- Research article
- Open Access
Combining sequence-based prediction methods and circular dichroism and infrared spectroscopic data to improve protein secondary structure determinations
© Lees and Janes; licensee BioMed Central Ltd. 2008
- Received: 13 June 2007
- Accepted: 15 January 2008
- Published: 15 January 2008
A number of sequence-based methods exist for protein secondary structure prediction. Protein secondary structures can also be determined experimentally from circular dichroism, and infrared spectroscopic data using empirical analysis methods. It has been proposed that comparable accuracy can be obtained from sequence-based predictions as from these biophysical measurements. Here we have examined the secondary structure determination accuracies of sequence prediction methods with the empirically determined values from the spectroscopic data on datasets of proteins for which both crystal structures and spectroscopic data are available.
In this study we show that the sequence prediction methods have accuracies nearly comparable to those of spectroscopic methods. However, we also demonstrate that combining the spectroscopic and sequences techniques produces significant overall improvements in secondary structure determinations. In addition, combining the extra information content available from synchrotron radiation circular dichroism data with sequence methods also shows improvements.
Combining sequence prediction with experimentally determined spectroscopic methods for protein secondary structure content significantly enhances the accuracy of the overall results obtained.
- Circular Dichroism
- Secondary Structure Prediction
- Position Specific Score Matrix
- Secondary Structure Content
- Secondary Structure Assignment
With the growing availability of a large number of new genome sequences, there is an ongoing interest in the structures of the proteins represented by the open reading frames in those genomes. Although there has been a growth in the number of crystal structures of proteins as the result of Structural Protemics programmes worldwide, their production has not kept pace with the sequencing effort. Furthermore, such programmes often produce small amounts of any given protein which are not enough for crystal structure analysis. However, these quantities are often sufficient for other biophysical studies.
Modern sequence-based prediction methods can provide information on the secondary structure content of these proteins without the need for producing any protein. Recent years have seen large improvements in the per-residue secondary structure prediction from sequence [1, 2], although they are still limited in their accuracies, especially when applied to certain classes of protein, most notably those with high β-sheet content.
Circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopies are commonly used techniques for secondary structure content determination, and these methods have also shown great advances in their accuracies , with the development of new reference data bases  and empirical methods [5–7]. The spectral data collected by these methods requires a relatively small amount of pure protein and can be obtained rapidly [8–11].
The pioneering work of Chou in predicting secondary structure from sequence was through an elaborate and elegant covariance matrix approach . In this study we have examined the performance of sequence based prediction methods using a neural network methodology, versus the experimentally-determined methods of CD and FTIR for assessing secondary structure content. In order to do this, we have used existing reference data sets available in the literature along with established analytical methods and demonstrated the synergies between the different strategies thereby proposing a new combined approach which will greatly improve the results obtained over any single technique that may find use in Structural Proteomics and other structural biology studies.
Secondary Structure Predictions from Sequences
Reported correlation coefficients for secondary structure content prediction by CD are typically in the range of r = 0.92–0.97 for α-helix (H), r = 0.80–0.90 for β-sheet (E) [4, 6]. The reported values vary because of differences in the reference dataset, secondary structure assignment and prediction algorithms used. The secondary structure content prediction for sequence prediction methods is generally not reported after publication of the crystal structure. The current highest reported secondary structure content prediction by sequence in the literature is r = 0.92 for α-helix and r = 0.81 for β-sheet . More recent methods have their performances available on the EVA website . However, currently r for secondary structure content prediction is not reported on this web-site.
GSEQ dataset 3-fold cross-validation.
Other (G, I, T, B, S, C)
Secondary Structure Content Prediction
RASP46 dataset cross-validation.
Other (G, I, T, B, S, C)
SP175 dataset cross-validation.
Other (G, I, T, B, S, C)
Spectroscopic Identification of Poor Sequence-Based Predictions
Another question that can be addressed is whether secondary structure content measurements can be used to aid with the overall accuracy of a sequence-based prediction method. We see a negative correlation between the Q2 scores and the abs difference between the content prediction from sequence and spectroscopy with r = -0.3 for α-helix and r = -0.5 for β-sheet from the RASP46 dataset and r = -0.4 for α-helix and r = -0.4 for β-sheet from the SP175 dataset.
The results above show that for the proteins in the RASP46 and SP175 reference datasets, a similar secondary structure content accuracy is obtained from the sequence prediction methods as from the experimentally-based methods. Combining the sequence and the cCD and FTIR approaches results in a significantly improved accuracy, especially for β-sheet-rich proteins. For SRCD data, combining this with the sequence prediction method, similarly there is a significant improvement over both these approaches when evaluated in isolation. Notably there is more information content available from the SRCD extended wavelength data which leads to improved accuracy in determining individual secondary structure components . Restricting the defined secondary structures to the three components, as is common practice for FTIR and secondary structure predictions, imposes identical limitation requirements on the SRCD data although many more components than these can be distinguished. The considerable improvement in α-helix over β-sheet and 'Other' reflects that this secondary structure component still provides the dominant spectral characteristics to SRCD spectra. Were FTIR data to be collected for the proteins of the SP175 dataset then this would most likely lead to further significant improvements when combined with the SRCD data and sequence prediction methods. In future, other methods such as Raman Optical Activity  could be incorporated into these combinations which would likely result in a further overall improvement.
There are many Structural Proteomics programmes producing proteins for crystal structure investigations. Of note, a number of these proteins prove difficult to obtain in the amounts necessary for such investigations. However, in these cases spectroscopic methods such as SRCD, notable because of the small amounts of material needed to obtain spectra, [8, 9] in combination with secondary structure sequence prediction methods could well provide significant insight into the protein structure. A further benefit arises from such a combined approach. Sequence-based methods can sometimes generate poor predictions for protein secondary structure content, which can go unrecognised and therefore lead to further inaccuracies as a result. The reliability of the prediction results can be tested when in combination with the experimental-based spectroscopic methods, thus providing an improved measure of reliability.
There is evidence that from SRCD data the higher information content enables fold information to be obtained from the data [ref.  and Wallace, personal communication]. Such fold information combined with a method for tertiary structure prediction such as Threader prediction techniques [15, 19] could offer a novel approach to modeling protein structures, potentially with an improved accuracy. The combination of sequence prediction methods with experimental spectroscopically-determined methods for secondary structure content offer a valuable addition for gaining information about protein structure, and maybe potentially an insight into function as a result.
Spectroscopic Datasets (RASP46 and SP175)
The RASP50 dataset consists of 50 composite CD and FTIR spectra . The spectra were obtained with kind permission of Dr. K. Oberg (MannKind Corporation). The CD spectra are in the range of 185–240 nm. The FTIR spectra were in the range of 1720-1500 cm-1 at 1 cm-1 intervals. Combining the two different methods was previously shown to give improvements in performance of secondary structure determination . The spectra were water baseline-subtracted but not side chain-subtracted, as described in the original publication . The area of each of the FTIR spectra was scaled to a total intensity of 663, so that the sum of the integrated areas under the CD and FTIR curves were roughly equal in all cases, ensuring that neither the CD nor FTIR information dominated in the secondary structure predictions. The RASP50 dataset was modified to produce the RASP46 data set, by removing four spectra as follows: The spectrum of rennin appeared to have a disordered spectrum and so was removed. The spectrum of ricin was removed because of uncertainties establishing a match between the PDB file and the protein sequence provided from the bioscience supplier . The spectrum of α-hemolysin was excluded since this membrane protein would be unsuitable for the sequence prediction methods implemented in this study which were designed for soluble proteins. The spectrum of insulin was removed because the 20 amino acid chain B sequence of the PDB file 1trz would not produce an output from the PSI-BLAST  program.
The SP175 dataset  was a synchrotron radiation circular dichroism (SRCD) dataset consisting of 72 spectra in the range of 175–240 nm. This dataset was used because it has been suggested that the higher information content in SRCD spectra relative to conventional CD (cCD) spectra, will produce more accurate secondary structure analyses [5, 17]. From this dataset the spectrum of jacalin was removed because the 18 residue B chain of PDB file 1ku8 would not produce a successful PSI-BLAST output. The CD spectra of pectate-lyase C and ferredoxin were not available when the current study was carried out, so these were also not included in the SP175 dataset used in this study. The CD spectra from both the RASP46 and SP175 datasets were expressed in Δε units.
Neural Network Derived Sequence Dataset (GSEQ)
The PISCES  server uses a combination of PSI-BLAST and structure-based alignments to determine sequence identities and was used to provide a non-redundant dataset for training and testing the sequence-to-secondary structure neural network (NN) predictor as follows: The 25% sequence identity and ≤ 2.5 Å resolution cutoff dataset was downloaded from the PISCES server . Any PDB files containing membrane proteins were removed from the dataset. The sequence database used for PSI-BLAST alignments was the UNIPROT_100 sequence dataset . This was filtered to remove low-complexity regions, transmembrane regions and coiled coil segments using the pfilt  algorithm. Position specific scoring matrix (PSSM) profiles were generated for the PISCES dataset sequences by running 3-iterations of PSI-BLAST with the -h option, the threshold for sequence inclusion in the next iteration, set to 0.001. It was noted that the PSI-BLAST algorithm should be run with the -v option, the upper limit value for the number of sequence matches in any run, above the default value of 500 since many proteins gave more than this number of matches. Possible homologues between the PISCES-derived dataset and the spectroscopy datasets were identified by running 5 iterations of PSI-BLAST on the RASP46 and SP175 proteins sequences using the UNIREF100 sequence dataset . After this, any proteins were removed from the PISCES-derived dataset if they had E-value scores ≤ 5.0 with any proteins in the RASP46 or SP175 datasets. Finally, any proteins of sequence length < 30 amino acids were removed from the dataset. The final dataset, which we designate GSEQ, contained 2984 protein chains. The PSSM profiles were scaled to be between 0 and 1 by the standard logistic function. An extra input for each residue was used to indicate if the central residue was passed the N- or C-terminus.
The secondary structure assignment scheme applied was that of α-helix (H), β-sheet and other (G, I, B, S, T, C), using the designation provided in the DSSP output file . This secondary structure assignment has previously been shown to be appropriate for both sequence based  and CD or FTIR spectroscopic prediction methods [5, 16].
Neural Network Architecture and Training
The PSI-PRED algorithm creates a simple NN architecture that has been shown to be amongst the best methods for secondary structure prediction from sequence . The NNs constructed in this work had identical architectures to that used in the original PSI-PRED paper . They consisted of 15 × 21 input, and 75 hidden neurons (units in the original work ) in the sequence-to-structure neural network and, separately, 4 × 15 inputs and 60 hidden neurons (units) in the structure-to-structure network. The training parameters for online back-propagation were also maintained at the same values (momentum = 0.9, learning rate = 0.005) as in the original PSI-PRED publication. The training parameters and network architecture were not optimised since the main purpose of this paper was to reveal relative differences and potential synergies between sequence-based and spectroscopic-based methods under similar conditions.
Training and testing were carried out using a 3-fold cross-validation of the network. Initially, during training 10% of the training data was kept aside as the validation set and not included in the training. Training was stopped when the performance of the validation set began to degrade relative to the training set. The GSEQ dataset was then used to assess the performance of the network by 3-fold cross validation as given in Table 1. The test sets in Tables 2 and 3, RASP46 and SP175, respectively, contained none of the training set of proteins.
Assessment of the Prediction Accuracy
Secondary structure content prediction methods are typically measured using the widely reported Pearsons correlation coefficient (r). Values of r range between +1 and -1 representing perfect positive and negative correlation respectively. Additionally either the root mean squared deviation (δ), or the absolute deviation (abs) are reported. When judging the performance of a method, high values of r and low values of δ indicate good performance. Corr is the Mathews correlation coefficient .
Assessment of the prediction accuracy on the RASP46 and SP175 datasets was carried out using the SIMPLS algorithm  combined with zeroing negative fractions and rescaling to 100%. The PDB files and corresponding secondary structure contents for the datasets were those described in the original publications which produced the datasets. The performance parameters for these datasets were assessed using full cross-validation. Sequence-to-structure predictions on the sequences were carried out by taking the average of the prediction of the three networks produced from the 3-fold cross-validation on the corresponding proteins in each of the experimental datasets.
We thank B.A. Wallace and members of the Wallace Lab of the School of Crystallography, Birkbeck College, University of London for useful discussions.
- Rost B: Protein secondary structure prediction continues to rise. J Struct Biol 2001, 134: 204–218. 10.1006/jsbi.2001.4336View ArticlePubMedGoogle Scholar
- Sen TZ, Cheng HT, Kloczkowski A, Jernigan RL: A consensus data mining secondary structure prediction by combining GOR V and fragment database mining. Protein Sci 2006, 15: 2499–2506. 10.1110/ps.062125306PubMed CentralView ArticlePubMedGoogle Scholar
- Janes RW: Bioinformatics analyses of circular dichroism protein reference databases. Bioinformatics 2005, 21: 4230–4238. 10.1093/bioinformatics/bti690View ArticlePubMedGoogle Scholar
- Lees JG, Miles AJ, Wien F, Wallace BA: A reference database for circular dichroism spectroscopy covering fold and secondary structure space. Bioinformatics 2006, 22: 1955–1962. 10.1093/bioinformatics/btl327View ArticlePubMedGoogle Scholar
- Lees JG, Miles AJ, Janes RW, Wallace BA: Novel methods for secondary structure determination using low wavelength (VUV) circular dichroism spectroscopic data. BMC Bioinformatics 2006, 7: 507. 10.1186/1471-2105-7-507PubMed CentralView ArticlePubMedGoogle Scholar
- Sreerama N, Woody RW: Computation and Analysis of Protein Circular Dichroism Spectra. Meth Enzymol 2004, 383: 318–351.View ArticlePubMedGoogle Scholar
- Whitmore L, Wallace BA: DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Res 2004, 32: W668-W673. 10.1093/nar/gkh371PubMed CentralView ArticlePubMedGoogle Scholar
- Miles AJ, Wein F, Lees JG, Rodger A, Janes RW, Wallace BA: Calibration and standardisation of synchrotron radiation circular dichroism and conventional circular dichroism spectrophotometers. Spectroscopy 2003, 17: 653–661.View ArticleGoogle Scholar
- Miles AJ, Wien F, Lees JG, Wallace BA: Calibration and standardisation of synchrotron radiation and conventional circular dichroism spectrometers. Part 2: Factors affecting magnitude and wavelength. Spectroscopy 2005, 19: 43–51.View ArticleGoogle Scholar
- Kelly SM, Jess TJ, Price NC: How to study proteins by circular dichroism. Biochim Biophys Acta 2005, 1751: 119–139.View ArticlePubMedGoogle Scholar
- Miles AJ, Wallace BA: Synchrotron radiation circular dichroism spectroscopy of proteins and applications in structural and functional genomics. Chem Soc Rev 2006, 35: 39–51. 10.1039/b316168bView ArticlePubMedGoogle Scholar
- Chou KC: A novel-approach to predicting protein structural classes in a (20–1)-d amino-acid-composition space. Proteins -Struc Func and Genetics 1995, 21: 319–344. 10.1002/prot.340210406View ArticleGoogle Scholar
- Chandonia JM, Karplus M: New methods for accurate prediction of protein secondary structure. Proteins 1999, 35: 293–306. 10.1002/(SICI)1097-0134(19990515)35:3<293::AID-PROT3>3.0.CO;2-LView ArticlePubMedGoogle Scholar
- Koh IYY, Eyrich VA, Marti-Renom MA, Przybylski D, Madhusudhan MS, Eswar N, Grana O, Pazos F, Valencia A, Sali A, Rost B: EVA: Evaluation of protein structure prediction servers. Nucleic Acids Res 2003, 31: 3311–3315. 10.1093/nar/gkg619PubMed CentralView ArticlePubMedGoogle Scholar
- Jones DT: Protein secondary structure prediction based on position-specific scoring matrices. J Mol Biol 1999, 292: 195–202. 10.1006/jmbi.1999.3091View ArticlePubMedGoogle Scholar
- Oberg KA, Ruysschaert JM, Goormaghtigh E: The optimization of protein secondary structure determination with infrared and circular dichroism spectra. Eur J Biochem 2004, 271: 2937–2948. 10.1111/j.1432-1033.2004.04220.xView ArticlePubMedGoogle Scholar
- Wallace BA, Janes RW: Synchrotron radiation circular dichroism spectroscopy of proteins: secondary structure, fold recognition and structural genomics. Curr Opin Chem Biol 2001, 5: 567–571. 10.1016/S1367-5931(00)00243-XView ArticlePubMedGoogle Scholar
- Zhu FJ, Isaacs NW, Hecht L, Tranter GE, Barron LD: Raman optical activity of proteins, carbohydrates and glycoproteins. Chirality 2006, 18: 103–115. 10.1002/chir.20225View ArticlePubMedGoogle Scholar
- Bryson K, McGuffin LJ, Marsden RL, Ward JJ, Sodhi JS, Jones DT: Protein structure prediction servers at University College London. Nucleic Acids Res 2005, 33: W36-W38. 10.1093/nar/gki410PubMed CentralView ArticlePubMedGoogle Scholar
- Oberg KA, Ruysschaert JM, Goormaghtigh E: Rationally selected basis proteins: a new approach to selecting proteins for spectroscopic secondary structure analysis. Protein Sci 2003, 12: 2015–2031. 10.1110/ps.0354703PubMed CentralView ArticlePubMedGoogle Scholar
- Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997, 25: 3389–3402. 10.1093/nar/25.17.3389PubMed CentralView ArticlePubMedGoogle Scholar
- Wang G, Dunbrack RL Jr: PISCES: a protein sequence culling server. Bioinformatics 2003, 19: 1589–1591. [http://dunbrack.fccc.edu/PISCES.php] 10.1093/bioinformatics/btg224View ArticlePubMedGoogle Scholar
- Bairoch A, Apweiler R, Wu CH, Barker WC, Boeckmann B, Ferro S, Gasteiger E, Huang HZ, Lopez R, Magrane M, Martin MJ, Natale DA, O'Donovan C, Redaschi N, Yeh LSL: The Universal Protein Resource (UniProt). Nucleic Acids Res 2005, 33: 154–159. 10.1093/nar/gki070View ArticleGoogle Scholar
- Jones DT, Swindells MB: Getting the most from PSI-BLAST. Trends Biochem Sci 2002, 27: 161–164. 10.1016/S0968-0004(01)02039-4View ArticlePubMedGoogle Scholar
- Kabsch W, Sander C: Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 1983, 22: 2577–2637. 10.1002/bip.360221211View ArticlePubMedGoogle Scholar
- Mathews BW: Comparison of the predicted and observed secondary structure of T4 phage lysozyme. Biochim Biophys Acta 1975, 405: 442–451.View ArticleGoogle Scholar
- DeJong S: SIMPLS – An alternative approach to partial least-squares regression. Chemometrics and Intelligent Lab Systems 1993, 18: 251–263. 10.1016/0169-7439(93)85002-XView ArticleGoogle Scholar
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