Automated backbone assignment of labeled proteins using the threshold accepting algorithm

The sequential assignment of backbone resonances is the first step in the structure determination of proteins by heteronuclear NMR. For larger proteins, an assignment strategy based on proton side-chain information is no longer suitable for the use in an automated procedure. Our program PASTA (Protein ASsignment by Threshold Accepting) is therefore designed to partially or fully automate the sequential assignment of proteins, based on the analysis of NMR backbone resonances plus C information. In order to overcome the problems caused by peak overlap and missing signals in an automated assignment process, PASTA uses threshold accepting, a combinatorial optimization strategy, which is superior to simulated annealing due to generally faster convergence and better solutions. The reliability of this algorithm is shown by reproducing the complete sequential backbone assignment of several proteins from published NMR data. The robustness of the algorithm against misassigned signals, noise, spectral overlap and missing peaks is shown by repeating the assignment with reduced sequential information and increased chemical shift tolerances. The performance of the program on real data is finally demonstrated with automatically picked peak lists of human nonpancreatic synovial phospholipase A₂, a protein with 124 residues.     

Resonance assignment strategies for the analysis of nmr spectra of proteins

Determination of the high resolution solution structure of a protein using nuclear magnetic resonance (NMR) spectroscopy requires that resonances observed in the NMR spectra be unequivocally assigned to individual nuclei of the protein. With the advent of modern, two-dimensional NMR techniques arose methodologies for assigning the¹H resonances based on 2D, homonuclear¹H NMR experiments. These include the sequential assignment strategy and the main chain directed strategy. These basic strategies have been extended to include newer 3D homonuclear experiments and 2D and 3D heteronuclear resolved and edited methods. Most recently a novel, conceptually new approach to the problem has been introduced that relies on heteronuclear, multidimensional so-called triple resonance experiments for both backbone and sidechain resonance assignments in proteins. This article reviews the evolution of strategies for the assignment of resonances of proteins.     

Ansig for Windows: An interactive computer program for semiautomatic assignment of protein NMR spectra

Assignment of NMR spectra is a prerequisite for structure determination of proteins using NMR. The time spent on the assignment is comparatively long compared to that spent on other parts in the protein structure determination process, but it can be shortened by using either interactive or fully automated computer programs. To benefit from the advantages of both types of program we have developed a version of the interactive assignment program ANSIG to include automatized, yet user-supervised, routines. The new program includes tools for (i) semiautomatic sequential assignment, (ii) plotting of distances from PDB structure files directly in NMR spectra and (iii) statistical analysis of distance restraint violations with the possibility to directly zoom to violated NOEs in NOESY spectra.     

PACES: Protein sequential assignment by computer-assisted exhaustive search

A crucial step in determining solution structures of proteins using nuclear magnetic resonance (NMR) spectroscopy is the process of sequential assignment, which correlates backbone resonances to corresponding residues in the primary sequence of a protein, today, typically using data from triple-resonance NMR experiments. Although the development of automated approaches for sequential assignment has greatly facilitated this process, the performance of these programs is usually less satisfactory for large proteins, especially in the cases of missing connectivity or severe chemical shift degeneracy. Here, we report the development of a novel computer-assisted method for sequential assignment, using an algorithm that conducts an exhaustive search of all spin systems both for establishing sequential connectivities and then for assignment. By running the program iteratively with user intervention after each cycle, ambiguities in the assignments can be eliminated efficiently and backbone resonances can be assigned rapidly. The efficiency and robustness of this approach have been tested with 27 proteins of sizes varying from 76 amino acids to 723 amino acids, and with data of varying qualities, using experimental data for three proteins, and published assignments modified with simulated noise for the other 24. The complexity of sequential assignment with regard to the size of the protein, the completeness of NMR data sets, and the uncertainty in resonance positions has been examined.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1023/A:1023589029301     

Sequence-specific NMR assignment of proteins by global fragment mapping with the program Mapper

A new program, Mapper, for semiautomatic sequence-specific NMR assignment in proteins is introduced. The program uses an input of short fragments of sequentially neighboring residues, which have been assembled based on sequential NMR connectivities and for which either the ¹³C and ¹³C chemical shifts or data on the amino acid type from other sources are known. Mapper then performs an exhaustive search for self-consistent simultaneous mappings of all these fragments onto the protein sequence. Compared to using only the individual mappings of the spectroscopically connected fragments, the global mapping adds a powerful new constraint, which results in resolving many otherwise intractable ambiguities. In an initial application, virtually complete sequence-specific assignments were obtained for a 110 kDa homooctameric protein, 7,8-dihydroneopterin aldolase from Staphylococcus aureus.     

A new approach for obtaining sequential assignment of large proteins

A novel NMR experiment for obtaining sequential assignment of large proteins and protein complexes is described. The proposed method takes full advantage of transverse relaxation optimized spectroscopy (TROSY) and utilizes spin-state-selection to distinguish between intraresidual and sequential connectivities in the HNCA-TROSY-type correlation experiment. Thus, the intra- and interresidual cross peaks can be identified without relaying magnetization via carbonyl carbon, which relaxes very rapidly at the high magnetic fields where TROSY is most efficient. In addition, the presented method enables measurement of several scalar and residual dipolar couplings, which can potentially be used for structure determination of large proteins.     

A modified strategy for sequence specific assignment of protein NMR spectra based on amino acid type selective experiments

The determination of the three-dimensional structure of a protein or the study of protein–ligand interactions requires the assignment of all relevant nuclei as an initial step. This is nowadays almost exclusively performed using triple-resonance experiments. The conventional strategy utilizes one or more pairs of three dimensional spectra to obtain redundant information and thus reliable assignments. Here, a modified strategy for obtaining sequence specific assignments based on two dimensional amino acid type selective triple-resonance experiments is proposed. These experiments can be recorded with good resolution in a relatively short time. They provide very specific and redundant information, in particular on sequential connectivities, that drastically increases the ease and reliability of the assignment procedure, done either manually or in an automated fashion. The new strategy is demonstrated with the protein domain PB1 from yeast CDC24p. Dedicated to Rüdiger Winter ( 06.04.2004)     

Use of biosynthetic fractional 13C-labeling for backbone NMR assignment of proteins

We describe a simple approach to classify amino acid residue types in NMR spectra of proteins for supporting the backbone resonance assignments. It makes use of the differences in biosynthetic pathways of the 20 amino acids in Escherichia coli. Therefore, it is distinct from the parameters routinely exploited in the backbone resonance assignment such as chemical shifts and spin topology information. The combination of biosynthetically directed fractional 13C-labeling and uniform 15N-labeling enables us to obtain both residue-type specific information and sequential connectivities from a single protein sample. The residue-type classification exploiting biosynthetic pathways can be used for accelerating the conventional backbone assignment procedure.     

Data requirements for reliable chemical shift assignments in deuterated proteins

The information required for chemical shift assignments in large deuterated proteins was investigated using a Monte Carlo approach (Hitchens et al., 2002). In particular, the consequences of missing amide resonances on the reliability of assignments derived from C and CO or from C and C chemical shifts was investigated. Missing amide resonances reduce both the number of correct assignments as well as the confidence in these assignments. More significantly, a number of undetectable errors can arise when as few as 9% of the amide resonances are missing from the spectra. However, the use of information from residue specific labeling as well as local and long-range distance constraints improves the reliability and extent of assignment. It is also shown that missing residues have only a minor effect on the assignment of protein-ligand complexes using C and CO chemical shifts and C inter-residue connectivity, provided that the known chemical shifts of the unliganded protein are utilized in the assignment process.     

Assignment of protein NMR spectra based on projections,multi-way decomposition and a fast correlation approach

We present an approach for the assignment of protein NMR resonances that combines established and new concepts: (a) Based on published reduced dimensionality methods, two 5-dimensional experiments are proposed. (b) Multi-way decomposition (PRODECOMP) applied simultaneously to all acquired NMR spectra provides the assignment of resonance frequencies under conditions of very low signal-to-noise. (c) Each resulting component characterizes all spin (1/2) nuclei in a (doubly-labeled) CbetaH(n)-CalphaH-C'-NH-CalphaH-CbetaH(n) fragment in an unambiguous manner, such that sequentially neighboring components have about four atoms in common. (d) A new routine (SHABBA) determines correlations for all component pairs based on the common nuclei; high correlation values yield sequential chains of a dozen or more components. (e) The potentially error-prone peak picking is delayed to the last step, where it helps to place the component chains within the protein sequence, and thus to achieve the final backbone assignment. The approach was validated by achieving complete backbone resonance assignments for ubiquitin.     

Tools for the automated assignment of high-resolution three-dimensional protein NMR spectra based on pattern recognition techniques

One of the major bottlenecks in the determination of proteinstructures by NMR is in the evaluation of the data produced by theexperiments. An important step in this process is assignment, where thepeaks in the spectra are assigned to specific spins within specificresidues. In this paper, we discuss a spin system assignment tool based onpattern recognition techniques. This tool employs user-specified templatesto search for patterns of peaks in the original spectra; these patterns maycorrespond to side-chain or backbone fragments. Multiple spectra willnormally be searched simultaneously to reduce the impact of noise. Thesearch generates a preliminary list of putative assignments, which arefiltered by a set of heuristic algorithms to produce the final results list.Each result contains a set of chemical shift values plus information aboutthe peaks found. The results may be used as input for combinatorialroutines, such as sequential assignment procedures, in place of peak lists.Two examples are presented, in which (i) HCCH-COSY and -TOCSY spectra arescanned for side-chain spin systems; and (ii) backbone spin systems aredetected in a set of spectra comprising HNCA, HN(CO)CA, HNCO, HN(CA)CO,CBCANH and CBCA(CO)NH.     

Resonance assignment of 13C/15N labeled solid proteins by two- and three-dimensional magic-angle-spinning NMR

The comprehensive structure determination of isotopically labeled proteins by solid-state NMR requires sequence-specific assignment of ¹³C and ¹⁵ N spectra. We describe several 2D and 3D MAS correlation techniques for resonance assignment and apply them, at 7.0 Tesla, to ¹³C and ¹⁵N labeled ubiquitin to examine the extent of resonance assignments in the solid state. Both interresidue and intraresidue assignments of the ¹³C and ¹⁵N resonances are addressed. The interresidue assignment was carried out by an N(CO)CA technique, which yields N_i-C_i–1 connectivities in protein backbones via two steps of dipolar-mediated coherence transfer. The intraresidue connectivities were obtained from a new 3D NCACB technique, which utilizes the well resolved C chemical shift to distinguish the different amino acids. Additional amino acid type assignment was provided by a ¹³C spin diffusion experiment, which exhibits ¹³C spin pairs as off-diagonal intensities in the 2D spectrum. To better resolve carbons with similar chemical shifts, we also performed a dipolar-mediated INADEQUATE experiment. By cross-referencing these spectra and exploiting the selective and extensive ¹³ C labeling approach, we assigned 25% of the amino acids in ubiquitin sequence-specifically and 47% of the residues to the amino acid types. The sensitivity and resolution of these experiments are evaluated, especially in the context of the selective and extensive ¹³C labeling approach.     

Evaluation of an algorithm for the automated sequential assignment of protein backbone resonances: A demonstration of the connectivity tracing assignment tools (CONTRAST) software package

Summary The peptide sequential assignment algorithm presented here was implemented as a macro within the CONnectivity TRacing ASsignment Tools (CONTRAST) computer software package. The algorithm provides a semi- or fully automated global means of sequentially assigning the NMR backbone resonances of proteins. The program's performance is demonstrated here by its analysis of realistic computer-generated data for III^Glc, a 168-residue signal-transducing protein of Escherichia coli [Pelton et al. (1991) Biochemistry, 30, 10043–10057]. Missing experimental data (19 resonances) were generated so that a complete assignment set could be tested. The algorithm produces sequential assignments from appropriate peak lists of nD NMR data. It quantifies the ambiguity of each assignment and provides ranked alternatives. A best first approach, in which high-scoring local assignments are made before and in preference to lower scoring assignments, is shown to be superior (in terms of the current set of CONTRAST scoring routines) to approaches such as simulated annealing that seek to maximize the combined scores of the individual assignments. The robustness of the algorithm was tested by evaluating the effects of imposed frequency imprecision (scatter), added false signals (noise), missing peaks (incomplete data), and variation in userdefined tolerances on the performance of the algorithm.     

Assigning large proteins in the solid state: a MAS NMR resonance assignment strategy using selectively and extensively <Superscript>13</Superscript>C-labelled proteins

In recent years, solid-state magic-angle spinning nuclear magnetic resonance spectroscopy (MAS NMR) has been growing into an important technique to study the structure of membrane proteins, amyloid fibrils and other protein preparations which do not form crystals or are insoluble. Currently, a key bottleneck is the assignment process due to the absence of the resolving power of proton chemical shifts. Particularly for large proteins (approximately >150 residues) it is difficult to obtain a full set of resonance assignments. In order to address this problem, we present an assignment method based upon samples prepared using [1,3-¹³C]- and [2-¹³C]-glycerol as the sole carbon source in the bacterial growth medium (so-called selectively and extensively labelled protein). Such samples give rise to higher quality spectra than uniformly [¹³C]-labelled protein samples, and have previously been used to obtain long-range restraints for use in structure calculations. Our method exploits the characteristic cross-peak patterns observed for the different amino acid types in ¹³C-¹³C correlation and 3D NCACX and NCOCX spectra. An in-depth analysis of the patterns and how they can be used to aid assignment is presented, using spectra of the chicken α-spectrin SH3 domain (62 residues), αB-crystallin (175 residues) and outer membrane protein G (OmpG, 281 residues) as examples. Using this procedure, over 90% of the Cα, Cβ, C′ and N resonances in the core domain of αB-crystallin and around 73% in the flanking domains could be assigned (excluding 24 residues at the extreme termini of the protein). Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A novel approach for sequential assignment of 1H, 13C, and 15N spectra of proteins: heteronuclear triple-resonance three-dimensional NMR spectroscopy. Application to calmodulin

A novel approach is described for obtaining sequential assignment of the backbone 1H, 13C, and 15N resonances of larger proteins. The approach is demonstrated for the protein calmodulin (16.7 kDa), uniformly (approximately 95%) labeled with 15N and 13C. Sequential assignment of the backbone residues by standard methods was not possible because of the very narrow chemical shift distribution range of both NH and C alpha H protons in this largely alpha-helical protein. We demonstrate that the combined use of four new types of heteronuclear 3D NMR spectra together with the previously described HOHAHA-HMQC 3D experiment [Marion, D., et al. (1989) Biochemistry 28, 6150-6156] can provide unambiguous sequential assignment of protein backbone resonances. Sequential connectivity is derived from one-bond J couplings and the procedure is therefore independent of the backbone conformation. All the new 3D NMR experiments use 1H detection and rely on multiple-step magnetization transfers via well-resolved one-bond J couplings, offering high sensitivity and requiring a total of only 9 days for the recording of all five 3D spectra. Because the combination of 3D spectra offers at least two and often three independent pathways for determining sequential connectivity, the new assignment procedure is easily automated. Complete assignments are reported for the proton, carbon, and nitrogen backbone resonances of calmodulin, complexed with calcium.     

IBIS--a tool for automated sequential assignment of protein spectra from triple resonance experiments

We have developed a tool for computer-assisted assignments of protein NMR spectra from triple resonance data. The program is designed to resemble established manual assignment procedures as closely as possible. IBIS exports its results in XEASY format. Thus, using IBIS the operator has continuous visual and accounting control over the progress of the assignment procedure. IBIS achieves complete assignments for those residues that exhibit sequential triple resonance connectivities within a few hours or days.     

Computer-assisted assignment of multidimensional NMR spectra of proteins: Application to 3D NOESY-HMQC and TOCSY-HMQC spectra

Summary An algorithm based on the technique of combinatorial minimization is used for the semi-automated assignment of multidimensional heteronuclear spectra. The program (ALFA) produces the best assignment compatible with the available input data. Even partially misleading or missing data do not seriously corrupt the final assignment. Ambiguous sequences of the possible assignment and all alternatives are indicated. The program can also use additional non-spectroscopic data to assist in the assignment procedure. For example, information from the X-ray structure of the protein and/or information about the secondary structure can be used. The assignment procedure was tested on spectra of mucous trypsin inhibitor, a protein of 107 residues.     

Test of circular dichroism (CD) methods for crambin and CD-assisted secondary structure prediction of its homologous toxins

Methods that analyze protein circular dichroism (CD) spectra for fractions of secondary structure are evaluated for the plant protein crambin, which has a known high-resolution crystal structure. In addition, a two-step secondary structure prediction scheme is presented and used for the toxins homologous to crambin, shown by others to have secondary structures similar to crambin. The test of CD spectral analysis methods with the protein crambin employed two computer programs and several CD basis sets. Crambin's crystal structure, known to 0.945A resolution (Hendrickson, W.A., Teeter, M.M. Nature 290:107-113, 1981), allows accurate evaluation of results. Analysis with the protein spectra basis sets (Provencher, S.W., Gl?ckner, J. Biochemistry 20:33-37, 1981) as modified (Manavalan, P., Johnson, W.C., Jr. Anal. Biochem. 167:76-85, 1987) agreed most closely with crambin's crystal structure. This method was then applied to the CD spectra of the membrane-active toxins homologous to crambin (alpha 1- and beta-purothionin, phoratoxin A and B, and viscotoxin A3 and B). The new program SEQ (pronounced "seek") was developed to assign the secondary structure along the protein chain in a hierarchical fashion and applied to the plant toxins. The method constrained the secondary structure fractions to those from CD analysis and combined standard statistical methods with amphipathic helix location. Both CD-arrived secondary structure percentages and sequence assignment indicate that the viscotoxins are structurally most similar to crambin. Purothionin's secondary structure was predicted to be fundamentally similar to crambin's with a difference at the start of the first helix. This assignment agreed with Raman and NMR analyses of purothionin and lends validity to the method presented here. Differences from the NMR in the CD secondary structure fraction analysis for phoratoxin suggest interference in the CD from tryptophan residues.     

Backbone assignment of proteins with known structure using residual dipolar couplings

A prerequisite for NMR studies of protein-ligand interactions or protein dynamics is the assignment of backbone resonances. Here we demonstrate that protein assignment can significantly be enhanced when experimental dipolar couplings (RDCs) are matched to values back-calculated from a known three-dimensional structure. In case of small proteins, the program MARS allows assignment of more than 90% of backbone resonances without the need for sequential connectivity information. For bigger proteins, we show that the combination of sequential connectivity information with RDC-matching enables more residues to be assigned reliably and backbone assignment to be more robust against missing data. Structural or dynamic deviations from the employed 3D coordinates do not lead to an increased error rate in RDC-supported assignment. RDC-enhanced assignment is particularly useful when chemical shifts and sequential connectivity only provide a few reliable assignments.