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Protein Fiber Linear Dichroism for Structure Determination and Kinetics in a Low-Volume, Low-Wavelength Couette Flow Cell

Timothy R. Dafforn ^*, Jacindra Rajendra , David J. Halsall , Louise C. Serpell  ^¶ and Alison Rodger 

^* Department of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom; Department of Chemistry, University of Warwick, Warwick CV4 7AL, United Kingdom; Department of Clinical Biochemistry and Department of Haematology, University of Cambridge, Cambridge Institute of Medical Research, Cambridge CB2 2XY, United Kingdom; and ^¶ Neurobiology Division, Medical Research Council Centre, Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom

Correspondence: Address reprint requests to Timothy R. Dafforn, Department of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK. E-mail: tim.dafforn{at}hotmail.com.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSION ACKNOWLEDGEMENTS REFERENCES

 
High-resolution structure determination of soluble globular proteins relies heavily on x-ray crystallography techniques. Such an approach is often ineffective for investigations into the structure of fibrous proteins as these proteins generally do not crystallize. Thus investigations into fibrous protein structure have relied on less direct methods such as x-ray fiber diffraction and circular dichroism. Ultraviolet linear dichroism has the potential to provide additional information on the structure of such biomolecular systems. However, existing systems are not optimized for the requirements of fibrous proteins. We have designed and built a low-volume (200 µL), low-wavelength (down to 180 nm), low-pathlength (100 µm), high-alignment flow-alignment system (couette) to perform ultraviolet linear dichroism studies on the fibers formed by a range of biomolecules. The apparatus has been tested using a number of proteins for which longer wavelength linear dichroism spectra had already been measured. The new couette cell has also been used to obtain data on two medically important protein fibers, the all-ß-sheet amyloid fibers of the Alzheimer's derived protein Aß and the long-chain assemblies of ₁-antitrypsin polymers.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSION ACKNOWLEDGEMENTS REFERENCES

 
Proteins are classified by their structural characteristics to be either globular, membrane bound, or fibrous. Globular proteins are usually highly aqueous soluble whereas membrane proteins are lipid soluble. They provide the majority of the enzymatic, regulatory, and signaling machinery required to maintain a cell. Fibrous proteins in general behave as structural elements both within cells forming the cytoskeleton or in the extracellular environment such as the basement membrane. Fibrous proteins have a range of mechanical properties tailored to their function. Collagen, for example, has, weight for weight, a tensile strength that exceeds that of steel. It is found in connective tissue where such strength is required to maintain tissue integrity under extreme loads (Fratzl et al., 1998; Prockop and Fertala, 1998). The high tensile strength of silk is used by insects for tasks as diverse as catching prey to protection of larval forms. Actin (Otterbein et al., 2001) and myosin, by way of contrast, combine in muscle fibers and perform complex ratchet-like conformational changes that underlie muscle contractions. (Geeves and Holmes, 1999). All these proteins have amino acid sequences that have evolved to form fibers of the correct characteristics. However, in some cases, normally soluble, globular proteins undergo considerable conformational change and polymerize to form ß-sheet dominated fibrils. These fibrils are deposited in the tissues leading to diseases collectively known as amyloidoses. Each disease is characterized by protein being deposited as amyloid fibrils. At least 20 different proteins are associated with deposition of such ß-sheet fibrils. These include Aß in Alzheimer's disease, islet amyloid polypeptide in some Type 2 diabetes patients, and prion in the transmissible spongiform encephalopathies. Parkinson's disease and Huntington's disease also involve the deposition of ß-sheet fibrils intracellularly. The proteins that form amyloid fibrils do not share any similarities in their native nonfibrillar structure, sequence, or function. Proteins as diverse as lysozyme, immunoglobulin light chain, and apolipoprotein A1 form fibrils in diseased states. The amyloid fibrils formed, however, share morphology and a common core structure (Sunde et al., 1997).

Fibrous proteins constitute the major portion of biomass in the natural world. However, our understanding of the conformations of the polypeptide chains that make up these fibers is limited. This is particularly apparent when compared to our knowledge of globular proteins. It is usually not possible to study the structure of fibrous proteins with conventional techniques used for globular proteins such as single crystal x-ray crystallography or NMR, since fibers are often both insoluble and heterogeneous in length. Instead, electron microscopy (EM) and x-ray fiber diffraction have been utilized to examine the morphology and appearance of the fibers, as well as any repeating structure.

In some cases it has been possible to combine the crystallographic information from a soluble form of a fiber-forming protein with fiber diffraction from a fibrous form (e.g., (Holmes et al., 1990; Rayment et al., 1993) to deduce structural information. The secondary structural elements making up fibers have also been probed using circular dichroism (CD), Fourier transform infrared spectroscopy and solid state NMR. To build up a model of the fibrous protein being studied, several of these techniques are often combined to give information on the spacing of individual elements within the fiber, and in some cases they can be combined to gain medium resolution structural information. As the structural study of protein fibers cannot rely on one technique to provide a complete description of the structure, it is all the more important to continue developing new techniques that give unique information on the fibers' construction.

One technique that has the potential to provide information on the structural arrangement of secondary structural elements within the fiber that is not obtainable by the methods so far mentioned is ultraviolet (UV) flow-oriented linear dichroism (LD). The LD signal is produced by measuring the difference in absorbance of linearly polarized light parallel and perpendicular to an orientation direction. LD signals can be positive and negative, positive signals being for transitions whose polarization is along the direction of orientation and negative for those perpendicular to it. These measurements are usually made using a CD spectropolarimeter adapted to produce linear polarized light for LD as opposed to circularly polarized light for CD.

LD has been used extensively to examine the structure of DNA, and in this case, the signal from the -^* transitions of the DNA bases is negative as the bases lie more perpendicular than parallel to the DNA helix axis. The use of LD to study protein structure (rather than, e.g., protein DNA complexes) has been limited to early work of Miki and Mihashi (1976), who investigated the signal of nucleotide bound to F-actin, work of Nordh and Nordén (1986) on tubulin, our work on the LD of peptide fibers (Pandya et al., 2000) and our recent work on the LD of proteins bound to liposomes. (Rodger et al., 2002a,b) The early measurements of actin and tubulin LD were restricted to the aromatic regions of the spectrum due to cell and instrument constraints. The LD of the far-UV region where one probes the amide n-^* (220 nm) and -^* transitions (208 nm only for the -helix and 195 nm for all motifs) has the possibility, like CD, to provide information on the backbone structure of proteins. In particular, LD has the potential to provide detailed information on the orientation of secondary structural elements with respect to the fiber axis. This information has the potential to fill an important gap in the puzzle that surrounds the structures of many fibrous proteins. This was illustrated by our previous work on the n-^* and the tail of the -^* transitions in the peptide fibers, which confirmed that the -helices ran parallel to the fiber axis. (Pandya et al., 2000) The potential importance of the use of linear dichroism in the study of the conformation of protein fibers has not been realized due to a number of factors including: the limited availability of couette flow cells or other effective orientation equipment; the limited wavelength range of published spectra, which imply that the backbone LD is not really accessible; and the substantial sample requirements of the previously available flow cells. However, our work on -helical peptide fibers does show the attraction of LD as a technique to study fibers: it gives information that is not readily available by other techniques, making development of an LD cell with characteristics required by biochemistry important.

In practice the most challenging aspect of LD experiments is the fact that the molecules in the sample must be aligned. This has been achieved in the past for proteins using a range of alignment methods, including magnetic fields, stretched films, squeezed gels, and shear flow (Rodger, 1993). We chose to use couette flow as we wished to collect data to as low wavelengths as possible. We present here data on a range of protein fibers that we have obtained with our recently constructed 50 µm annular gap calcium fluoride optics couette flow cell that is illustrated in Fig. 1. To interpret the LD spectra, it is essential to have at least approximate assignments of transition polarizations. Fig. 2 summarized the approximate transition polarizations of the chromophores relevant to the systems studied in this article.

View larger version (25K): [in this window] [in a new window]   FIGURE 1  Schematic of couette flow LD and picture of the 50 µM annual gap CaF2 couette flow cell.

View larger version (18K): [in this window] [in a new window]   FIGURE 2  The orientations of the various polarization moments in (a) -helix, (b) ß-sheet, (c) poly proline type II, (d) tryptophan, (e) tyrosine, and (f) adenosine chromophores. Arrows indicate transition moment polarizations, and the approximate wavelength maxima of the transitions is indicated near the arrows. In some cases, the common notation for the transition is also given. Wavelengths in parentheses indicate the intensity of this transition is weak.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSION ACKNOWLEDGEMENTS REFERENCES

Human plasma ₁-antitrypsin was incubated at 60°C for 3 h in 50 mM TrisHCl 100 mM KCl pH 7.6 to produce polymers (Dafforn et al., 1999; Sivasothy et al., 2000; Lomas et al., 1992). Residual monomeric ₁-antitrypsin was then removed by incubation at 95°C for 1 h.

Synthetic Aß_1-42 peptides were obtained from Bachem, St. Helens, UK, and incubated for several months in water at 10 mg/mL at 4°C to produce Aß_1-42 fibrils.

Acid soluble type I collagen was dissolved initially in 100 mM acetic acid. Any insoluble residue was removed by centrifugation at 16,000 x g for 15 min. The soluble fraction was then dialyzed against 100 mM phosphate buffer at pH 7.4 for 24 h at 4°C.

Spectroscopic experiments
CD measurements were performed using either a Jasco (Tokyo, Japan) J-810 or J-715 spectropolarimeter in 50 mM phosphate buffer pH 7.4 at 20°C using a 0.05 cm pathlength quartz cuvette and averaged over five scans of response time = 1 s.

LD measurements were performed using a Jasco J-715 spectropolarimeter adapted for LD spectroscopy. Samples were placed in 50 mM phosphate buffer pH 7.4 at 20°C and aligned in the light beam using custom-made couette cells (Rodger et al., 2002a,b). The cell (Fig. 1) consists of a cylindrical cross section sleeve with a cylinder mounted centrally with respect to its circular face on a rotating spindle within the sleeve. The spindle center and the center of the circular cross section of the sleeve are aligned so the cylinder is able to rotate freely within the sleeve. The gap formed between the cylinder and the sleeve is filled with the sample, and upon rotation of the inner cylinder a shear force is induced across the sample. This configuration allows the use of smaller sample volumes (200 µl) than has previously been possible (the previous cell required 2 ml of sample). The light beam is incident radially on the cell and the ones used in this work have two windows on the outer cylinder for the light to pass through. The protein data presented in this paper were all collected with the couette flow cell described in Rodger et al. (2002b). This cell, which is illustrated in Fig. 1, was constructed by Crystal Precision Optics (Rugby, UK). The central rotating cylinder and the entry and exit windows are made of calcium fluoride. This should extend the cell wavelength further into the UV than is obtainable with quartz. Further, the annular gap between the rotating cylinder and windows (see schematic on Fig. 1) is 50 µm (one-tenth that of existing cells (Rodger, 1993)), which reduces the concentration of nonanalyte molecules (e.g., buffers, Cl^-) in the light beam. Comparison of this cell with previous, long-pathlength cells shows that the increase in the shear force across the 50 µm annular gap compared with the 500 µm annular gap counteracts the expected Beer-Lambert loss of signal due to smaller pathlength and less sample being in the light beam (data not shown). A combination of smaller "wasted" sample volume below the windows and the smaller annular gap reduce the required sample volume by approximately an order of magnitude in the CaF₂ cell, which is attractive for studies of biomacromolecules. The voltage applied to the motor that rotates the spindle and hence the CaF₂ cylinder is controlled electronically to allow the sample solution to be maintained with the highest possible degree of alignment without inducing turbulent flow and Taylor vortices.

The shear rate was determined by applying the equations from Nordén et al. (1991) which are based on the work of Taylor (1963). If the inner cylinder rotates:

(1)

where is the angular velocity (d/dt) and R_i and R_o are the radii of the inner and outer cylinders respectively. This approximates to

(2)

If = 1000 rpm or alternatively 1000 x 2/60 = 105 s^-1; R_o = 1.5 cm, 500 µm = 0.05 cm annular gap makes G 3000 s^-1.

All spectra were calculated as an average of five measurements.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSION ACKNOWLEDGEMENTS REFERENCES

 
The 50 µm CaF₂ couette cell was used to measure the LD spectra of four fibrous proteins: collagen type 1; actin, for which LD data exists in the literature thus allowing direct validation of the new cell against older systems; the Alzheimer's related protein Aß_1-42; and ₁-antitrypsin. Before the LD spectrum of each sample was recorded, each was subjected to analysis by CD (Fig. 3). The samples were also examined using negative stain electron microscopy (data not shown). These techniques when combined are able to determine whether the sample is fibrillar and hence amenable to alignment, and also to confirm that the secondary structural content of each fiber is as expected from the literature (see Table 1).

View larger version (21K): [in this window] [in a new window]   FIGURE 3  CD spectra of (a) collagen type I (0.75 mg/ml), (b) F-actin (0.75 mg/ml), (c) Aß1-42 (0.32 mg/ml), and (d) 1-antitrypsin (0.72 mg/ml) in a 0.5 mm pathlength cuvette.

View larger version (24K): [in this window] [in a new window]   FIGURE 4  LD spectra of the backbone region of (a) collagen type I (1.2 mg/ml), (b) F-actin (0.05mg/ml) with insert of the aromatic region collected at 0.31mg/ml the concentration, (c) Aß1-42 (0.32mg/ml), and (d) 1-antitrypsin (1.2 mg/ml) in the 50 µm CaF2 couette cell.

The significant number of aromatic residues in F-actin (F actin: 1.1% Trp, 3.2% Phe, 4.2% Tyr) as well as the bound ADP (1 molecule per actin subunit), all have the potential to produce an LD signal in the "aromatic region". The LD spectrum for F-actin, shown as an inset in Fig. 4 b, has significant features in the aromatic region between 250 and 300 nm in accord with the literature (Miki and Mihashi, 1976). The two positive maximum at 285 and 290 nm are due to the ADP (all polymerized actin should have ADP bound; nonpolymerized is probably ATP), which must be oriented with its plane more parallel than perpendicular to the fiber axis, whereas the 295 nm and 265 nm negative maxima correspond to the tryptophans on average being more perpendicular to the fiber axis.

Actin polymerization monitored by LD
The observation of a significant LD signal for F-actin at 205 nm in the 50 µm CaF₂ LD cell allowed us to follow the polymerization of actin. G-actin polymerization was triggered by the addition of KCl (100 mM) and MgCl₂ (2 mM). The kinetics plot obtained (Fig. 5) shows an increase in LD signal over a period of 500 s that conformed to initial lag followed by an increase to saturation. This sort of trace is consistent with those observed by other methods (Millonig et al., 1988; Nishida and Sakai, 1983), and shows the potential of LD for probing fiber formation kinetics—the real advantage of LD being that it only gives a signal for the fibers.

View larger version (18K): [in this window] [in a new window]   FIGURE 5  Polymerization of actin (0.3 mg/ml) followed by monitoring the LD signal at 205 nm for 500 s. Polymerization was induced by addition of KCl and MgCl2 to a concentration of 100 mM and 2 mM, respectively, at t = 0 s. At t = 500 s, an excess of KCl and MgCl2 was added (with resulting dilution of the actin and hence immediate decrease of the LD signal) to ensure that the reaction had gone to completion.

Aß_1-24 and ₁-antitrypsin

A CD spectrum of Aß_1-42 shows a classical ß-sheet CD spectrum (Fig. 3) with a negative maximum at 216 nm and a positive maximum at 197 nm. This is in agreement with previous structural studies (Serpell, 2000) that show that these fibers contain predominantly ß-structure. Visualization of the samples by EM shows the presence of ordered fibers with an average width of 80 Å. Amyloid fibers typically have a cross ß-x-ray fiber diffraction pattern that indicates the ß strands run perpendicular to the fiber axis.

The LD spectrum of Aß_1-42 (Fig. 4) is dominated by a single strong positive maximum at 205 nm with very low signal intensity in the n-^* region of the spectrum (220 nm) that may be due exclusively to background scattering. No evidence for an LD signal within the aromatic region (250–300 nm) was observed despite almost 10% of residues within the Aß_1-42 being either tyrosines or phenylalanines. This indicates that the conformations of the aromatic residues are either not ordered with respect to the ß-sheet structure or average to close to the magic angle of 54.7° (Rodger and Nordén, 1997), information that had not previously been available.

X-ray crystal structures of the monomeric form of ₁-antitrypsin show it to have a mixed /ß-structure; this is confirmed to be the case in the polymeric form by observation of an /ß-CD spectrum (Fig. 3). The corresponding LD spectrum (Fig. 4) shows a negative signal at 220 nm and below. The spectrum is qualitatively similar in the backbone region but opposite in sign from that of F-actin, which is also a mixed /ß-protein. The signal-to-noise is much worse, suggesting shorter or less rigid fibers have been formed; this is confirmed by EM studies of the polymers that show them to take the form of "beads on a string" rather than the rigid rods of actin. The schematics of Fig. 6 indicating the orientations of the -helices and ß-strands in these two proteins are consistent with these opposite spectra. In both cases, the ß-strands are a mix of orientations. The near UV part of the LD spectrum of ₁-antitrypsin shows a single negative minima at 258 nm, which is likely to correspond to the relatively high proportion of phenylalanines (₁-antitrypsin: 0.7% Trp, 6.5% Phe, 1.4% Tyr) in the protein being well-aligned with respect to one another so that the short axis of the phenol group is perpendicular to the fiber axis.

View larger version (58K): [in this window] [in a new window]   FIGURE 6  Stereo diagrams of positions of secondary structure motifs in (a) F-actin and (b) -antitrypsin fibers. In each case, the coils of the top schematic show -helices and the ribbons of the bottom schematic show ß-sheets.

	CONCLUSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSION ACKNOWLEDGEMENTS REFERENCES

Some general structure/LD rules can be summarized from our data:

1. Circular dichroism gives a useful handle on the percentages of secondary structure motifs in the fiber.
   
2. The aromatic region is useful if there is a detectable signal, and where different chromophores have different absorbance wavelengths it should be possible to analyze the data quantitatively to determine their relative orientations. A matrix method-type approach would be most useful (Bayley et al., 1969; Besley and Hirst, 1999).
   
3. The 220 nm LD is only significant for -helices, and a negative signal means the average -helix orientation is more parallel than perpendicular to the fiber axis.
   
4. For the -helix, a dip in the spectrum is usually observed at 210 nm with a sign opposite from the 220 nm n-^* signal. This is the long wavelength -^* component that is unique to the -helix. Below 200 nm, a signal of the same sign as the n-^* transition is expected (cf. Fig. 2).
   
5. The 195-200 nm region is dominated by -^* transitions. For poly proline type II motifs, one expects a signal polarized parallel to the helix axis (which in the case of collagen is parallel to the fiber axis, so positive in sign) above 200 nm and one of the opposite sign below 200 nm.
   
6. ß-sheets have one significant band at 200 nm. If it is positive in sign, then the peptide backbone of the ß-strands are more perpendicular than parallel to the fiber axis (cf. Fig. 2).
   
7. Mixed /ß structures are dominated by the -helix LD down to 210 nm. The ß-sheet is probably dominant below this, leading to signals at a slightly longer wavelength than the -helix's lower -^* component.
   

	ACKNOWLEDGEMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSION ACKNOWLEDGEMENTS REFERENCES

 
T.D. is a Medical Research Council Career Development Fellow. L.S. is a Wellcome Trust Research Career Development Fellow and thanks the Laboratory of Molecular Biology (Cambridge) for the use of the electron microscope.

A.R. is funded by the Engineering and Physical Sciences Research Council (GR/M91105 and GR/R40869/01).

Submitted on June 2, 2003; accepted for publication August 21, 2003.

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