Protein Conformational Analysis Research Articles

Generation of a substructure library for the description and classification of protein secondary structure. II. Application to spectra–structure correlations in fourier transform infrared spectroscopy

Fourier transform infrared spectroscopy has become well known as a sensitive and informative tool for studying secondary structure in proteins. Present analysis of the conformation-sensitive amide I region in protein infrared spectra, when combined with band narrowing techniques, provides more information concerning protein secondary structure than can be meaningfully interpreted. This is due in part to limited models for secondary structure. Using the algorithm described in the previous paper of this series, we have generated a library of substructures for several trypsin-like serine proteases. This library was used as a basis for spectra-structure correlations with infrared spectra in the amide I' region, for five homologous proteins for which spectra were collected. Use of the substructure library has allowed correlations not previously possible with template-based methods of protein conformational analysis.

Determination of calcium ions tightly bound to proteins

A rapid and sensitive procedure is described for the determination of calcium ions tightly bound to proteins using high-performance gel filtration chromatography, followed by the destabilization of the protein conformation and fluorimetric analysis with Quin-2. With this method, contaminating, unbound calcium can be eliminated simultaneously and one can determine the content of a calcium ion in a protein utilizing less than 200 pmol of the protein.

Secondary structure analysis of the scrapie-associated protein PrP 27-30 in water by infrared spectroscopy

A protease-resistant form of the protein PrP (PrP-res) accumulates in tissues of mammals infected with scrapie, Creutzfeldt-Jakob disease, and related transmissible neurodegenerative diseases. This abnormal form of PrP can aggregate into insoluble amyloid-like fibrils and plaques and has been identified as the major component of brain fractions enriched for scrapie infectivity. Using a recently developed technique in Fourier transform infrared spectroscopy which allows protein conformational analysis in aqueous media, we have studied the secondary structure of the proteinase K resistant core of PrP-res (PrP-res 27-30) as it exists in highly infectious fibril preparations. Second-derivative analysis of the infrared spectra has enabled us to quantitate the relative amounts of different secondary structures in the PrP-res aggregates. The analysis indicated that PrP-res 27-30 is predominantly composed of beta-sheet (47%), which is consistent with its amyloid-like properties. In addition, significant amounts of turn (31%) and alpha-helix (17%) were identified, indicating that amyloid-like fibrils need not be exclusively beta-sheet. The infrared-based secondary structure compositions were then used as constraints to improve the theoretical localization of the secondary structures within PrP-res 27-30.

Monitoring of protein conformation by high-performance size-exclusion liquid chromatography and scanning diode array second-derivative UV absorption spectroscopy

Genetic methods now allow the rapid production of mutant proteins for structure-function analysis. To properly interpret any change in biologic activity resulting from modification in primary sequence, it is essential to monitor conformational changes resulting from mutations. Several methods allow low-resolution protein conformational analysis. One method, second-derivative UV absorption spectroscopy, is particularly useful for proteins containing tyrosine and/or tryptophan residues. Using high-performance size-exclusion liquid chromatography and scanning diode array detection we have demonstrated that it is possible to monitor the degree of aggregation as well as conformational perturbation for a series of interleukin-2 structural mutants. Furthermore, the combination of high-performance liquid chromatography and second-derivative UV absorption spectroscopy avoids a potential artifactual contribution in non-chromatographic analysis due to protein aggregation.

A carcinoembryonic antigen (CEA) binding protein from ascites influences CEA uptake by macrophages

A variant of CEA which is less readily endocytosed by macrophages has been isolated from malignant ascites. In vivo , CEA is cleared more slowly by the liver ( t 1 2 = 15.1 minutes ) than CEAs isolated from hepatic metastases ( t 1 2 = 3.1 minutes ). In vitro , rat and human Kupffer cells and rat alveolar macrophages endocytose this CEA less effectively. This slow clearing form of CEA is associated with a smaller (45kD) acidic glycoprotein (CORA) with which it forms a stable complex. CORA can be visualized on reducing gels but not on non reducing gels or by HPLC run under non reducing conditions. This suggests a non-covalent complex between the two glycoproteins. Analysis of protein conformation by circular dichroism revealed changes in the ascites CEA consistent with binding of CORA to the molecule. Western blot showed that CORA crossreacts with antisera to α 1-acid glycoprotein and double immunodiffusion demonstrated cross-reactivity but not identity. Sequencing of CNBr peptides showed sequence homology with α 1-acid glycoprotein but areas of unique sequence were also found. It is suggested that binding of CORA to CEA blocks the macrophage receptor binding of CEA.

Microcomputer analyzed initial rate kinetics of the benzene enhanced unfolding of myoglobin: A biophysical chemistry experiment

This paper descries an inexpensive biophysical chemistry experiment, designed to be completed in one lab period, that introduces students to the subject of globular protein conformation and microcomputer analysis of initial rate data for the unfolding of proteins appropriate for an upper-division physics or biochemistry lab.

Determination of an empirical energy function for protein conformational analysis by energy embedding

AbstractIt is quite easy to propose an empirical potential for conformational analysis such that given crystal structures lie near local minima. What is much more difficult, is to devise a function such that the native structure lies near a relatively deep local minimum, at least in some neighborhood of the native in conformation space. An algorithm is presented for finding such a potential acting on proteins where each amino acid residue is represented by a single point. When the given structure is either an α‐helical, β‐strand, or hairpin bend segment of pancreatic trypsin inhibitor, the resulting potential function in each case possesses a deep minimum within 0.10 Å of the native conformation. The improved energy embedding algorithm locates a marginally better minimum in each case only 0.1–1.3 Å away from the respective native state. In other words, this potential function guides a conformational search toward structures very close to the native over a wide range of conformation space.

Distance-constraint approach to protein folding. I. Statistical analysis of protein conformations in terms of distances between residues

A statistical analysis of protein conformations in terms of the distance between residues, represented by their Cα atoms, is presented. We consider four factors that contribute to the determination of the distancedi,i+k between a given pair ofith and(i+k)th residues in the native conformation of a globular protein: (1) the distancek along the chain, (2) the size of the protein, (3) the conformational states of theith to(i+k)th residues, and (4) the amino acid types of the and(i+k)th residues. In order to account for the dependence on the distancek along the chain, the statistics are taken for three ranges, viz., short, medium, and long ranges (k≤8; 9≤k≤20; andk≥21; respectively). In the statistics of short-range distances, a mean distanceDk and its standard deviationSk are calculated for each value ofk, with and without taking into account the conformational states of all residues fromi toi+k (factors 1 and 3). As an Appendix, the relations for converting from the distances between residues into other conformational parameters are discussed. In the statistics of long-range distances, a reduced distanced*ij (the actual distance divided by the radius of gyration) is used to scale the data so that they become independent of protein size, and then a mean reduced distanceDl (aμ, aν) and its standard deviation σl (aμ, aν) are calculated for each amino acid pair (aμ, aν) (factors 2 and 4). The effect of the neighboring residues along the chain on the value of the distanced*ij is explored by a linear regression analysis between the actual reduced distanced*ij and the mean value over theDl for all possible pairs of residues in the two segments of the (i−2)th to the (i+2)th and the (j−2)th to the (j+2)th residues. The effect is assessed in terms of the tangentAl (aμ, aν) of the calculated regression line for each amino acid pair (aμ, aν). In the statistics of medium-range distances, only factors 1 and 4 are considered, to simplify the analysis. The scaled distancedi,i+k†=(di,i+k-Dk)/Sk is used to eliminate the dependence onk, the distance along the chain. The propertiesDm (aμ, aν), σm (aμ, aν) andAm (aμ, aν) corresponding toDl (aμ, aν), σl (aμ, aν), andAl (aμ, aν), and also calculated for each amino acid pair (aμ, aν). The results are interpreted as follows: the smaller values ofDl (aμ, aν) andDm (aμ, aν) indicate a preference of the pair (aμ, aν) for a contact (e.g., pairs between hydrophobic amino acids, and pairs of Cys with aromatic amino acids), and the larger values of these quantities indicate a preference for distant mutual location (e.g., pairs between strong hydrophilic amino acids); the smaller values of σl (aμ, aν) and σm (aμ, aν) indicate a strong preference for either contact or noncontact (e.g., pairs between hydrophobic amino acids, and pairs between strong hydrophobic and hydrophilic amino acids, respectively), and the larger values of these quantities indicate the ambivalent/neutral nature of the preference for contact and noncontact (e.g., pairs containing Ser or Thr); the smaller values ofAl (aμ, aν) andAm (aμ, aν) indicate that the distance of an (aμ, aν) pair is determined independently of the amino acid character of the neighboring residues along the chain (e.g., some pairs of Cys or Met with other amino acids) and the larger values of these quantities indicare that such amino acid character contributes strongly to the determination of the distance (e.g., pairs containing Ser or Thr, and pairs between amino acids with small side chains). The difference between the statistics for the long- and medium-range distances is also discussed; the former reflect the difference between the hydrophobic and hydrophilic character of the residues, but the latter cannot be easily interpretable only in terms of hydrophobicity and hydrophilicity. The data analyzed here are used in the optimization of an object function to compute protein conformation in a subsequent paper.

A b i n i t i o studies of structural features not easily amenable to experiment. 23. Molecular structures and conformational analysis of the dipeptide N-acetyl-N′-methyl glycyl amide and the significance of local geometries for peptide structures

The molecular structures of four conformations of N-acetyl–N′-methyl glycyl amide were refined by geometrically unconstrained ab initio gradient relaxation on the 4–21G level. The most stable form I contains a seven-membered ring closed by hydrogen bonding. A second local minimum II is less than 1 kcal/mol above I and represents the fully extended form with a five-membered hydrogen bonded ring. The two other minima refined, III and IV, are open forms which are 4–5 kcal/mol less stable than I. The refined geometries make it possible to estimate the significance of local geometries, in contrast to standard geometry, in the various conformations. It is found that bond distances in different conformations can vary by up to 0.02 Å, and important backbone bond angles can vary by up to 7°. Except for the symmetrical form II, small deviations from amide planarity (H–N–C = 0 angles of 3–10°) are the rule, even though the equilibrium structure of the unperturbed amide group in 4–21G space is planar. It can be concluded that local geometry relaxations at different points of the potential energy surface of a peptide system can amount to several Kcal/mol per residue and should be an important aspect of protein conformational analysis.

Evaluation of Phenylalanine and Tyrosine Raman Lines for the Amino Acid Ratio

Raman spectroscopy has been used increasingly to determine the conformation of the peptide backbone and the side chain microenvironment of some amino acid residues. Because of the relative uncertainty about the absolute magnitude of intensity, Raman spectroscopy has been used primarily for qualitative evaluation of properties of proteins. There have, however, been some attempts to use Raman spectroscopy for quantitative purposes in the analysis of protein conformations.

Analysis of ribosomal protein conformation in Bacillus subtilis by reductive methylation : Identification of Proteins with different Conformation in Monosomes and Polysomes

Analysis of ribosomal protein conformation in Bacillus subtilis by reductive methylation : Identification of Proteins with different Conformation in Monosomes and Polysomes

Determination of protein secondary structure in solution by vacuum ultraviolet circular dichroism

A new approach is presented for evaluating the secondary structure of globular proteins in solution by analysis of circular dichroism spectra. Two major improvements are introduced. First, extension of the ultraviolet region down to 165 nm. Extension of the spectral region to the vacuum ultraviolet allows observation of new characteristic bands, which improves substantially the analysis of protein conformation. Secondly, new reference spectra that contain all main protein conformations, including two sets of (β-turns, are taken into account. One set of β-turns representative of all main types of turns, contains the sequences (Ala 2-Gly 2) n, l-Pro- d-Ala, N-acetyl- l-Pro-Gly- l-Leu-OH. A comparison of the results of the analysis with one β-turn and with a set of turns indicates that there is essentially no major improvement; thus one β-turn can be taken as the basis spectrum. In addition, the reference spectra include the β-form represented by polypeptide chains composed of alternating hydrophobic and hydrophilic amino acids, which is more closely related to anti-parallel β-pleated sheet structures in proteins. It appears possible to determine the content of antiparallel and of parallel β-pleated sheets with the present basic β-spectrum. The circular dichroism of aromatic side-chain chromophores was measured in the vacuum ultraviolet in order to take their contribution into account. It appears that for proteins with higher aromatic amino acid content and with weak circular dichroism amide bands, it is necessary to take into account a correction for the aromatic side-chain contribution. The conformation of 13 proteins representative of all main classes of globular proteins was determined from circular dichroism data between 165 nm and 250 nm at 0.2 nm intervals. In particular, the secondary structure of the β-rich class of proteins (concanavalin A, Bence-Jones protein V REI, prealbumin, superoxide dismutase), the α/β class of proteins (lactate dehydrogenase, triose phosphate isomerase, subtilisin, glyceraldehyde-3-phosphate dehydrogenase, flavodoxin and pyruvate kinase) and the (α + β) class of proteins (lysozyme and cytochrome c) were quantitatively characterized in solution. The reliability of the method was also assessed by comparing the data obtained with those available from X-ray crystallography. In general, a good agreement was found between the present circular dichroism data and published X-ray data, except for rubredoxin. By including all types of secondary structure and by enlarging substantially the spectral region, our method permits analysis of the conformation of a variety of proteins, such as nucleic acid-binding proteins, immunoglobulins and β-pleated sheet-rich proteins. It offers new possibilities for investigating the dynamics of protein conformation in solution.

Circular dichroic analysis of protein conformation: Inclusion of the β-turns

The mean residue ellipticity, [θ], at any wavelength, λ, of a protein in aqueous solution is expressed as [θ] λ = f H [θ] H x (1 − k n ) + f β[θ] β + f t [θ] t + f R [θ] R with two constraints: 1 ≥ f j ≥ 0 and Σf j = 1. The subscripts H, β, t, and R refer to the helix, β-form, β-turn, and unordered form. The fractions, f j ′s, of 15 proteins are based on X-ray crystallography, f t refers to the net β-turn after cancelling those residues having dihedral angles of opposite sign. The [ θ] H x of an infinite helix and its chain-length dependence factor, k, were computed from the myoglobin data (Chen et al., 1974, Biochemistry, 13, 3350). The average number of residues per helical segment, n , for 15 proteins was about 10, which can be used for proteins of unknown structure. The reference spectra of other three structural elements are computed by a least-squares method. Once the reference spectra are chosen, the same equation above can be used to estimate the fractions of the secondary structure of a portein from its CD data points between 190 and 240 nm at 1-nm intervals. The computed helical content is usually good to excellent (concanavalin A is a notable exception). Inclusion of the β-turn in the analysis improves the correlation for the estimates of the β-form, but the computed β t values are not significantly correlated with the X-ray results. Matrix formulation proves the equivalence of the least-squares method and the integral curve-fitting.

Circular dichrosim of chromaffin granule proteins in situ: analysis of turbidity effects and protein conformation.

The circular dichroism spectra for proteins in situ in catecholamine secretory vesicles (chromaffin granules) is presented together with an analysis of protein conformation and turbidity effects on the spectra. The calculational analysis has resolved scattering and absorption effects in the turbid suspension spectra using a coated-sphere scattering model which allows for different materials in its shell and core. The intrinsic conformation of the proteins in situ was estimated by an iterative procedure with various trial protein conformations, for the chromaffin granules both intact and after release of their contents. The resulting average secondary structures (within about 10%) are: (25% alpha helix, 15% beta structure) for the membrane proteins and (15% alpha helix, 5% beta structure) for the soluble contents. The protein conformation did not change with osmotic release of the granule's contents. Consequently, if chromogranins are involved in a catecholamine storage complex, this is not reflected in any detectable change in their average secondary structure.

Conformatonal analyss of bovine erythrocuprein from circular dichroism and infra-red spectra

1. 1. The analysis of protein conformation from circular dichroism (C.D.) spectra by curve-fitting and matrix-rank determination is examined with reference to spectra of bovine crythrocuprcin. 2. 2. Poly-α-amino-acid reference spectra are not applicable to bovine erythrocuprein C.D. spectra in so far as they give least-square fits with negative percentages of secondary structure, in contrast to reference spectra derived from the C.D. spectra of the proteins ribonuclease, lysozyme, and myoglobin. The latter indicate the presence of a small percentage of α-helix and preponderance of unordered structure over β-structure in the protein. 3. 3. The presence of these structural modes and the preponderance of unordered structure is supported by the infra-red spectrum of the holoprotein in the amide I region. 4. 4. Matrix-rank analysis of a set of five independent C.D. spectra of bovine erythrocuprein in the far ultra-violet supports the three-component fit of the spectra by least squares.