Raman spectra of polyglycines

The laser-excited Raman spectrum of helical polyglycine II has been obtained. Oligomers of polyglycine are in the planar zigzag conformation and their Raman spectra are indicative of the spectrum of polyglycine I. The Raman spectra of polyglycines have bands complementary to the infrared which are sensitive to the conformation of the chain. The spectra of the oligomers have bands sensitive to the length of the polyglycine. The Raman spectra of di- and triglycine in aqueous solution suggest the conformation is neither planar nor helical.     

Raman spectroscopic study of mechanically deformed poly-L-alanine

Raman spectroscopy has been used in investigating the conformational transitions of poly-L -alanine (PLA) induced by mechanical deformation. We see evidence of the alpha-helical, antiparallel beta-sheet, and a disordered conformation in PLA. The disordered conformation has not been discussed in previous infrared and X-ray diffraction investigations and may have local order similar to the left-handed 3₁ poly glycine helix. The amide III mode in the Raman spectrum of PLA is more sensitive than the amide I and II modes to changes in secondary structure of the polypeptide chain. Several lines below 1200 cm^?1 are conformationally sensitive and may generally be useful in the analysis of Raman spectra of proteins. A line at 909 cm^?1 decreases in intensity after deformation of PLA. In general only weak scattering is observed around 900 cm^?1 in the Raman spectra of antiparallel beta-sheet polypeptides. The Raman spectra of the amide N–H deuterated PLA and poly-L -leucine (PLL) in the alpha-helical conformation and poly-L -valine (PLV) in the beta-sheet conformation are presented. Splitting is observed in the amide III mode of PLV and the components of this mode are assigned. The Raman spectrum of an alpha-helical random copolymer of L -leucine and L -glutamic acid is shown to be consistent with the spectra of other alphahelical polypeptides.     

The twilight zone between protein order and disorder

The amino acid composition of intrinsically disordered proteins and protein segments characteristically differs from that of ordered proteins. This observation forms the basis of several disorder prediction methods. These, however, usually perform worse for smaller proteins (or segments) than for larger ones. We show that the regions of amino acid composition space corresponding to ordered and disordered proteins overlap with each other, and the extent of the overlap (the “twilight zone”) is larger for short than for long chains. To explain this finding, we used two-dimensional lattice model proteins containing hydrophobic, polar, and charged monomers and revealed the relation among chain length, amino acid composition, and disorder. Because the number of chain configurations exponentially grows with chain length, a larger fraction of longer chains can reach a low-energy, ordered state than do shorter chains. The amount of information carried by the amino acid composition about whether a protein or segment is (dis)ordered grows with increasing chain length. Smaller proteins rely more on specific interactions for stability, which limits the possible accuracy of disorder prediction methods. For proteins in the “twilight zone”, size can determine order, as illustrated by the example of two-state homodimers.     

Quantitative IR spectrophotometry of peptide compounds in water (H2O) solutions. III. Estimation of the protein secondary structure

Infrared spectra of 13 globular proteins have been obtained in the 1800-1480-cm-1 region for H2O solutions. A method for estimating protein secondary structure from the ir spectrum has been developed. The method can also be used for estimating polypeptide and fibrous protein conformation. For the globular and fibrous proteins and polypeptides analyzed, the correlation coefficients between the ir and x-ray estimates of ordered helix, disordered helix, ordered beta-structure, disordered beta-structure, turns, and remainder were 0.98, 0.80, 0.99, 0.87, 0.90, and 0.92 respectively.     

Circular dichroism of the “random” polypeptide chain

The circular dichroism (CD) spectrum of an unordered polypeptide chain does not correspond, as has been assumed heretofore, to that of a charged chain such as poly-L -glutamic acid or poly-L -lysine. The latter have been shown to have locally ordered structures with characteristic CD spectra. We have now obtained CD spectra of the unordered forms of the above synthetic, polypeptides, as well as of two fibrous proteins (collagen and feather keratin) and a globular protein (myoglobin). These spectra are all similar to that of unordered polyproline, having a negative band in the vicinity of 2000 mμ and no additional bands at longer wavelengths. The lack of structural uniqueness of the unordered polypeptide chain is emphasized by these studies.     

Molecular dynamics simulations of conformation and chain length dependent terahertz spectra of alanine polypeptides

Terahertz absorption spectra of alanine polypeptides in water were simulated with classical molecular dynamics at 310 K. Vibrational modes and oscillator strengths were calculated based on a quasi-harmonic approximation. Absorption spectra of Ala_n (n = 5, 15, 30) with different chain lengths and Ala₁₅ in coiled and helical conformations were studied in 10–40 cm^? 1 bandwidth. Simulation results indicated both the chain length and the conformation have significant influences on THz spectra of alanine polypeptides. With the increase of chain length, the average THz absorption intensity increases. Compared with the helical Ala₁₅ polypeptide, the THz spectra of coiled one shows stronger absorption peaks. These results were explained from different numbers of hydrogen bonds formed between polypeptides and the surrounding water molecules.     

Low-frequency infrared bands and chain conformations of polypeptides

Infrared spectra of polypeptides were measured in the region of 1800–400 cm^?1. For the α-helical form, disordered form, and antiparallel-chain β-form, amide V band- arising from N-H out-of-plane bending models were observed at 610–620, around 650, and 700–705 cm^?1, respectively, and amide V′ bands arising from N-D out-of-plane bending modes were observed at 455–465, around 510, and a 515–530 cm^?1, respectively. These correlations are useful for conformation diagnoses, particularly for copolyamino-acids or proteins which are not oriented. The nature of low-frequency amide bands are discussed with reference to potential energy distributions calculated for the α-helical form and β form.     

Raman spectroscopic study of tropomyosin denaturation

Raman spectra of the pH denaturation of tropomyosin are presented. In the native state tropomyosin has an alpha-helical content of nearly 90%, but this value drops rapidly as the pH is raised above 9.5. The Raman spectrum of the native state is characterized by a strong amide I line appearing at 1655 cm^?1, very weak scattering in the amide III region around 1250 cm^?1, and a medium-intensity line at 940 cm^?1. When the protein is pH-denatured, a strong amide III line appears at 1254 cm^?1 and the 940 cm^?1 line becomes weak. The intensities of the latter two lines are a sensitive measure of the alpha-helical and disordered chain content. These results are consistent with the helix-to-coil studies of the polypeptides. The Raman spectra of α-casein and prothrombin, proteins thought to have little or no ordered secondary structure, are investigated. The amide III regions of both spectra display strong lines at 1254 cm^?1 and only weak scattering is observed at 940 cm^?1, features characteristic of the denatured tropomyosin spectrum. The amide I mode of α-casein appears at 1668 cm^?1, in agreement with the previously reported spectra of disordered polypeptides, poly-L -glutamic acid and poly-L -lysine at pH 7.0 and mechanically deformed poly-L -alanine.     

A computer simulation of functional group contributions to free energy in water and a DPPC lipid bilayer

A series of all-atom molecular dynamics simulations has been performed to evaluate the contributions of various functional groups to the free energy of solvation in water and a dipalmitoylphospatidylcholine lipid bilayer membrane and to the free energies of solute transfer (Delta(DeltaG(o))X) from water into the ordered-chain interior of the bilayer. Free energies for mutations of the alpha-H atom in p-toluic acid to six different substituents (-CH3, -Cl, -OCH3, -CN, -OH, -COOH) were calculated by a combined thermodynamic integration and perturbation method and compared to literature results from vapor pressure measurements, partition coefficients, and membrane transport experiments. Convergence of the calculated free energies was indicated by substantial declines in standard deviations for the calculated free energies with increased simulation length, by the independence of the ensemble-averaged Boltzmann factors to simulation length, and the weak dependence of hysteresis effects on simulation length over two different simulation lengths and starting from different initial configurations. Calculated values of Delta(DeltaG(o))X correlate linearly with corresponding values obtained from lipid bilayer transport experiments with a slope of 1.1 and from measurements of partition coefficients between water and hexadecane or decadiene, with slopes of 1.1 and 0.9, respectively. Van der Waals interactions between the functional group of interest and the acyl chains in the ordered chain region account for more than 95% of the overall potential energy of interaction. These results support the view that the ordered chain region within the bilayer interior is the barrier domain for transport and that solvation interactions within this region resemble those occurring in a nonpolar hydrocarbon.     

Structural details of crystalline cellulose from higher plants

It is commonly assumed that cellulose from higher plants contains the Ialpha and Ibeta crystalline allomorphs together with surface and disordered chains. For cellulose Ialpha, the evidence for its presence in higher plants is restricted to the C-4 signals in the solid-state (13)C NMR spectrum, which match those of crystalline cellulose Ialpha from algal sources. Algal cellulose Ialpha can be converted to the Ibeta form by high-temperature annealing. We used this approach to generate cellulose samples differing in Ibeta content from flax fibers and celery collenchyma, which respectively are representative of textile (secondary-wall) and primary-wall cellulose. It was then possible to isolate the detailed spectral contributions of the surface, Ibeta and Ialpha-like phases from linear combinations of the observed (13)C NMR and FTIR spectra. The (13)C NMR spectra resembled those of highly crystalline tunicate or algal cellulose Ibeta and Ialpha, with slight differences implying increased disorder and minor conformational discrepancies. The FTIR spectrum of the Ibeta form was closely similar to its more crystalline counterparts, but the FTIR spectrum of the Ialpha form was not. In addition to increased bandwith indicative of lower order, it showed substantial differences in the profile of hydroxyl stretching bands. These results confirm that higher plants synthesize cellulose Ibeta but show that the Ialpha-like chains, although conformationally quite similar to crystalline algal cellulose Ialpha, sit in a different hydrogen-bonding environment in higher plants. The differences are presumably occasioned by the small diameter of the crystallites and the influence of the crystallite surface on chain packing.     

Vibrational neutron spectroscopy of collagen and model polypeptides.

A pulsed source neutron spectrometer has been used to measure vibrational spectra (20-4000 cm-1) of dry and hydrated type I collagen fibers, and of two model polypeptides, polyproline II and (prolyl-prolyl-glycine)10, at temperatures of 30 and 120 K. the collagen spectra provide the first high resolution neutron views of the proton-dominated modes of a protein over a wide energy range from the low frequency phonon region to the rich spectrum of localized high frequency modes. Several bands show a level of fine structure approaching that of optical data. The principal features of the spectra are assigned. A difference spectrum is obtained for protein associated water, which displays an acoustic peak similar to pure ice and a librational band shifted to lower frequency by the influence of the protein. Hydrogen-weighted densities of states are extracted for collagen and the model polypeptides, and compared with published calculations. Proton mean-square displacements are calculated from Debye-Waller factors measured in parallel quasi-elastic neutron-scattering experiments. Combined with the collagen density of states function, these yield an effective mass of 14.5 a.m.u. for the low frequency harmonic oscillators, indicating that the extended atom approximation, which simplifies analyses of low frequency protein dynamics, is appropriate.     

Structural analysis of biological aliphatic compounds using surface-enhanced Fourier transform Raman spectroscopy

The surface-enhanced Raman scattering (SERS) technique for Fourier transform Raman spectrometry is employed to reveal the chemical structure of biological aliphatic compounds consisting of folded, long aliphatic chains. The structural analysis is performed via the measurements of the accordion-vibration modes generated in the ordered, long aliphatic chain. The SERS spectra after subtraction of a background spectrum give segment lengths that are almost perfectly consistent with the chemical structures studied by mass spectrometry. The agreement of the SERS results with those of mass spectrometry suggests the positions of kinks in the long hydrocarbon chain. The combination technique of SERS and mass spectrometry is useful to discuss the structure of folded, long biological lipids.     

Mechanism and dynamics of conformational ordering in xanthan polysaccharide

The thermally induced order-disorder transition of xanthan (extracellular bacterial polysaccharide from Xanthomonas campestris) has been investigated by optical rotation, differential scanning calorimetry, stopped-flow reaction kinetics and low-angle laser light scattering, and the results have been analysed in terms of Zimm -Bragg helix-coil transition theory. The reciprocal of the transition midpoint temperature (Tm) varies linearly with the logarithm of cation (K+) the salt dependence of Tm, is in agreement with Manning polyelectrolyte theory the ordered structure. The associated increase in cation binding, calculated from the salt dependence of tm, is in agreement with the Manning polyelectrolyte theory for one of the candidate structures from X-ray diffraction, a 5(1) single helix stabilized by packing of side-chains along the polymer backbone, but not for the alternative double-helix structure that has also been proposed. At each salt concentration, the two fundamental parameters of the Zimm -Bragg theory, s and sigma, were calculated. The equilibrium constant for growth of the ordered structure (s) is derived directly from calorimetric measurement of transition enthalpy (delta Hcal ), and sigma, which quantifies the relative instability of the helix nucleus, is derived from the ratio of delta Hcal to the apparent transition enthalpy (delta Happ ) obtained by van't Hoff analysis of the optical rotation data. The temperature course of conformational ordering calculated theoretically is in good quantitative agreement with experimental results from both optical rotation and scanning calorimetry. The calculated average length of stable, ordered chain-sequences increases with decreasing temperature, but equals or exceeds the total chain length from light scattering only at temperatures more than approximately equal to 70 K below Tm, suggesting that ordered and disordered regions may co-exist within the same xanthan molecule. Consistent with this interpretation, the observed rate of conformational ordering increases sharply under conditions where the starting solution for dynamic measurements is partially ordered, suggesting that ordered sequences within each chain may act as helix nuclei for adjacent disordered regions, so that helix growth, rather than the slower nucleation process, becomes rate limiting.     

SPINE-D: accurate prediction of short and long disordered regions by a single neural-network based method

Short and long disordered regions of proteins have different preference for different amino acid residues. Different methods often have to be trained to predict them separately. In this study, we developed a single neural-network-based technique called SPINE-D that makes a three-state prediction first (ordered residues and disordered residues in short and long disordered regions) and reduces it into a two-state prediction afterwards. SPINE-D was tested on various sets composed of different combinations of Disprot annotated proteins and proteins directly from the PDB annotated for disorder by missing coordinates in X-ray determined structures. While disorder annotations are different according to Disprot and X-ray approaches, SPINE-D's prediction accuracy and ability to predict disorder are relatively independent of how the method was trained and what type of annotation was employed but strongly depend on the balance in the relative populations of ordered and disordered residues in short and long disordered regions in the test set. With greater than 85% overall specificity for detecting residues in both short and long disordered regions, the residues in long disordered regions are easier to predict at 81% sensitivity in a balanced test dataset with 56.5% ordered residues but more challenging (at 65% sensitivity) in a test dataset with 90% ordered residues. Compared to eleven other methods, SPINE-D yields the highest area under the curve (AUC), the highest Mathews correlation coefficient for residue-based prediction, and the lowest mean square error in predicting disorder contents of proteins for an independent test set with 329 proteins. In particular, SPINE-D is comparable to a meta predictor in predicting disordered residues in long disordered regions and superior in short disordered regions. SPINE-D participated in CASP 9 blind prediction and is one of the top servers according to the official ranking. In addition, SPINE-D was examined for prediction of functional molecular recognition motifs in several case studies.     

Functional anthology of intrinsic disorder. 2. Cellular components, domains, technical terms, developmental processes, and coding sequence diversities correlated with long disordered regions

Biologically active proteins without stable ordered structure (i.e., intrinsically disordered proteins) are attracting increased attention. Functional repertoires of ordered and disordered proteins are very different, and the ability to differentiate whether a given function is associated with intrinsic disorder or with a well-folded protein is crucial for modern protein science. However, there is a large gap between the number of proteins experimentally confirmed to be disordered and their actual number in nature. As a result, studies of functional properties of confirmed disordered proteins, while helpful in revealing the functional diversity of protein disorder, provide only a limited view. To overcome this problem, a bioinformatics approach for comprehensive study of functional roles of protein disorder was proposed in the first paper of this series (Xie, H.; Vucetic, S.; Iakoucheva, L. M.; Oldfield, C. J.; Dunker, A. K.; Obradovic, Z.; Uversky, V. N. Functional anthology of intrinsic disorder. 1. Biological processes and functions of proteins with long disordered regions. J. Proteome Res. 2007, 5, 1882-1898). Applying this novel approach to Swiss-Prot sequences and functional keywords, we found over 238 and 302 keywords to be strongly positively or negatively correlated, respectively, with long intrinsically disordered regions. This paper describes approximately 90 Swiss-Prot keywords attributed to the cellular components, domains, technical terms, developmental processes, and coding sequence diversities possessing strong positive and negative correlation with long disordered regions.     

Effects of denaturants on the dynamics of loop formation in polypeptides

Quenching of the triplet state of tryptophan by close contact with cysteine has been used to measure the reaction-limited and diffusion-limited rates of loop formation in disordered polypeptides having the sequence cys-(ala-gly-gln)j-trp (j=1-9). The decrease in the length-dependence of the reaction-limited rate for short chains in aqueous buffer, previously attributed to chain stiffness, is not observed at high concentrations of chemical denaturant (6 M GdmCl and 8 M urea), showing that denaturants increase chain flexibility. For long chains, both reaction-limited and diffusion-limited rates are significantly smaller in denaturant and exhibit a steeper length dependence. The results can be explained using end-to-end distributions from a wormlike chain model in which excluded volume interactions are incorporated by associating a 0.4-0.5 nm diameter hard sphere with the end of each virtual peptide bond. Fitting the data with this model shows that the denaturants reduce the persistence length from approximately 0.6 nm to approximately 0.4 nm, only slightly greater than the length of a peptide bond. The same model also describes the reported length dependence for the radii of gyration of chemically denatured proteins containing 50-400 residues. The end-to-end diffusion coefficients obtained from the diffusion-limited rates are smaller than the sum of the monomer diffusion coefficients and exhibit significant temperature dependence, suggesting that diffusion is slowed by internal friction arising from barriers to backbone conformational changes.     

Vacuum ultraviolet spectroscopy of poly-α-amino acids

Vacuum ultraviolet spectra are reported for a series of poly-α-amino acids which were film cast onto lithium fluoride discs. The solvents used for film casting were chosen such that chain conformations representative of those normally found for polypeptides were produced. Comparison of the spectra obtained suggests that the peptide group has at least one conformationally sensitive transition in the vacuum ultraviolet region at ～165 nm. For the β, or extended, form of the polymers, the intensity of this transition appears to be greater than that found for the monomer units, whereas the formation of a more compact helix results in a reduction in the intensity of this transition relative to that found for the monomers.     

Effect of temperature on the circular dichroism spectra of polypeptides in the extended state

The circular dichroism (CD) spectra of poly-L -proline and of poly-L -glutamic acid and poly-L -lysine in their charged states have been studied as a function of temperature. The variation of CD spectra with temperature is inconsistent with the assignment of the spectrum of such charged polypetides to an unordered chain conformation, but does support our earlier assignment to a locally ordered structure—what we have called the extended helix conformation. These results also strengthen our previous assignment of the CD spectrum of an unordered chain, and indicate that three conformational states (α-helix, extended helix, and unordered) should be incorporated in our thinking about conformational transitions in polypeptides.     

Insect crossbridges, relaxed by spin-labeled nucleotide, show well-ordered 90 degrees state by X-ray diffraction and electron microscopy, but spectra of electron paramagnetic resonance probes report disorder.

The structure of glycerinated Lethocerus insect flight muscle fibers, relaxed by spin-labeled ATP and vanadate (Vi), was examined using X-ray diffraction, electron microscopy and electron paramagnetic resonance (e.p.r.) spectra. We obtained excellent relaxation of MgATP quality as determined by mechanical criteria, using vanadate trapping of 2' spin-labeled 3' deoxyATP at 3 degree C. In rigor fibers, when the diphosphate analog is bound in the absence of Vi, the probes on myosin heads are well-ordered, in agreement with electron microscopic and X-ray patterns showing that myosin heads are ordered when attached strongly to actin. In relaxed muscle, however, e.p.r. spectra report orientational disorder of bound (Vi-trapped) spin-labeled nucleotide, while electron microscopic and X-ray patterns both show well-ordered bridges at a uniform 90 degrees angle to the filament axis. The spin-labeled nucleotide orientation is highly disordered, but not completely isotropic; the slight anisotropy observed in probe spectra is consistent with a shift of approximately 10% of probes from angles close to 0 degrees to angles close to 90 degrees. Measurements of probe mobility suggest that the interaction between probe and protein remains as tight in relaxed fibers as in rigor, and thus that the disorder in relaxed fibers arises from disorders of (or within) the protein and not from disorder of the probe relative to the protein. Fixation of the relaxed fibers with glutaraldehyde did not alter any aspect of the spectrum of the Vi-trapped analog, including the slight order observed, showing that the extensive inter- and intra-molecular cross-linking of the first step of sample preparation for electron microscopy had not altered relaxed crossbridge orientations. Two models that may reconcile the apparently disparate results obtained on relaxed fibers are presented: (1) a rigid myosin head could possess considerable disorder in the regular array about the thick filament; or (2) the nucleotide site could be on a disordered, probably distal, domain of myosin, while a more proximal region is well ordered on the thick filament backbone. Our findings suggest that when e.p.r. probes signal disorder of a local site or domain, this is complementary, not contradictory, to signals of general order. The e.p.r. spectra show that a portion of the myosin molecule can be disordered at the same time as the X-ray diffraction and electron microscopy show the bulk of myosin head mass to be uniformly oriented and regularly arrayed.     

SPINE-D: Accurate Prediction of Short and Long Disordered Regions by a Single Neural-Network Based Method

Abstract

Short and long disordered regions of proteins have different preference for different amino acid residues. Different methods often have to be trained to predict them separately. In this study, we developed a single neural-network-based technique called SPINE-D that makes a three-state prediction first (ordered residues and disordered residues in short and long disordered regions) and reduces it into a two-state prediction afterwards. SPINE-D was tested on various sets composed of different combinations of Disprot annotated proteins and proteins directly from the PDB annotated for disorder by missing coordinates in X-ray determined structures. While disorder annotations are different according to Disprot and X-ray approaches, SPINE-D's prediction accuracy and ability to predict disorder are relatively independent of how the method was trained and what type of annotation was employed but strongly depend on the balance in the relative populations of ordered and disordered residues in short and long disordered regions in the test set. With greater than 85% overall specificity for detecting residues in both short and long disordered regions, the residues in long disordered regions are easier to predict at 81% sensitivity in a balanced test dataset with 56.5% ordered residues but more challenging (at 65% sensitivity) in a test dataset with 90% ordered residues. Compared to eleven other methods, SPINE-D yields the highest area under the curve (AUC), the highest Mathews correlation coefficient for residue-based prediction, and the lowest mean square error in predicting disorder contents of proteins for an independent test set with 329 proteins. In particular, SPINE-D is comparable to a meta predictor in predicting disordered residues in long disordered regions and superior in short disordered regions. SPINE-D participated in CASP 9 blind prediction and is one of the top servers according to the official ranking. In addition, SPINE-D was examined for prediction of functional molecular recognition motifs in several case studies. The server and databases are available at http://sparks.informatics.iupui.edu/.