Determination Of Protein Secondary Structure Research Articles

Environmental features are important in determining protein secondary structure.

We have investigated amino acid features that determine secondary structure: (1) the solvent accessibility of each side chain, and (2) the interaction of each side chain with others one to four residues apart. Solvent accessibility is a simple model that distinguishes residue environment. The pairwise interactions represent a simple model of local side chain to side chain interactions. To test the importance of these features we developed an algorithm to separate alpha-helices, beta-strands, and "other" structure. Single residue and pairwise probabilities were determined for 25,141 samples from proteins with <30% homology. Combining the features of solvent accessibility with pairwise probabilities allows us to distinguish the three structures after cross validation at the 82.0% level. We gain 1.4% to 2.0% accuracy by optimizing the propensities, demonstrating that probabilities do not necessarily reflect propensities. Optimization of residue exposures, weights of all probabilities, and propensities increased accuracy to 84.0%.

A Simple Approach to Membrane Protein Secondary Structure and Topology based on NMR Spectroscopy

This paper describes a simple, qualitative approach for the determination of membrane protein secondary structure and topology in lipid bilayer membranes. The approach is based on the observation of wheel-like resonance patterns observed in the NMR 1H- 15N/ 15N polarization inversion with spin exchange at the magic angle (PISEMA) and 1H/ 15N heteronuclear correlation (HETCOR) spectra of membrane proteins in oriented lipid bilayers. These patterns, named Pisa wheels, have been previously shown to reflect helical wheel projections of residues that are characteristic of α-helices associated with membranes. This study extends the analysis of these patterns to β-strands associated with membranes and demonstrates that, as for the case of α-helices, Pisa wheels are extremely sensitive to the tilt, rotation, and twist of β-strands in the membrane. Therefore, the Pisa wheels provide a sensitive, visually accessible, qualitative index of membrane protein secondary structure and topology.

Estimation of Protein Secondary Structure from Circular Dichroism Spectra: Inclusion of Denatured Proteins with Native Proteins in the Analysis

We have expanded our reference set of proteins used in the estimation of protein secondary structure by CD spectroscopy from 29 to 37 proteins by including 3 additional globular proteins with known X-ray structure and 5 denatured proteins. We have also modified the self-consistent method for analyzing protein CD spectra, SELCON3, by including a new selection criterion developed by W. C. Johnson, Jr. (Proteins Struct. Funct. Genet. 35, 307–312, 1999). The secondary structure corresponding to the denatured proteins was approximated to be 90% unordered, owing to the spectral similarity of the denatured proteins and unordered structures. We examined the thermal denaturation of ribonuclease T1 by CD using both the original and expanded sets of reference proteins and obtained more consistent results with the expanded set. The expanded set of reference proteins will be helpful for the determination of protein secondary structure from protein CD spectra with higher reliability, especially of proteins with significant unordered structure content and/or in the course of denaturation.

Determination of protein secondary structure and solvent accessibility using site-directed fluorescence labeling. Studies of T4 lysozyme using the fluorescent probe monobromobimane.

We report an investigation of how much protein structural information could be obtained using a site-directed fluorescence labeling (SDFL) strategy. In our experiments, we used 21 consecutive single-cysteine substitution mutants in T4 lysozyme (residues T115-K135), located in a helix-turn-helix motif. The mutants were labeled with the fluorescent probe monobromobimane and subjected to an array of fluorescence measurements. Thermal stability measurements show that introduction of the label is substantially perturbing only when it is located at buried residue sites. At buried sites (solvent surface accessibility of <40 A(2)), the destabilizations are between 3 and 5.5 kcal/mol, whereas at more exposed sites, DeltaDeltaG values of < or = 1.5 kcal/mol are obtained. Of all the fluorescence parameters that were explored (excitation lambda(max), emission lambda(max), fluorescence lifetime, quantum yield, and steady-state anisotropy), the emission lambda(max) and the steady-state anisotropy values most accurately reflect the solvent surface accessibility at each site as calculated from the crystal structure of cysteine-less T4 lysozyme. The parameters we identify allow the classification of each site as buried, partially buried, or exposed. We find that the variations in these parameters as a function of residue number reflect the sequence-specific secondary structure, the determination of which is a key step for modeling a protein of unknown structure.

Recognition of an Essential Adenine at a Protein−RNA Interface: Comparison of the Contributions of Hydrogen Bonds and a Stacking Interaction

Recognition of an Essential Adenine at a Protein−RNA Interface: Comparison of the Contributions of Hydrogen Bonds and a Stacking Interaction

Hydrolysis of haemoglobin surveyed by infrared spectroscopy: I. solvent effect on the secondary structure of haemoglobin

The hydrolysis of bovine haemoglobin in an acetic acid/sodium acetate buffer enables to produce peptides of major importance in biomedical research. The global objective is to survey this reaction by infrared spectroscopy. This article concerns the first step: the evaluation by spectroscopy of the effect on protein secondary structure of the addition of ethanol in the buffer.Conformational changes are related to solvent–protein interactions as the protein folding is driven by the entropy of removing hydrophobic groups from contact with the solvent. Therefore, the stability of haemoglobin in an ethanol–water mixture results in a competition between the water structure, which is strengthened by the presence of the alcohol, and the solubility of hydrophobic residues. Previous non infrared experiments, based on mass spectrometry for example, have been reported for the investigation of the denaturation of haemoglobin by an organic solvent.The use of vibrational spectroscopy for protein secondary structure determination has proved its efficiency. We focus here on the study of the denaturing of haemoglobin in a water medium by addition of ethanol. As our investigations deal with very low concentrated haemoglobin, we apply a technique that uses films dried from dilute solution. Although it is not generally accepted that the protein conformation is retained when the solvent evaporates off, we validate this method comparing some results to previous infrared study made at upper concentrations with liquid sampling. We observe that Fourier Transform Infrared (FTIR) Spectroscopy, combined with few mathematical treatments, permits to estimate that haemoglobin remains in a native form unless a concentration of more or less 20% of ethanol is reached. For greater values modifications are perceptible on the infrared spectra.

Use of infrared spectroscopy to assess secondary structure of human growth hormone within biodegradable microspheres

The purpose of this study was to test the utility of infrared (IR) spectroscopy to determine protein secondary structure in biodegradable microspheres. Encapsulation of proteins within biodegradable polymers, [e.g. poly(lactic‐co‐glycolic acid) (PLGA)] for controlled drug release has recently been the subject of intense research effort. The ability to assess protein integrity after microsphere production is necessary to successfully produce microspheres that release native proteins. We used IR spectroscopy, a noninvasive method–as opposed to conventional organic solvent extraction or in vitro release at elevated temperature–to assess the secondary structure of recombinant human growth hormone (rhGH) within dry and rehydrated microspheres. PLGA microspheres containing rhGH with different excipients were prepared by a conventional double‐emulsion method. The protein IR spectra indicated that the encapsulation process could perturb the structure of rhGH and that excipients could inhibit this damage to varying degrees. A strong positive correlation was found between intensity of the dominant α‐helical band in the spectra of rhGH in rehydrated microspheres and the percent monomer released from microspheres during incubation in buffer. We also studied microspheres prepared with zinc‐precipitated rhGH. The addition of Zn2+ during microsphere processing partially inhibited protein unfolding and fostered complete refolding of rhGH upon rehydration. In conclusion, IR spectroscopy can serve as a valuable tool to assess protein structure within both dried and rehydrated microspheres.

UV resonance Raman-selective amide vibrational enhancement: quantitative methodology for determining protein secondary structure.

We have directly determined the amide band resonance Raman spectra of the "average" pure alpha-helix, beta-sheet, and unordered secondary structures by exciting within the amide pi-->pi* transitions at 206.5 nm. The Raman spectra are dominated by the amide bands of the peptide backbone. We have empirically determined the average pure alpha-helix, beta-sheet, and unordered resonance Raman spectra from the amide resonance Raman spectra of 13 proteins with well-known X-ray crystal structures. We demonstrate that we can simultaneously utilize the amide I, II, and III bands and the Calpha-H amide bending vibrations of these average secondary structure spectra to directly determine protein secondary structure. The UV Raman method appears to be complementary, and in some cases superior, to the existing methods, such as CD, VCD, and absorption spectroscopy. In addition, the spectra are immune to the light-scattering artifacts that plague CD, VCD, and IR absorption measurements. Thus, it will be possible to examine proteins in micelles and other scattering media.

Applications of a New 206.5-nm Continuous-Wave Laser Source: UV Raman Determination of Protein Secondary Structure and CVD Diamond Material Properties

We demonstrate the utility of a new 206.5-nm continuous-wave UV laser excitation source for spectroscopic studies of proteins and CVD diamond. Excitation at 206.5 nm is obtained by intracavity frequency doubling the 413-nm line of a krypton-ion laser. We use this excitation to excite resonance Raman spectra within the π → π amide transition of the protein peptide backbone. The 206.5-nm excitation resonance enhances the protein amide vibrational modes. We use these high signal-to-noise spectral data to determine protein secondary structure. We also demonstrate the utility of this source to excite CVD and gem-quality diamond within its electronic bandgap. The diamond Raman spectra have very high signal-to-noise ratios and show no interfering broad-band luminescence. Excitation within the diamond bandgap also gives rise to narrow photoluminescence peaks from diamond defects. These features have previously been observed only by cathodoluminescence measurements. This new continuous-wave UV source is superior to the previous pulsed sources, because it avoids nonlinear optical phenomena and thermal sample damage; Photoluminescence.

The Use and Misuse of FTIR Spectroscopy in the Determination of Protein Structure

Fourier transform infrared (FTIR) spectroscopy is an established tool for the structural characterization of proteins. However, many potential pitfalls exist for the unwary investigator. In this review we critically assess the application of FTIR spectroscopy to the determination of protein structure by (1) outlining the principles underlying protein secondary structure determination by FTIR spectroscopy, (2) highlighting the situations in which FTIR spectroscopy should be considered the technique of choice, (3) discussing the manner in which experiments should be conducted to derive as much physiologically relevant information as possible, and (4) outlining current methods for the determination of secondary structure from infrared spectra of proteins.

Determination of protein secondary structure by Fourier transform infrared spectroscopy: A critical assessment

Determination of protein secondary structure by Fourier transform infrared spectroscopy: A critical assessment

Artifacts Associated with the Determination of Protein Secondary Structure by ATR-IR Spectroscopy

The last decade has seen a dramatic increase in the application of FT-IR spectroscopy to biological problems. As well as transmission methods, many specialized techniques are increasingly being applied to biological structural studies, the most popular being ATR spectroscopy. While ATR-IR spectroscopy has been applied to conformational studies of lipids and membrane-associated proteins and peptides for some time, it has only recently been applied to soluble proteins. Two assumptions are made in the analysis of soluble proteins as films. First, it is assumed that drying of films does not result in irreversible structural alterations. Second, it is assumed that rehydration of the film results in the same degree of protein/solvent interaction as in solution, or that reduced solvent interaction in films does not produce structural alterations. In this note we compare spectra of representative proteins as films deposited on ATR elements and in solution. Our results show that significant differences are apparent between solution and film spectra.

An unusual peptide conformation may precipitate amyloid formation in Alzheimer's disease: application of solid-state NMR to the determination of protein secondary structure

The formation of insoluble proteinaceous deposits is characteristic of many diseases which are collectively known as amyloidosis. There is very little molecular-level structural information available regarding the amyloid deposits due to the fact that the constituent proteins are insoluble and noncrystalline. Therefore, traditional protein structure determination methods such as solution NMR and X-ray crystallography are not applicable. We report herein the application of the solid-state NMR technique rotational resonance (R2) to the accurate measurement of carbon-to-carbon distances in the amyloid formed from a synthetic fragment (H2N-LeuMetValGlyGlyValValIleAla-CO2H) of the amyloid-forming protein of Alzheimer's disease (AD). This sequence has been implicated in the initiation of amyloid formation. Two distances measured by R2 indicate that an unusual structure, probably involving a cis amide bond, is present in the aggregated peptide amyloid. This structure is incompatible with the accepted models of fibril structure. A relationship between this structure and the stability of the amyloid is proposed.

Determination of protein secondary structure using factor analysis of infrared spectra

A method is presented for determining the secondary structural composition of a protein in aqueous solution from its infrared spectrum. A factor analysis approach is used to analyze the infrared spectra of 18 proteins whose crystal structures are known from X-ray studies. Factor analysis followed by multiple linear regression identifies those eigenspectra that correlate with the variation in properties described by the calibration set. The properties of interest in this study are % alpha-helix, % beta-sheet, and % turns. In the analysis of an unknown, the factor loadings required to reproduce its spectrum are substituted in the regression equation for each property to predict its secondary structural composition. The accuracy of the method was determined by removing each standard, in turn, from the calibration set and using a calibration set generated from the remainder to predict its composition. By this method we obtain standard errors of prediction of 3.9% for alpha-helix, 8.3% for beta-sheet, and 6.6% for turns. The method may also be applied to the spectra of proteins in 2H2O. The method has important advantages over those currently in use for the quantitative analysis of the infrared spectra of proteins. Manipulation of the spectrum is kept to a minimum, no curve-fitting is necessary, and the several amide I band components need not be assigned.

How to determine protein secondary structure in solution by Raman spectroscopy: practical guide and test case DNase I

For many proteins available in large (milligram) quantities, a three-dimensional structure determination by X-ray or NMR methods is very difficult, impossible, or too costly. In these cases, spectroscopic determination of secondary structure content can be a valuable source of partial information about protein structure in solution. In particular, Raman spectroscopy can be used to determine to fair accuracy the helix and sheet content of a globular protein. However, technical difficulties have hampered the routine application of the method: (1) The large background signal of aqueous solvent in the amide I region is difficult to subtract accurately. (2) The reference data set of Raman spectra of proteins with known crystal structure is incomplete, and the assignment of secondary structure in a known crystal structure is not unambiguous. (3) The mathematical problem of extracting structure information from the spectra is ill determined; i.e., there are many apparently satisfactory solutions for a given spectrum. We have now partly solved and partly sidestepped these problems by improving and simplifying existing methods. Here, we give a step-by-step outline of a procedure intended for routine determination of the percentage of a-helix and @-sheet from the amide I Raman spectra of proteins in solution. Its main features are (a) an uncom- plicated procedure for solvent subtraction, aided by use of a divided spinning cell technique, (b) a numerically stable data handling algorithm, and (c) a clear statement of expected accuracy. In our hands, using the reference spectra of Williams (1 983), helix content can be determined to an accuracy of 6 percentage points (largest error 12%) and @-sheet content to an accuracy of 5 percentage points (largest error 7%). However, the experimental distinction between parallel and antiparallel 0-sheet does not appear possible without a significant expansion of the set of reference proteins. As a test we have measured the Raman spectrum of DNase I, a known structure treated as unknown, and derive 14% a-helix and 22% 0-sheet content, compared to X-ray derived values of 20% helix and 25% sheet (hydrogen bonds per 100 residues). The error, -6% for helix and -3% for sheet content, is typical. The method can be a tool for checking the structural purity of genetically engineered proteins, detecting major structural alterations of mutant proteins, and providing a priori information as input to predictions of protein structure.

Secondary structure prediction and determination of proteins — a review

The rapid increase in sequence data in combination with a greater understanding of the forces regulating protein structure has been the impetus for an upsurge in the development of theoretical prediction methods. These methods have afforded protein chemists the ability to identify and quantify the various secondary structures along the protein chain. Concurrently, various physico-chemical techniques have been developed such as nuclear Overhauser enhancement n.m.r. and laser Raman spectroscopy. In addition, traditional methods such as infrared and circular dichroism spectroscopy have been refined. Although both predictive and physico-chemical techniques are limited in the types of secondary structure they are capable of determining, they have provided valuable information with regards to protein folding and topology in the absence of X-ray data, and have formed the basis for the development of improved methods for secondary structure determination. This paper reviews some of the predictive and physico-chemical methods presently used to determine protein secondary structure.

Application of a modified computer algorithm in determining potential antigenic determinants associated with the AIDS virus glycoprotein

A computer program was developed for use on an Apple IIe that utilized the parameters developed by Hopp and Woods ( T. P. Hopp and K. R. Woods, 1983, Mol. Immunol. 20, 483–489) for predicting the hydrophilic regions of a given protein. This program will produce a listing of the hydrophilic sequence averages and graphically illustrates the peak areas. The hydrophilic averages over a hexapeptide length can be used to predict protein structure. In conjunction with the Chou-Fasman predictive scheme for protein secondary structure determination, the possible antigenic determinants for the envelope glycoprotein of three viruses isolated from patients with acquired immunodeficiency syndrome (AIDS) were predicted. These predicted determinants could be used to generate synthetic peptides that represent a potential vaccine preparation or in developing a diagnostic assay that specifically detects the agent.

An examination of various algorithms in the determination of protein secondary structure from circular dichroism data

An examination of various algorithms in the determination of protein secondary structure from circular dichroism data

A new method for determining protein secondary structure by laser raman spectroscopy applied to FD phage

A new method is presented for estimating the secondary structure of proteins from the intensity distribution of the laser Raman amide I spectrum. We have applied this method to the coat protein of the fd filamentous phage.

The estimation of protein secondary structure by laser raman spectroscopy from the amide III′ intensity distribution

The determination of protein secondary structure in aqueous solution has undergone an important advance [ 11. It is now possible to estimate fractions of bi-H-bonded andmonoH-bonded helix, antiparallel P-sheet, parallel P-sheet, and P-turn to within 25% of X-ray diffraction estimates with -70% confidence, whereas before, using circular dichroism (CD) [ 21 or Raman spectroscopy [3], only fractions of total helix and P-sheet were estimated, to within -8%. This improvement has come about mostly through the generation of computer-subtracted difference laser Raman spectra, and specifically through the subtraction of the water spectrum from the amide I spectrum of proteins. Here we present evidence that this general approach can be applied to the amide III’ region of the laser Raman spectrum of proteins in D20 as well, and that estimates of the structure of solvent exchanged regions of the protein can thereby be obtained. When a protein is dissolved in D,O, exposed amide N-H groups exchange with the solvent, and the .amide III vibrations shift to a lower frequency from between 1200 and 1320 cm-’ to what are termed the amide III’ vibrations between 900 and 1000 cm-‘. While several investigations have pursued analyzing the amide III region of the vibrational spectrum for protein secondary structure determination [3-71 little has been done with the amide III’ region. We believe that there are some practical advantages to using the amide III’ over the amide III region for structural studies: (i) Bands due to helix in the amide III region are not very intense relative to the corresponding nonhelical bands (R. W. W., A. K. D., W. L. P., in preparation). (ii) CH deformation modes are strong in the amide III helix region. The amide III’ region seems to be less affected by these kinds of problems. Both helical and S-sheet polypeptides show strong amide III’ bands in DzO. The C-C stretching bands in this region are also conformationally sensitive, and may constructively reinforce the structurally corresponding amide III’ bands. Here, the spectrum of each protein in DzO was subtracted from the corresponding spectrum of the protein in Hz0 so that bands unaffected by deuteration of the amide nitrogen are removed. The resulting difference spectrum represents the true contours of the amide III’ spectrum. Four of these amide III’ difference spectra [6] were then used to calculate the spectra of helix and P-sheet by a procedure similar that in [2] for CD spectra. We have performed these same calculations on the watersubtracted amide I spectra of these proteins in HzO, a method developed more fully in [ 1 ] so that a direct comparison can be made between the results of these two methods.