilization of the enzyme upon substrate binding. cancer | heme enzymes | immunomodulation | indoleamine 2,3-dioxygenase

A standardized protocol enabling rapid NMR data collection for high-quality protein structure determination is presented that allows one to capitalize on high spectrometer sensitivity: a set of five G-matrix Fourier transform NMR experiments for resonance assignment based on highly resolved 4D and 5D spectral information is acquired in conjunction with a single simultaneous 3D [sup.15]N, [sup.13][C.sup.aliphatic],[sup.13][C.sup.aromatic]-resolved [[sup.1]H,[sup.1]H]-NOESY spectrum providing [sup.1]H-[sup.1]H upper distance limit constraints. The protocol was integrated with methodology for semiautomated data analysis and used to solve eight NMR protein structures of the Northeast Structural Genomics Consortium pipeline. The molecular masses of the hypothetical target proteins ranged from 9 to 20 kDa with an average of [approximately equal to] 14 kDa. Between 1 and 9 days of instrument time were invested per structure, which is less than [approximately equal to] 10-25% of the measurement time routinely required to date with conventional approaches. The protocol presented here effectively removes data collection as a bottleneck for high-throughput solution structure determination of proteins up to at least [approximately equal to] 20 kDa, while concurrently providing spectra that are highly amenable to fast and robust analysis. G-matrix Fourier transform projection NMR | NMR structure determination | structural genomics

Salicylic acid (SA) is a critical signal for the activation of plant defense responses against pathogen infections. We recently identified SA-binding protein 2 (SABP2) from tobacco as a protein that displays high affinity for SA and plays a crucial role in the activation of systemic acquired resistance to plant pathogens. Here we report the crystal structures of SABP2, alone and in complex with SA at up to 2.1-[Angstrom] resolution. The structures confirm that SABP2 is a member of the [alpha]/[beta] hydrolase superfamily of enzymes, with Ser-81, His-238, and Asp-210 as the catalytic triad. SA is bound in the active site and is completely shielded from the solvent, consistent with the high affinity of this compound for SABP2. Our biochemical studies reveal that SABP2 has strong esterase activity with methyl salicylate as the substrate, and that SA is a potent product inhibitor of this catalysis. Modeling of SABP2 with MeSA in the active site is consistent with all these biochemical observations. Our results suggest that SABP2 may be required to convert MeSA to SA as part of the signal transduction pathways that activate systemic acquired resistance and perhaps local defense responses as well. salicylic acid | salicylic-acid-binding protein | systemic acquired resistance | [alpha]/[beta] hydrolase

A summary of new methods of automation for determining 3D structures of proteins from Nuclear Magnetic Resonance (NMR), are explained. Manual methods of protein data analysis often involve using laboratory specific, or even user-specific, rules of interpretation and validation.

The RImA class of enzymes ([RImA.sub.1] and [RImA.sup.II]) catalyzes N1-methylation of a guanine base (G745 in Gram-negative and G748 in Grampositive bacteria) of hairpin 35 of 23S rRNA. We have determined the crystal structure of Escherichia coli [RImA.sub.1] at 2.8-[Angstrom] resolution, providing 3D structure information for the RImA class of RNA methyltransferases. The dimeric protein structure exhibits features that provide new insights into its molecular function. Each [RImA.sub.1] molecule has a Zn-binding domain, responsible for specific recognition and binding of its rRNA substrate, and a methyltransferase domain. The asymmetric [RImA.sub.1] dimer observed in the crystal structure has a well defined W-shaped RNA-binding cleft. Two S-adenosyl-L-methionine substrate molecules are located at the two valleys of the W-shaped RNA-binding cleft. The unique shape of the RNA-binding cleft, different from that of known RNA-binding proteins, is highly specific and structurally complements the 3D structure of hairpin 35 of bacterial 23S rRNA. Apart from the hairpin 35, parts of hairpins 33 and 34 also interact with the [RImA.sub.1] dimer.

Regions of polypeptide chain of bovine pancreatic ribonuclease A have been identified. Results demonstrate the presence of residues in the first alpha-helix and the first beta-sheet of the protein stabilised in the oxidative regeneration pathway of RNase A.

A detailed nuclear magnetic resonance (NMR) study of the structure and dynamics of the major cold-shock protein (CspA) in the absence of nucleic acid ligands to better grasp the mechanisms of nucleic acid recognition in organisms is presented. In particular, nearly complete 1H 13C and 15N resonance assignments for and a refined solution NMR structure of Escherichia coli CspA are presented. These coordinates furnish an improved description of the beta-sheet core, of the backbone structure of the interstrand polypeptide loops and of many side chain conformations in the solution structure.

The atomic resolution structure of the amino terminal of alpha-tropomyosin (alpha-TM) is investigated through circular dichroism and two-dimensional proton nuclear magnetic resonance spectroscopic studies of a chimeric protein. The protein chimera contains the 14 terminal residues of the rabbit alpha-TM and the rest of the peptide is from a transcription factor of a yeast. The results coupled with intrachain constraint studies were used to construct a three-dimensional conformation of the chimeric protein.

Automated analysis of resonance assignments generated through triple-resonance nuclear magnetic resonance spectroscopy of mutant and wild-type proteins of the bovine pancreatic ribonuclease A found a similarity in overall chain folds and structural perturbations near the mutation sites. Comparison of the entropy and enthalpy of unfolding of the two types reveal that part of the destabilization of the mutant protein come from entropic and enthalpic changes during the folded state.

Analyses of locations and structural distortions following the removal of the Cys65-Cys72 disulfide bond were done as a means of determining the structural differences between the three-dimensional folding intermediate and the wt RNase A. Removal of disulfide bonds results in an increase in 1H/2H exchange rates for backbone amides located throughout the 'protein fold.

Nuclear magnetic resonance and circular dichroism spectroscopy techniques were used to test the hypothesis that the Z-domain of staphylococcal protein A (SpA) undergoes helix unwinding when SpA binds to the constant region (Fc) of immunoglobulin G. The results show that the Z-domain in an unbound state has the structure of an antiparallel three-helical bundle, and that this helical structure is not disrupted during binding with Fc.

Examination of the conformational dynamics of human type-alpha transforming growth factor, hTGF alpha, in aqueous solution at neutral pH by nitrogen-15 relaxation measurements reveals the interconversions between multiple protein conformation on the microsecond time scale. Disulfide bond isomerization in the solution structure of hTGF alpha, partial unfolding of the core beta-sheet and hinge-binding motions between the C- and N-terminal subdomains account for these dynamic fluctuations.

Synthesis of a minimAl homeodomain helps analyze the sequence specificity needs of homeodomain structure and DNA binding activity. Examination of a minimAl homeodomain, Ala-Msx, shows that consensus residues of homeodomain have enough sequence information to identify specific DNA. Nonconsensus residues associated with N-terminal arm are essential for high DNA binding and stabilization of homeodomain structure.

Solution nuclear magnetic resonance structure analysis of major cold shock protein (CspA) from Escherichia coli reveals the beta-barrel structure consisting of five antiparallel beta chains with a densely clustered hydrophobic core. The 24-base DNA sequence binding to CspA is homologous to the noncoding regulatory fragment of CspA's mRNA transcript.

An improved strategy for examining sequence-specific resonance assignments was used for an 8.2-kDa engineered area of the cell wall protein A of Staphylococcus aureus with emphasis on proton and nitrogen resonance assignments. The amino acid, asparagine and glutamine spin systems were determined with a combination of HCCNH-TOCSY, 2D 2QF-COSY, TOCSY and N-HSQC data. The aliphatic resonances of residue i to the amide and nitrogen resonances of residue i + 1 and their sequential connections were determined by the HCC(CO)-NH-TOCSY data. The proton and nitrogen resonance assignments for the Z-domain were identified by the HCCNH-TOCSY and HCC(CO)NH-TOCSY spectra.

The structural formation of the human type-alpha transforming growth factor (hTGF-alpha) at pH 6.5 and at 10 degrees centigrade was analyzed. Proton-detected heteronuclear NMR coordinates were employed to define the locations and amplitudes of the pH-induced structural variations. The heteronuclear NMR data and the homonuclear Overhauser effect spectroscopy (NOESY) data presented information on the structural changes that took place when the solution was changed from neutral to acidic pH.

Distance geometry calculations and restrained energy minimization were used to analyze nuclear magnetic resonance (NMR) data in determining the solution structure of murine epidermal growth factor (mEGF) at pH 3.1 and 28 degrees Celsius. The derived structure resembles mEGF molecular structure previously reported. In addition, internal motion in several regions of the mEGF molecule was detected by analysis of proton NMR data.

The article presents a new methodology, which combines the electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) techniques for determining the structure of the homodimer proteins. The new method is shown to be extremely accurate and efficient.

The article discusses a methodology, which employs the unassigned NOESY data for determining the NMR structure of the various accurate automated proteins. The models generated in the study are shown to be extremely accurate and reliable.

A two-dimensional C(omega-1)-C-selective-H(omega-2) spectroscopic technique can measure hydrogen homonuclear coupling constants in carbon-13-enriched molecules. The method aims at overcoming the cross-peak overlapping in couplingconstant measurements of large peptides and small proteins by using a conformation-independent magnetization transfer pathway; by separating the relevant cross-peak components; and by exploiting the variations in nuclear spins among amino acids with one or more beta-protons. It may also be used to measure homonuclear coupling constants of protein-bound ligands with carbon-13 tags.

The solution NMR structure of the C-terminal 126 residue NlpC/P60 domain of Escherichia coli Spr is presented, which corresponds to residues 63-188 in the Spr precursor. The solution NMR structure determination of E. coli is performed following standard protocols of the Northeast Structural Genomics Consortium.

A high-throughput NMR methodology (FAST-NMR) to annotate the biological function of novel proteins through the structural and sequence analysis of protein-ligand interactions is described. The CPASS (Comparison of Protein Active Site Structures) software and database are used to compare the active site with proteins of known function and the methodology is demonstrated by using unannotated protein SAV1430 from Staphylococcus aureus.

The correlation among the NMR spectral quality and the effectiveness in structure identification by x-ray crystallography is evaluated for 159 prokaryotic and eukaryotic proteins that are already checked to evade the proteins that give either polydisperse or aggregated illustrations. The results show that both NMR and the X-ray crystallography methods give similar structural details and both can be used to get optimum accuracy.

The new NMR quality assessment scores, including Recall, Precision, and F-measure scores, which quickly provide global measures of the goodness-of-fit of the three-dimensional (3D) structures with NOESY peak lists from information retrieval statistics are described. It is found that the sensitivity of the F-measure is improved using a scaled Fold Discriminating Power (DP) score.

Research has been conducted on antibacterial peptide microcin J25. The authors have demonstrated that this peptide is a 21-residue lariat protoknot which consists of cyclic segment followed by a linear segment that loops back and threads through the cyclic segment.

Results demonstrate that in solution, residues 270-279 are parallel, linear helices in alpha-tropomyosin. Data indicate that the parallel helices are exposed and exhibit flexibility around residue Y267 and the ends, which facilitate binding to troponin T and complex formation with the N-terminus of tropomyosin and actin.

Author(s): Yuanpeng J. Huang [1]; Gaetano T. Montelione [1] Enzymes are proteins that catalyse chemical processes by lowering the energy barriers between substrate and product, thus increasing the rate of [...]

NMR relaxation (Tsub1, Tsub2, NOE) experiments are useful for studying the internal dynamics of proteins. Direct maping of spectral density functions is another approach, althoug the most common approach is based on the 'model-free' formalism. A complete set of model-free parameters consistent with experimental data is determined graphically, and applied to published data to show the effects of experimental uncertainties on motional parameters.