Infrared Analysis of Peptides and Proteins - American Chemical Society

Chapter 6

Determination of Secondary Structure in Protein Aggregates Using Attenuated Total Reflectance FTIR A. L. Fink, S. Seshadri, R. Khurana, and K. A. Oberg

Downloaded by MONASH UNIV on April 23, 2013 | http://pubs.acs.org Publication Date: December 3, 1999 | doi: 10.1021/bk-2000-0750.ch006

Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064

Protein aggregation is a major problem in many fields. Currently the mechanism of formation and the structure of protein aggregates are poorly understood. The insoluble nature of protein aggregates limits the number of techniques which can be used to ascertain the conformation of the aggregated protein. Attenuated total reflectance (ATR) FTIR is one method which can readily provide information about the secondary structure content of both soluble and insoluble proteins. ATR-FTIR was used to examine the structure of inclusion bodies, folding aggregates, amorphous precipitates and amyloid fibrils. A common feature of the aggregated proteins is the presence of additional β -structure compared to the native conformation.

Protein aggregates are a major problem, not only for many protein chemists performing basic research, but the aggregation of proteins can present significant technical and economic problems in the biotechnology and pharmaceutical industries, and lead to lethal and debilitating situations when present in the body in the form of a protein deposition disease. The intrinsic insoluble nature of protein deposits (as in amorphous aggregates, inclusion bodies, amyloid fibrils, etc.) places severe restrictions on the availability of methods for ascertaining the structure of the material. FTIR spectroscopy, especially in the attenuated total reflectance (ATR) mode, is well suited for determining structural features of proteins. Proteins in the form of solutions, thin films (hydrated or dry, from solutions or precipitates), solids (including lyophilized or spray-dried powders), or suspensions of precipitates (e. g. inclusion bodies, amyloid fibrils), can be used for ATR-FTIR analysis. After a brief discussion of some of the key features of protein aggregation, this chapter focuses on the use of ATR-FTIR in the determination of the structure of

132

© 2000 American Chemical Society

In Infrared Analysis of Peptides and Proteins; Singh, B.; ACS Symposium Series; American Chemical Society: Washington, DC, 1999.

133 protein aggregates, and in investigations to unravel the underlying molecular mechanisms of protein aggregation.


Protein Aggregation Protein aggregation is now recognized as a major problem in many fields and has become the focus of increasing research efforts (1-5). Substantial evidence is accumulating to support the hypothesis that the key precursors of protein aggregation are partially-folded intermediates, which may arise either during the folding of newly synthesized proteins, as with inclusion bodies, for example, or from the native state, as appears likely for at least some extracellular amyloid deposits (1,5-14), Circumstances that lead to the population of partially-folded intermediates, especially if their concentration is high, are thus likely to lead to aggregation; these include mutations, or environmental conditions, which produce differential destabilization of the native state relative to the partially-folded intermediate. Furthermore, the characteristics and properties of the intermediates may be significantly different from those of the native (and unfolded) conformation (15). Protein aggregation is conveniently classified into ordered and disordered deposits. Amyloid fibrils (both in vivo and in vitro) are examples of ordered aggregates, whereas inclusion bodies are examples of in vivo disordered aggregates. Corresponding disordered in vitro aggregates are folding aggregates, formed during the refolding of denaturant-unfolded protein at high protein concentrations, or under weakly native conditions at high protein concentration. Native, folded proteins may aggregate under certain conditions, most notably salting out and isoelectric precipitation. Such precipitates of native protein are readily distinguished from "pathological" aggregates by their solubility in buffer under native-like conditions. In contrast, "pathological" aggregates dissolve/dissociate only in the presence of high concentration of dénaturant or detergent. Using ATR-FTIR, we have shown that the native conformation is retained in "salting out" precipitates (Figure I ).

1700

1680

1660

1640

1620

1600

1

Wavenumber crrr

Figure 1. Second derivative spectrum of the amide I region of interleukin-2: the solid line is for the native protein the dotted line represents the spectrum for ammonium-sulfate-precipitated interleukin-2. In Infrared Analysis of Peptides and Proteins; Singh, B.; ACS Symposium Series; American Chemical Society: Washington, DC, 1999.


134 Inclusion bodies and other aggregates formed during protein folding have been assumed to arise from hydrophobic interactions of the unfolded or denatured states, whereas amyloid fibrils and other extracellular aggregates have been assumed to arise from native-like conformations in a process analogous to the polymerization of hemoglobin S (3). However, if aggregation arises from specific partially-folded intermediates, then one would expect that aggregation will be favored by factors and conditions that favor population of these intermediates. Indeed, this is usually found to be the case. Furthermore, the characteristics and properties of the intermediates may be significantly different from those of the native and unfolded conformations. There are many unanswered questions relating to protein aggregation, including details of the mechanism of aggregation and the underlying kinetics, the structure of the aggregates, the factors which discriminate between ordered aggregates (amyloid) and disordered aggregates (inclusion bodies, folding aggregates, amorphous deposits), the nature of the specific intermolecular interactions, and how aggregation may be prevented. Problems Due To Protein Aggregation. Several dozen protein deposition diseases are known (16). The most familiar include the amyloid diseases such as Alzheimer's disease, and prion diseases such as bovine spongiform encephalopathy (BSE - Mad Cow disease), and Creutzfeldt-Jacob disease (CJD) in humans). In both amyloid and prion diseases the aggregated protein is usually in the form of ordered fibrils. Amyloid fibril formation has been observed to arise from both peptides and proteins. Several protein deposition diseases involve non-ordered protein deposits, such as light-chain deposition disease and cataracts. A number of neurological diseases, e. g. Parkinson's disease, involve deposited proteins in the form of "inclusions". Classical inclusion body formation is very common when proteins are overexpressed, especially in bacteria; these inclusion bodies are usually highly homogeneous. Protein aggregation is also a problem in the storage or delivery of protein drugs, and in the lyophilization and rehydration of pharmaceutical proteins. Attenuated Total Reflectance (ATR) FTIR In ATR-FTIR, the sample is placed in contact with the surface of material having a high refractive index, known as the internal reflection element (IRE). The* IRE is usually made of germanium or zinc selenide. When infrared radiation penetrates the IRE at an angle beyond the critical angle of incidence, total internal reflection of this incident radiation produces an evanescent (standing) wave at the boundary between the IRE and the sample (Figure 2). This reflected beam penetrates beyond the crystal, and can be absorbed by materials in contact with its surface. The depth of penetration varies, depending on the material of the IRE and the wavelength. Since the IR beam is not transmitted through the sample, the spectra are unaffected by turbidity. Several different A T R cell designs are available; we favor out-ofcompartment, horizontal trapezoidal-shaped IREs, which may be used in either a trough or flow-cell configuration. ATR may be used with samples in solution (17) as a thin-film (starting with either solution or suspension), as a suspension or in the dry solid state. Interactions between the protein and the IRE surface (which may perturb the protein conformation) are minimal with suspensions of aggregated proteins. t



135

ATR has been used to analyze spectra of soluble and insoluble proteins and peptides (including inclusion bodies and fibrils) and for solid (powdered) proteins/peptides (13,18,19). Details of the A T R method have been published (13,17,20,21). The simplest way in which to study samples of aggregated proteins is as a thin film deposited from a suspension. There are few or no interfering bands from either solvents or dispersion media and the limited sample preparation avoids conditions which could affect the conformation of the material. Typically 50 to 100 μΐ of 0.5 to 1 mg/ml solution or suspension (i.e. 10-200 μg of protein) is placed on the IRE and the sample is dried down using nitrogen gas or dry air or a vacuum. The thin films have been shown to maintain protein molecules in a hydrated state and to conserve their 3D structure (17,22,23). The limited penetration depth in thin film ATR is a substantial advantage in that it minimizes the contribution of any liquid water present. A T R samples containing materials of high refractive index may result in significant shifts in the observed band positions, making comparisons between samples difficult. Our analysis is usually focused on the amide I region (1600-1700 cm" region, corresponding to C=0 stretch), which gives information about the secondary structure (17,24), although we also have used the amide II and III regions (13,17). Quantitative analysis of secondary structure is determined either from assignment of deconvoluted bands (following second derivative and Fourier self-deconvolution) or from partial least-squares multivariate analysis (17). The fine structure in the amide I band is most readily seen in the form of Fourier self-deconvoluted or second derivative spectra, or curve-fit component bands. 1

Data Analysis. Two general methods have been used to analyze the IR spectra of proteins to ascertain their secondary structure: deconvolution (resolution enhancement) and assignment of the component bands, or factor analysis, using pattern recognition methods. The fundamental difficulty encountered in the analysis of the amide I spectra arises from the fact that the widths of these component bands are usually greater than the separation between the maxima of adjacent peaks.


136

The preferred method to deconvolute the amide I region is as follows: first both FSD and second derivative spectra are run to determine the positions of the components. A robust analysis can only be assured if both methods give identical peak locations. Curve fitting can be done using the band positions derived from the FSD and second derivative spectra to fit the raw spectrum to a combination of GaussianLorentzian peaks. The area under each peak is then used to compute the percentage of the individual component contributing to the amide I region; it is assumed that the extinction coefficients are identical for each type of secondary structure, and consequently that component peak area is directly proportional to the fraction of secondary structure accounting for that component.


Partially-folded Intermediates As Aggregation Precursors Most partially-folded intermediates have a strong propensity to aggregate (25). In studies on partially-folded intermediates we have observed that their aggregation is usually accompanied by an increase in β-structure. For example, the helical apomyoglobin forms three distinct ensembles of partially-folded intermediates at low pH, A i , A , and A (15). The least structured of these intermediates shows half as much α-helix as the native state, and aggregates at protein concentrations above ~ 0.25 mg/ml as determined by dynamic light and small-angle X-ray scattering. Thinfilm ATR-FTIR spectra of the soluble aggregated intermediate show increasing amounts of β-structure as the protein concentration is increased (Figure 3). At higher protein concentrations the intermediate precipitates; the precipitated material also shows a large β-sheet component in the ATR-FTIR spectrum. The data clearly demonstrate that as the concentration of partially-folded intermediates increases the intermediates associate, initially to form soluble aggregates, and at higher protein concentrations to form insoluble precipitates, in which the aggregated material is substantially enriched in β-sheet or extended chain conformation. 2

3

1700

1680

1690

1640

1620

1600

Wavaiunbcrcnr

1

Figure 3. Increasing protein concentration leads to aggregation of an apomyoglobin partially-folded intermediate: the dotted line is the amide I spectrum of the monomeric intermediate, the solid line corresponds to the soluble aggregate: note the substantial increase in β-structure shown by the shoulder at 1627 cm' . 1


137


Aggregation Can Intermediates

Induce

Secondary

Structure

Into

Partially-Folded

Recently, in studies on partially-folded intermediates of staphylococcal nuclease (SNase) we have discovered a rather interesting phenomenon, namely that aggregation of a partially-folded intermediate may induce substantial additional secondary structure in the protein (26-27). Staphylococcal nuclease, like many proteins (25,28), forms partially-folded intermediates at low pH and low ionic strength (29). Under conditions of pH < 3.5, 100 mM sodium sulfate or 0.5 M K G , and low protein concentrations (

Infrared Analysis of Peptides and Proteins - American Chemical Society

Recommend Documents