J. Biol. Macromol., 9 (1) 23-30 (2009)

ArticleAmyloid fibrillation rate differs greatly among single-disulfide variants of hen lysozyme
Ryohei Kono1, Tetsuro Fujisawa2,3, Kazuyuki Akasaka1,4, Hideki Tachibana1,41Graduate School of Biology-Oriented Science and Technology, Kinki University, 930, Nishimitani,
Kinokawa, Wakayama 649-6493, Japan, 2Department of Biomolecular Science, Graduate School ofEngineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan, 3RIKEN SPring-8 center, 1-1-1 Kouto,
Sayo, Hyogo 679-5148, Japan, 4High Pressure Protein Research Center, Institute of Advanced Technology,
Kinki University, 930 Nishimitani, Kinokawa, Wakayama 649-6493, Japan.
Received February 2, 2009; Accepted February 9, 2009
Four molecular species of hen lysozyme single-disulfide variants, each retaining one of the four
native disulfide bonds (Cys6-Cys127, Cys30-Cys115, Cys64-Cys80, or Cys76-Cys94) spontaneously
form fibrils that are morphologically similar to the amyloid protofibril of hen lysozyme all-disulfidedeficient variant. The fibrillation rate of the single-disulfide variants, however, differs among eachother by as much as three orders of magnitude. Rapid-fibrillating species are those which havepreviously been shown to have submolecular structures comparatively stable and localized. The slowfibrillating species retains the Cys6-Cys127 disulfide bond, indicating that the N- and C-terminal
regions should be sterically separated from each other for efficient fibrillation to occur. The
proximity of N- and C-terminal regions imposed by the Cys6-Cys127 disulfide bond was also shown
to result in a fibril of a slightly decreased diameter.
Keywords: Hen Lysozyme; Disulfide bond; Amyloid fibril; Unfolded state
Introduction
Amyloid fibrillation is generally believed totake place via partially unfolded conformer ofprotein [1]. A comparative survey of amyloidogenic
conformation of globular proteins and that ofnatively unfolded proteins has shown that it isrelatively unfolded one and shares structuralproperties with a pre-molten globule state [2]. In
order to clarify the property of the amyloidogenic
conformation, it is helpful to study the fibrillation of
a set of protein variants with various extent of
intramolecular structures existing at different
peptide regions [3].
Formation of disulfide bond is generally
coupled with that of protein structure [4]. A seriesof disulfide variants of hen lysozyme have been
constructed and their intramolecular structures werecharacterized [5-8]. Among these, the 0SS variant,which lacks all the four disulfide bonds and isnearly unfolded under native solution
Correspondence to Hideki Tachibana.
Tel & Fax: +81-736-77-0345 ext.4110
E-mail: tachi887@waka.kindai.ac.jp

conditions, has been shown to spontaneously form
amyloid fibrils [9].
Here, four molecular species of singledisulfide variants, 1SS, each having one of the four
native disulfide bonds in hen lysozyme, Cys6Cys127, Cys30-Cys115, Cys64-Cys80, and Cys76Cys94 (Fig. 1), were employed to find how
individual native disulfide bonds and accompanying
submolecular structures affect the fibrillation
process. All the four 1SS variants are relatively
unfolded, belonging to the pre-molten globule state[2], and have only unstable submolecular structuresof differing size and stability [6]. The results show
highly differential fibrillation kinetics, acceleration
or deceleration altogether by three orders ofmagnitude, among the four 1SS variants, and aslight change in the fibril
The abbreviations used are: 0SS, hen lysozyme
variant which lacks all the four disulfide bonds;
1SS (6-127) ((30-115), (64-80), (76-94)), henlysozyme single-disulfide variant which retains thedisulfide bond Cys6-Cys127 (Cys30-Cys115,
Cys64-Cys80, Cys76-Cys94, respectively); β2m,
β2-microglobulin

24

Amyloid fibrillation of lysozyme single-disulfide variants

structure of the slow-fibrillating 1SS variant.

Fig. 1. Disulfide bridging patterns in the four 1SS
variants. C, A, and S denote cysteinyl, alanyl, andseryl residues, respectively. Thin lines indicate
disulfide bridging between cysteinyl residues. WT
hen lysozyme and 0SS are also shown for reference.
The Cys6-Cys127 bond connects extreme N- andC-terminal regions; Cys30-Cys115 resides in the αdomain in the native structure of WT lysozyme andconnects α-helices B (αB) and D (αD); Cys64Cys80 resides in the β-domain that contains
β1, β2, and β3 strands; Cys76-Cys94 is located at
the interface between the α- and β-domains.
Materials and Methods
Hen lysozyme 1SS variants were produced in
E. coli and chromatographically purified as
described previously [6,10]. For fibril formation,
freeze-dried 0SS powder was firstly dissolved inpure water. Then, the solution was mixed rapidly
with buffer and salt solutions to a desired final
composition to start the fibrillation reaction. Atomic
force microscopy (AFM) observations were carriedout on an SPI-3800 scanning probe microscope(Seiko Instruments Inc.) under a cyclic contact
mode as described [11]. High-performance size

exclusion chromatography (SEC) was carried out
on a TSK GSW column (7.5 mm i.d. × 7.5 cm,
Tosoh; a nominal Mr exclusion limit of 1 × 105)with an elution solution of 20 mM sodium acetate,
pH 4.0, 10 mM sodium sulfate used at a flow rateof 1.0 ml/min. Far-UV CD spectra were taken on a
J-820 spectropolarimeter (Japan Specroscopic) witha cell of 0.1-mm optical path length temperaturecontrolled at 25 °C as described [12]. Small-angle
X-ray Scattering (SAXS) measurements of the
fibril solutions were carried out at RIKEN
structural biology beamline I (BL45XU) at SPring8 in Harima, Japan, with X-ray beam of 0.9-Å
wavelength and a camera length of 2245 mm and atypical exposure time of 1 sec with a temperaturecontrolled (25 °C) cell of 50 µL capacity [13].
Results
Single-disulfide variants form amyloid-like fibrils
morphologically similar to the 0SS fibril
The all-disulfide-deficient variant of hen
lysozyme, 0SS, spontaneously forms amyloid fibrils
under mildly acidic conditions [9]. The 0SS fibril isabout 2 nm in height when observed under atomic
force microscopy, and has a nodular and winding
morphology typical of amyloid protofibrils [11].
Here, we found that four molecular species of hen
lysozyme 1SS variants also formed fibrils
spontaneously (Fig. 2). Morphologies of the 1SS
fibrils as observed under AFM were similar to eachother as well as to that of 0SS fibril, and theirheight was about 2 nm in common. In close
inspection, however, minor differences existed suchas frequent occurrence of ring forms in 1SS (30115) and short assemblies in 1SS (64-80).

Fig. 2. AFM images of the fibrils of the 1SS-variants and 0SS. Fibrils were formed by incubating 5 mg ml–1
each variant protein in 20 mM sodium acetate, pH 4.0, 50 mM NaCl at 25 °C for four days (1SS (6-127)) or
one day (1SS (30-115), 1SS (64-80), 1SS (76-94), and 0SS). All images are 1 µm × 1 µm in an observation
window size.
Fibrillation kinetics varies greatly among the four
The fibrillation kinetics of each 1SS variant
1SS variants
was monitored with high-performance SEC (Fig. 3).

R. Kono et al.

The 1SS variants, initially existed in their monomer
states and eluted at the total bed volume of the SEC
column, spontaneously self-associated into polymer
forms, which eluted early around the void volume of
the column, well-separated from the monomer peak.
The progress of the association was quite different
among the 1SS variants. It took tens of hours in one
variant, while minutes in some others (Fig. 4). The

25

apparent association rate constant (Table 1)
differed widely by more than three orders ofmagnitude among the four 1SS variants: High in
1SS (30-115) and 1SS (64-80), intermediate in 1SS(76-94), which was similar to 0SS control (apparent
association rate constant of 1.3 hr–1), and very low
in 1SS (6-127).

Fig. 3. Changes in SEC elution profiles of the 1SS variants with the progress of fibrillation. Each 1SS
solution, 4 mg ml–1 in 20 mM sodium acetate, pH 4.0, 50 mM NaCl, was incubated at 25 °C. At each
incubation period labeled to each profile aliquot of the solution containing ten µg protein was subjected to
SEC. P and M denote polymer and monomer peaks, respectively.

Amyloid fibrillation of lysozyme single-disulfide variants

26

Fig. 4. Time course of the increase in polymer fraction in each 1SS fibrillation reaction. The fractionalamount of polymer and that of monomer were quantified from the area of the elution peaks shown in Fig. 3:
1SS (6-127) (circle), 1SS (30-115) (up triangle), 1SS (64-80) (down triangle), 1SS (76-94) (square). As acontrol, those for 0SS (filled circle) were included. Apparent association rate constant was obtained with aregression of the time course to a single-exponential function of time (full lines for 1SS variants, and a dottedline for 0SS). Data points at up to 168 hrs (inset) were included in the regression for 1SS (6-127).
Table 1. Fibrillation properties and thermodynamic parameters of the 1SS variants.
Variant

Apparent association rate
constant(hr-1)

Fibril diametera(nm)

Intrinsic stability of
submolecular structureb(kJ mol-1)

1SS (6-127)0.010

6.9 ± 0.2

−17.9

1SS (30-115)

178.2 ± 0.2

−16.6

1SS (64-80)

147.8 ± 0.1

−15.2

1SS (76-94)

1.0

8.0 ± 0.1

−18.7

Diameter = 2 × 2 × (Cross-sectional radius of gyration at infinite dilution).
Mean and SD for three fibril samples are shown.
b
Taken from ref [6].
aThe association of each of the 1SS variants
was accompanied by β-structure formation: Themean residue ellipticity at 217 nm deepened from
−7,000 ~ −8,000 deg cm2 dmol-1 in the monomericstate to −12,000 ~ −15,000 deg cm2 dmol-1 in thepolymeric state (not shown). The diversity in the
kinetic rate of β-structure formation among the four

1SS variants closely paralleled with that in the rateof the increase in the polymer fraction as observedin SEC: rapid in 1SS (30-115) and 1SS (64-80),
and very slow in 1SS (6-127).
Slow-fibrillating 1SS variant has a slightly
decreased fibril diameter

R. Kono et al.

It is frequently noted that the width of fibrils
as seen under AFM does not necessarily represent
the actual diameter of the fibril in solution becauseof the mechanism of scanning-probing itself.
Instead of the width, the height under AFM is
usually taken to represent the diameter of the fibril.
All the four 1SS variants and 0SS employed here
showed a fibril height of about 2 nm in common
under AFM. However, the highly differential
fibrillation rate observed among the 1SS variants
prompted us to investigate possible differences infibril morphology in high resolution with SAXS
measurements (Fig. 5).

Fig. 5. Cross-sectional Guinier plots of SAXS
profile of the 1SS variant fibrils. Cross-sectional
radius of gyration of fibril at infinite dilution is
obtained from the slope of the cross-sectional
Guinier plots, ln(S × I(S)/C) vs S2, by using data
points in the region, 20 × 10-6 A2 < S2 < 55 × 10-6
A2 , shown in dotted lines: S, momentum transfer(=2(sin(θ))/λ, where 2θ is a scattering angle and λthe wavelength); I(S), scattering intensity; C,protein weight concentration. The plots weredisplaced along the ordinate for clarity. Plotsymbols are the same as in Fig. 4. There was noclear protein concentration dependency in I(S)/C.
Fibrils were formed by incubating 5 mg ml–1 protein
in 20 mM sodium acetate, pH 4.0, 50 mM sodium
chloride at 25°C for 75 days.
Information about fibril radii can be obtained
from cross-sectional Guinier plots of SAXS
intensity [14]. A slightly shallow slope of the plotfor 1SS (6-127) fibril as compared to those of theother three 1SS variants indicates that the fibril
diameter is decreased specifically for this variant.
This is not due to the comparatively high amount of
unassociated monomers of 1SS (6-127) coexistingwith its fibrils (Fig. 3), since 1SS (6-127) fibril

27

solutions of different incubation periods (3 and 75days), and therefore of different amounts ofcoexisting monomers, gave the decreased value ofthe cross-sectional radii that are closely similar toeach other, which is rationalized from much higher
contribution to scattering of high-molecular weight
fibrils compared to monomers. Quantitatively(Table 1), while the other three 1SS fibrils has adiameter of about 8 nm in common, 1SS (6-127)
has a slightly decreased diameter of about 7 nm.
We note that both of these values are quite larger
than the value, 2 nm, observed as a height of fibrils
under AFM. The fibrils may be so flexible as to be
flattened under AFM.
Discussion
Intricate effects of single disulfide bondincorporation on the fibrillation of lysozyme
The present study showed highly differential
effect of the incorporation of different single
disulfide bonds on the formation of protofibrils of
the intrinsically unfolded hen lysozyme alldisulfide-deficient variant. Although the effect waslarge on the apparent association rate, all the fourlysozyme 1SS variants fibrillate spontaneously tofibrils of nearly the same morphology in low
resolution. In this respect, the effects were not so
profound compared with those reported for β2microglobulin (β2m) [15,16], which contains asingle disulfide bond in its native form. The fibril of
β2m single-disulfide form is thick, tight andstraight, quite different from the thin, curledfilaments of the disulfide-absent (reduced) protein,although the morphology changes with pH and saltconcentration as well [17-19]. The less profoundeffect observed here for lysozyme is probably due
to the fact that all the four lysozyme 1SS variants
only contain unstable submolecular structures [6],close in structural level to intrinsically unfolded
0SS and distant from the fully structured 4SS native
form. Interestingly, WT lysozyme is known to form
thick and straight fibrils under harsh solution
conditions that lead partial proteolysis or under theaid of seeding [20,21], similar to β2m WT 1SS
form. Thus, the present 0SS to 1SS variant systemof lysozyme, and possibly 2SS and 3SS variant
systems to be undertaken in future, are suited toinvestigate intricate mechanistic details of the effect28

Amyloid fibrillation of lysozyme single-disulfide variantsof the introduction of disulfide bonds on fibrillation
at various levels of protein structures.
Relationship between the fibrillation efficiency andthe stability of submolecular structures
The present study has shown that 1SS (30115) and 1SS (64-80) fibrillates rapidly while 1SS(6-127) very slowly, compared with 0SS
fibrillation. Simple interpretation of the kinetic
result is that in the transition state between
monomeric and fibrillar states of 0SS, residue 30 issterically near to residue 115, residue 64 is also
near to residue 80, but residue 6 is at a distancefrom residue 127. In energetic terms, however, thepresent finding that two of the four 1SS variants
fibrillate faster than 0SS is surprising since
introduction of native disulfide bonds is thought tostabilize native structures and therefore disfavor
amyloid fibrillation, which is generally thought toinvolve misfolded structures.
We have previously analyzed the denaturantconcentration dependence of an effective
concentration of protein cysteinyl thiol groups thatparticipate in each native disulfide bond, and shownthat only local and unstable submolecular structures
exist in all of the four lysozyme 1SS variants [6].
When the thermodynamic parameter values
obtained previously for each 1SS variant arecompared with the rate of amyloid fibrillation
obtained here, we clearly see that the rapidfibrillating species have comparatively highintrinsic stability of submolecular structures: 1SS(30-115) and 1SS (64-80) rank second and first,
respectively, in the stability. (Note that the stabilityof the submolecular structures in Table 1 is shownwith the unfolding free energy change: less negativevalues indicate higher stability) On the other hand,
the moderate- and slow-fibrillating species, 1SS(76-94) and 1SS (6-127), rank fourth and third,
respectively, in the intrinsic stability. Thus,
relatively stable protein submolecular structures
which are formed coupled with native disulfidebond formation are able to enhance the fibrillation.
It is interesting that the two disulfide bonds, Cys30Cys115 and Cys64-Cys80, that enhance fibrillation,
have also been shown to be important for theformation of native secondary structures spatiallyclose to them [8]. Thus, it appears that starting froma completely unfolded state, submolecular structureformation proceeds to a certain structural level in
which
both
monomolecularfolding
andintermolecular fibrillation are favored.
Separation between N- and C-terminal regions isnecessary for efficient fibrillation
The fibrillation is decelerated in 1SS (6-127)
by two orders of magnitude, while it is accelerated
in 1SS (30-115) by one order of magnitude.
Considering the previous results that the 2SS
variant containing both of these two disulfide bonds
establishes the native tertiary structure similar tothat of the α-domain of WT lysozyme [7,22], themutually opposing effect observed here on
fibrillation of these two disulfide bonds isremarkable, and emphasizes the differential
mechanism between the native folding and amyloid
fibrillation of lysozyme. The extremely slow
fibrillation of 1SS (6-127) cannot be explained with
its relatively low intrinsic stability alone. It is mostprobably due to the fact that it has the largest sizeof substructure among the 1SS variants [6]. Such alarge structure should be on the pathway to lead
global native folding and therefore depresses
fibrillation. The inhibitory role of Cys6-Cys127
disulfide bond, which connects N- and C-terminal
regions of lysozyme, is reminiscent of the role of Nand C-terminal β-strands of β2m that protect native
folding against fibrillation [23]. The depressed
fibrillation and the slight but significant decrease in
the fibril diameter specifically observed in 1SS (6127) indicate that N- and C-terminal regions aresterically separated from each other in a naturalform 0SS fibrils. The imposed proximity of N- andC-terminal regions in 1SS (6-127) on the one handstrongly decelerates fibrillation, and on the other
hand forces protein peptide segments to change
their steric arrangement in fibrils.
It may be argued that the extremely slow
fibrillation of 1SS (6-127) is related with a possibly
long nucleation period involving a nearly first-order
reaction of conformational (or covalent-bond)change of protein as a rate-determining step. In sucha case, the length of a lag period and the overall
association rate will not strongly depend on the total
protein concentration, as actually observed for the
fibrillation of WT lysozyme [24]. We repeated thesame sort of fibrillation reaction as shown in Fig. 3
at various 1SS variant concentrations. The

R. Kono et al.

association rate did strongly increase in powers of
the total protein concentration with an exponent of
two to three (not shown). The linear dependence oflogarithm of the association rate on that of total
protein concentration observed for all the four 1SS
variants and 0SS conforms to an existing theory of“nucleation-elongation”
type
polymerization
reaction [25]. Thus, there does not exist serious
difference in the spontaneous nucleation mechanism
among the 1SS variants. The high variation in theapparent association rate among them derives fromthe difference in the intrinsic rate for the elementary
step of binding of monomers to polymer ends anddevelopment of intermolecular β-structure.
Acknowledgements
We thank Mr. Hiroki Shimoura for his help in
SEC experiments. This work was supported by
Grant-in-Aid for Scientific Research 17570132 andby the Academic Frontier Program from the
Ministry of Education, Culture, Sports, Science,
and Technology of Japan.
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Communicated by Masayuki Oda