The first group showed a thermodynamic and kinetic stability similar to wild-type ribonuclease A, whereas both stabilities of the variants in the second group were greatly decreased, sugg
Trang 1Probing the unfolding region of ribonuclease A by site-directed
mutagenesis
Jens Ko¨ditz, Renate Ulbrich-Hofmann and Ulrich Arnold
Department of Biochemistry and Biotechnology, Martin-Luther University Halle-Wittenberg, Halle, Germany
Ribonuclease A contains two exposed loop regions, around
Ala20 and Asn34 Only the loop around Ala20 is sufficiently
flexible even under native conditions to allow cleavage by
nonspecific proteases In contrast, the loop around Asn34
(together with the adjacent b-sheet around Thr45) is the first
region of the ribonuclease A molecule that becomes
sus-ceptible to thermolysin and trypsin under unfolding
condi-tions This second region therefore has been suggested to be
involved in early steps of unfolding and was designated as
the unfolding region of the ribonuclease A molecule
Con-sequently, modifications in this region should have a great
impact on the unfolding and, thus, on the thermodynamic
stability Also, if the Ala20 loop contributes to the stability of
the ribonuclease A molecule, rigidification of this flexible
region should stabilize the entire protein molecule We
sub-stituted several residues in both regions without any
dra-matic effects on the native conformation and catalytic
activity As a result of their remarkably differing stability, the variants fell into two groups carrying the mutations: (a) A20P, S21P, A20P/S21P, S21L, or N34D; (b) L35S, L35A, F46Y, K31A/R33S, L35S/F46Y, L35A/F46Y, or K31A/ R33S/F46Y The first group showed a thermodynamic and kinetic stability similar to wild-type ribonuclease A, whereas both stabilities of the variants in the second group were greatly decreased, suggesting that the decrease in DG can be mainly attributed to an increased unfolding rate Although rigidification of the Ala20 loop by introduction of proline did not result in stabilization, disturbance of the network of hydrogen bonds and hydrophobic interactions that interlock the proposed unfolding region dramatically destabilized the ribonuclease A molecule
Keywords: limited proteolysis; local unfolding; protein engineering; ribonuclease A; stability
In contrast with the ensemble of conformational species in
the unfolded state [1], the native state of proteins is generally
characterized by a uniform overall global conformation [2]
Whereas larger proteins that consist of structural subunits
or domains often behave very complexly during the
processes of unfolding and refolding, most small proteins
can be considered as a single unit [3] Apart from local
fluctuations of the protein structure in the native state,
the protein molecule unfolds highly co-operatively when
exposed to denaturing conditions The stability of the
natively folded protein molecule is not determined by a
single feature, but a number of internal and external factors
contribute to the formation and stabilization of the native
protein structure [4] Studies on a large variety of proteins
led to the assumption that confined regions of the 3D
protein structure are crucial for the conservation of its folded, native state A local disruption of the most labile region, which was referred to as unfolding region, was postulated to initiate the co-operative unfolding of the protein molecule [5,6] This assumption was supported by the identification of a region in the neutral protease from Bacillus stearothermophilus that responds most sensitively
to changes in the amino-acid composition by site-directed mutagenesis [7–9] Consistent with this critical region, a
weak point in ArthrobacterD-xylose isomerase was postu-lated based on results from proteolysis experiments under thermal denaturation [10] More recently, Machius et al [11] deduced a weak region in a-amylase and Gaseidnes
et al [12] identified a weak spot or a nucleation site for unfolding in chitinase by mutational analysis Because of the decreased number of hydrogen bonds, loop regions at the surface of the protein molecule are candidates for such
unfolding regions In fact, loops that are tethered by irregular hydrogen bonds or hydrophobic patches were found to be crucial for either the folding or unfolding of proteins (for a review see [13])
Ribonuclease A (RNase A) is a small, compact, and rather stable enzyme which is cross-linked by four disulfide bonds [14] Nevertheless, even under native conditions the loop region around Ala20 is highly flexible [15], which leads
to proteolytic cleavage by non-specific proteases In con-trast, in spite of increased mobility detected for residues 37–42 by NMR [16], the flexibility of the loop around Asn34, which contains potential cleavage sites for trypsin and thermolysin, is obviously not sufficient to allow
Correspondence to U Arnold, Department of Biochemistry and
Bio-technology, Martin-Luther University Halle-Wittenberg,
Kurt-Mothes Strasse 3, 06120 Halle, Germany Fax: +49 3455527303,
Tel.: +49 3455524865, E-mail: arnold@biochemtech.uni-halle.de
Abbreviations: 6-FAM-dArU(dA) 2 -6-TAMRA,
6-carboxyfluorescein-dArU(dA) 2 -6-carboxytetramethylrhodamine; GdnHCl, guanidine
hydrochloride; RNase A, ribonuclease A.
Enzymes: bovine pancreatic ribonuclease A (EC 3.1.27.5); thermolysin
(EC 3.4.24.27)
Note: a website is available at http://www.biochemtech.uni-halle.de/
biotech
(Received 30 June 2004, revised 27 August 2004,
accepted 3 September 2004)
Trang 2cleavage by these proteases As soon as the protein molecule
starts to unfold globally, however, the RNase A molecule
becomes susceptible to these proteases, too The primary
cleavage sites were found in the loop region around Asn34
and the adjacent b-strand around Thr45, suggesting this
section as the unfolding region [17]
To investigate the contribution of the two loop regions to
the stability of the entire RNase A molecule, we replaced
several amino-acid residues by site-directed mutagenesis
(A20P, S21P, A20P/S21P, S21L, N34D, L35S, L35A,
F46Y, K31A/R33S, L35A/F46Y, L35S/F46Y, and K31A/
R33S/F46Y) and studied the effect of the mutations on the
thermodynamic and kinetic stability The similarity of the
impact on both the thermodynamic and kinetic stabilities
suggests a predominant effect on the native state by these
mutations
Experimental procedures
Proteins and chemicals
RNase A from Sigma (St Louis, MO, USA) was purified on
a Mono S FPLC column (Amersham Biosciences, Uppsala,
Sweden) Thermolysin (from Calbiochem, Schwalbach,
Germany) was used without further purification
Oligonu-cleotides were from MWG Biotech (Ebersberg, Germany)
and restriction enzymes AvaI, BsmI, EcoRI, HindIII, NdeI,
and SacI were from New England Biolabs (Frankfurt/
Main, Germany) Growth media were from Difco
Laboratories (Detroit, MI, USA) Escherichia coli
strains XL-1 Blue and BL21(DE3) were from Stratagene
(Heidelberg, Germany) 6-Carboxyfluorescein-dArU(dA)2
-6-carboxytetramethylrhodamine (6-FAM-dArU(dA)2
Technologies (Coralville, IA, USA) All other chemicals
were of purest grade commercially available
Site-directed mutagenesis
The RNase A variants A20P, S21P, and A20P/S21P had
been produced previously [18] For other variants, the
rnase A gene in the plasmid pBXR [19], a gift from
R T Raines (University of Wisconsin, Madison, WI,
USA), was modified by use of the QuikChangeTM
site-directed mutagenesis kit (Stratagene) to obtain the mutations N34D, K31A/R33S, L35A, L35S, and F46Y For the mutations K31A/R33S/F46Y, L35A/F46Y, and L35S/F46Y, site-directed mutagenesis was started from the rnase A genes that carry the mutations for K31A/R33S, L35A, or L35S using the oligonucleotides for the F46Y mutation The oligonucleotides and the introduced restric-tion sites to facilitate the selecrestric-tion of positive clones are shown in Table 1 The mutations were verified by DNA sequencing as described by Sanger et al [20] (SequiTherm-ExcelTMLongReadTMDNA sequencing kit, Biozym, Hess, Oldendorf, Germany, and Li-COR 4000 DNA-sequencer, MWG Biotech, Ebersberg, Germany) The plasmids carrying the correct DNA sequence were each transformed into E coli expression host strain BL21(DE3)
Expression, renaturation, and purification
of the enzyme variants The experimental procedure was performed as described previously [18] Briefly, cultures of E coli strain BL21(DE3) that had been transformed with a plasmid directing the expression of the corresponding RNase A variant were grown in terrific broth containing
50 lgÆmL)1 kanamycin [variants A20P, S21P, and A20P/S21P in vector pET 26b(+)] or 400 lgÆmL)1 ampicillin [the other variants in vector pET 22b(+)] at
37C to an A600 of 2 Gene expression was induced by
1 mM isopropyl thio-a-D-galactoside, and cells were grown additionally for 4 h before being harvested Cell lysis was performed by treatment with lysozyme and homogenization with a Gaulin homogenizer The inclu-sion bodies were isolated by centrifugation followed by resolubilization (20 mM Tris/HCl, 7M guanidine hydro-chloride (GdnHCl), 100 mM dithiothreitol, 10 mM EDTA, pH 8.0) and dialysis of the protein solution against 20 mM acetic acid Precipitates formed during dialysis were removed by centrifugation After renatura-tion of the protein [100 mM Tris/HCl, pH 8.5,
100 mM NaCl, 1 mM glutathione (reduced), 0.2 mM glutathione (oxidized), 10 mM EDTA at room tempera-ture for 24 h], it was purified on a Mono S column (50 mM Tris/HCl, pH 7.5, with a linear gradient of 0–500 mM NaCl)
Table 1 Oligonucleotides for site-directed mutagenesis The replaced nucleotides are bold-face and the introduced restriction sites are underlined.
rev 5¢-CA GTA GTT GGA GCT CAG GGC GGC GGA AGT GCT GGA G-3¢
N34D fw 5¢-C CAG ATG ATG AAG AGC CGG GAC CTG ACC AAA GAT CGA TGC-3¢ No restriction site
rev 5¢-GCA TCG ATC TTT GGT CAG GTC CCG GCT CTT CAT CAT CTG G-3¢
K31A/R33S fw 5¢-C TGT AAC CAG ATG ATG GCG AGC TCG AAC CTG ACC AAA GAT C3¢ SacI
rev 5¢-G ATC TTT GGT CAG GTT CGA GCT CGC CAT CAT CTG GTT ACA G-3¢
L35A fw 5¢-G ATG ATG AAG AGC CGG AAT GCC ACC AAA GAT CGA TGC AAG C-3¢ BsmI
rev 5¢-G CTT GCA TCG ATC TTT GGT GGC ATT CCG GCT CTT CAT CAT C-3¢
L35S fw 5¢-G ATG ATG AAG AGC CGG AAT TCC ACC AAA GAT CGA TGC AAG C-3¢ EcoRI
rev 5¢-G CTT GCA TCG ATC TTT GGT GGA ATT CCG GCT CTT CAT CAT C-3¢
rev 5¢-C CAG GGA CTC GTG CAC ATA TGT GTT CAC TGG CTT GC-3¢
Trang 3Determination of the protein concentration
The protein concentration of RNase A and the F46Y-free
variants was determined using the molar absorption
coef-ficient of 9800M )1ẳcm)1 at 278 nm [21] For the
F46Y-containing RNase A variants, a molar absorption
coefficient of 11 300M )1ẳcm)1 at 278 nm, determined as
described by Thannhauser et al [22], was used
For activity measurements, the concentration of the
RNase stock solutions was determined by use of the BCA
protein assay kit (Pierce, Bonn, Germany) with BSA as
calibration standard according to the instructions of the
manufacturer The absorbance of the samples was measured
at 560 nm after incubation at 37C for 30 min using a
micro plate reader MR 7000 (Dynatech, Denkendorf,
Germany)
Activity assay
Values of kcat/Kmof wild-type RNase A and its variants
were determined at 25C in 100 mMMes/NaOH, pH 6.0,
containing 100 mM NaCl, 50 nM 6-FAM-dArU(dA)2
-6-TAMRA, and 0.25Ờ0.5 ngẳmL)1RNase A as described
by Kelemen et al [23] The increase in fluorescence emission
at 515 nm (band width 10 nm), on excitation at 490 nm
(band width 1 nm), was followed in a 1ở 0.4 cm
fluores-cence cuvette using a Fluoro-Max-2 spectrometer (Jobin
Yvon, Grasbrunn, Germany)
The values of kcat/Kmwere determined using the
follow-ing equation:
kcat=KmỬ mv
đFend FstartỡơE
where mvis the initial velocity calculated from the linear
increase in the fluorescence signal, Fstartis the signal of the
substrate before the addition of enzyme, Fendis the signal
after cleavage of all substrate, and [E] is the concentration of
RNase A
CD spectroscopy
CD spectra of RNase A and its variants were recorded in
10 mMTris/HCl, pH 8.0, containing 1Ờ2 mgẳmL)1RNase
on a CD spectrometer 62 ADS (Aviv, Piscataway, NJ,
USA) at 25C Cuvettes of 1 cm and 0.01 cm path length
were used for CD spectroscopy in the near-UV region (250Ờ
340 nm) and in the far-UV region (200Ờ260 nm),
respect-ively
GdnHCl-induced transition curves
GdnHCl-induced transition curves of RNase A and its
variants were obtained by fluorescence spectroscopy on a
Fluoro-Max-2 spectrometer (Jobin Yvon) at 25C using
a cuvette of 1ở 0.4 cm Protein concentration was
50 lgẳmL)1in 50 mMTris/HCl, pH 8.0, containing 0Ờ6M
GdnHCl After equilibration, the fluorescence signal was
recorded at 303 nm and averaged over 40 s The band width
was 1 nm for excitation at 278 nm and 10 nm for emission
To calculate values of [D]50% (the concentration of
denaturant [D] at which 50% of the protein is denatured)
and mDG(the measure of the dependence of the free energy
on denaturant concentration) the linear function,
DGơDỬ DGwater mDGơD
was used where DG[D]is the free energy of unfolding at a given denaturant concentration, and DGwater is the free energy of unfolding in the absence of denaturant [24] The fluorescence signals y were fitted by nonlinear regression using the program SIGMA PLOT as described
by Santoro & Bolen [25] with the modification by Clarke
& Fersht [26],
DGơDỬ mDGđơD50% ơDỡ leading to the equation;
yỬđyN
0ợ mNơDỡ ợ đyD0ợ mDơDỡ expđmDG đơD50%ơDỡ
RT
1ợ expđmDG đơD50%ơD
where yN0and yD0are the intercepts, and mNand mDthe slopes in the pre-transition and post-transition region, respectively, in the y vs [D] graph The fraction of native protein (fN) was calculated from the fitted values using equation;
fNỬ yD y
yD yN with yDỬ yD0 + mD[D] and yNỬ yN0+ mN[D], where
yNand yDare the signals of the native and the denatured protein as a function of the denaturant concentration The effect of mutations on the free energy was calculated as described by Clarke & Fersht [26],
DDGơDỬ DGơD DG0ơD
Ử mDGđơD50% ơDỡ m0DGđơD050% ơDỡ where DDG[D] is the change in the free energy on mutation at a defined concentration of denaturant, DG[D],
mDG, and [D]50% are the values for wild-type RNase A, and DGằ[D], mằDG, and [Dằ]50% are the values of the respective variant
Thermal transition curves Thermal transition curves of wild-type, F46Y-RNase A, and L35A/F46Y-RNase A were obtained by measuring the absorbance at 287 nm and 25Ờ80C after equilibration using a U-2000 spectrophotometer (Hitachi, Tokyo, Japan) and a water-jacketed cuvette (1 cm) connected to a WK14 thermostat (Colora, Lorch, Germany) The protein concen-tration was 0.5Ờ1.0 mgẳmL)1in 50 mMTris/HCl, pH 8.0, containing 100 mMNaCl
The signal of absorbance was fitted as described by Santoro & Bolen [25] to obtain the transition midpoint Tm
By use of the vanỖt Hoff equation,
dđln KDỡ dđ1=Tỡ Ử
DH R
DHm, the free enthalpy at Tm, was calculated
Using a value of 9.4 kJẳK)1ẳmol)1for DCpof wild-type RNase A [27], DGT can be calculated by the GibbsỜ Helmholtz equation;
Trang 4DGT¼ DHm 1 T
Tm
DCp ðTm TÞ þ T ln T
Tm
Values of DDG25Cwere estimated by the relation
DDGT¼ DGT DG0T where DGTand DG¢Tare the values of wild-type RNase A
and its variant, respectively
Proteolysis
Proteolysis was carried out at 35.0–57.5C with final
concentrations of 0.1 mgÆmL)1wild-type RNase A or its
variants and 0.2 mgÆmL)1 thermolysin in 50 mM Tris/
HCl, pH 8.0, containing 1 mM CaCl2 In a typical
experiment, 20 lL thermolysin solution (2 mgÆmL)1 in
50 mM Tris/HCl, pH 8.0, containing 10 mM CaCl2) were
mixed with 160 lL 50 mM Tris/HCl, pH 8.0, and
equilibrated at a defined temperature The reaction was
started by addition of 20 lL RNase solution (1 mgÆmL)1
in 50 mMTris/HCl, pH 8.0) After defined time intervals,
samples of 20 lL were withdrawn, mixed immediately
with 5 lL 50 mM EDTA, dried under nitrogen, and
analyzed by SDS/PAGE
SDS/PAGE and determination of the rate constants
of unfolding (kU)
Electrophoresis was carried out on a Midget
Electro-phoresis Unit (Hoefer, San Francisco, CA, USA) as
described by Laemmli [28] using 10% and 15%
acryl-amide for stacking and separating gels, respectively The
gels were stained with Coomassie Brillant Blue R 250
After being stained, the gels were evaluated at 560 nm
using a densitometer CD 60 (Desaga, Heidelberg,
Germany)
The rate constants of proteolysis (kp) were calculated
from the decrease in the peak areas of the intact RNase
band as a function of time of proteolysis, which followed
pseudo-first-order kinetics The determination of kp was
performed at least twice If the protease can degrade
the unfolded protein only and the unfolding reaction is the
rate-limiting step for proteolysis, as was the case in our
experiments, kpcorresponds to the rate constant of
unfold-ing, kU[27,29]
From the linear function ln(kU/T) vs 1/T in the Eyring
plot, the kUvalues at 25C were calculated On the basis of
the Eyring equation,
DG# ¼ RT ln K
h
ln kU T
where DG#U, K, h, and R are free activation energy for the
unfolding reaction, Boltzmann’s, Planck’s, and the gas
constant, the change in activation energy for unfolding
DDG#Uon mutation is given by,
DDG# ¼ DG# DG0#U¼ RT lnk
0 U
kU where k¢Uis the rate constant for the variant and kUthe rate
constant of the wild-type enzyme [26]
Results
Design of the RNase A variants Analysis by use of the program FIRST [30] (http:// firstweb.asu.edu/) indicates the highest flexibility of the peptide backbone of native RNase A at the N-terminus and in the loop region between helices I and II (around Ala20), followed by the region from the end of helix II spanning the loop to the adjacent b-strand (Lys31–Phe46; Fig 1), which had also shown low stability in both refolding [31] and unfolding [17] experiments We replaced various amino-acid residues as both single and multiple mutations in the two loop regions to investigate their contribution to the overall stability of the RNase A molecule To maintain RNase A folding and activity, we refrained from dramatic interference with the protein structure such as charge-reversal mutations or the intro-duction or deletion of disulfide bonds
In the Ala20 loop region the exposed, proteolytically sensitive residues Ala20 and/or Ser21 were replaced by proline to rigidify the flexible loop As a control, Ser21 was replaced by leucine to introduce a cleavage site for thermolysin, which was also used to determine the unfolding rate constants of RNase A Thus, both the local unfolding
of this loop (via the cleavage at Ala20–Leu21) and the global unfolding of the RNase A molecule (via the cleavage
at Asn34–Leu35/Thr45–Phe46) can be detected In the proposed unfolding region, we selected residues with side chains found to be involved in intramolecular interactions (analysis using the programWHAT IF[32]) (Table 2) Lys31, Arg33, Leu35, and Phe46 (Fig 1) were replaced as both single and multiple mutations (K31A/R33S, L35S, L35A, F46Y, L35S/F46Y, L35A/F46Y, and K31A/R33S/F46Y)
As the crystal structure reveals, the side chains of these residues are involved in intramolecular interactions that form either a hydrophobic patch (Leu35 and Phe46 with Met29 and Met30; Fig 2A) or a hydrogen bond network (Arg33, Fig 2B) Furthermore, these residues had proven
to be crucial in the proteolytic degradation of the RNase A molecule on unfolding [17] As a control for replacing
Fig 1 Tertiary structure of RNase A The model (7rsa) was taken from the Brookhaven protein databank and drawn with Swiss PDB -VIEWER v3.7 The replaced residues are marked in red for the region around Ala20 and green for the proposed unfolding region (Lys31– Phe46).
Trang 5solvent-exposed amino-acid residues, Asn34 was replaced
by aspartate
Expression, renaturation, and purification
All RNase A variants were expressed as inclusion bodies
Even though they differed in their tendency to form
aggregates during renaturation, all variants could be
obtained in sufficient amounts (up to 30 mgÆL)1 culture
medium) The purified proteins proved to be homogeneous
by SDS/PAGE and rechromatography on a Mono S column
CD spectra
As detected by CD spectroscopy, all RNase A variants revealed a tertiary and secondary structure comparable to that of wild-type RNase A (not shown) with a marginal disturbance of the secondary structure in A20P/S21P-RNase A An increased signal in the CD spectra in the near-UV region of F46Y containing RNase A variants is attributed to the introduction of the additional tyrosine Activity
Enzymatic activity provides a sensitive measure of the impact of modifications on the native structure of an enzyme [33] The kcat/Km values for RNase A and its variants, determined with 6-FAM-dArU(dA)2-6-TAMRA
as substrate [23], revealed that all RNase A variants are active (Table 3) However, while the RNase A variants with mutations in the Ala20 loop region as well as N34D-RNase A and L35A-N34D-RNase A showed an activity compar-able to that of wild-type RNase A, the variants with mutations in the unfolding region (except for N34D and L35A) showed a more significant decrease in the kcat/Km values, with the lowest activity ( 20%) for L35S/F46Y-RNase A and L35A/F46Y-L35S/F46Y-RNase A
Thermodynamic stability
To study the effect of the mutations on the thermodynamic stability of the RNase A molecule, GdnHCl-induced
Table 2 Relative solvent accessibility of amino acid residues of wild-type RNase A The relative accessibility was calculated using the program WHAT IF [32] and relates the accessibility of the side chain of the residue in the protein to the accessibility in a Gly-XXX-Gly peptide in vacuum which
is a good approximation for the accessibility in the unfolded state of the protein.
Residue
Relative
accessibility
(%) Known side chain interactions and effects by modification
Met29 18 Hydrophobic core with Met30, Leu35, and Phe46
Met30 0 Hydrophobic core with Met29, Leu35, and Phe46
Lys31 76 No interactions; K31C slightly decreases T m [44]
Ser32 66 No interactions; S32C slightly decreases T m [44]
Arg33 23 H bonds with the backbone of Arg10 and Met13
Asn34 48 No interactions; attached carbohydrate moiety in the related RNase B increases T m by 1.5 C [27]
Leu35 9 Hydrophobic core with Met29, Met30, and Phe46
Thr36 12 No interactions but in proximity to Met30, Tyr97, and the disulfide bond Cys40–Cys95
Lys37 60 No interactions
Asp38 68 No interactions; D38R decreases T m by 4 C [52]
Arg39 69 No interactions
Cys40 11 Disulfid bond with Cys95; C40A/C95A decreases T m by 20 C [53]
Lys41 21 P1 subsite; H bond to the side chain of Asn44; K41R strongly decreases the activity but does not affect T m [54];
a chemical crosslink K7–K41 increases both DG(H2O)and DG#U by about 12 kJ mol)1[51]
Pro42 43 No interactions; P42A does not affect the thermodynamic stability [55]
Val43 44 No interactions
Asn44 3 H bond with Gln11 and Lys41
Thr45 8 B1 subsite; T45G decreases T m by 10 C [56]
Phe46 0 Hydrophobic core with Met29, Met30, and Leu35; exchange by Leu, Val, Glu, Lys, or Ala greatly decreases
DG(H2O)[42,43]
Fig 2 Tertiary structure of the unfolding region of wild-type RNase A.
The model (7rsa) was taken from the Brookhaven protein databank
and drawn with Swiss PDB - VIEWER v3.7 (A) Hydrophobic cluster
formed by residues Phe46, Leu35, Met29 and Met30 The ribbon at
positions Phe46 and Leu35 is marked in green (B) Hydrogen bonds
between the side chains of Arg33 and the backbone of Met13 and
Arg10 and the hydrogen bond between the side chain of Arg10 and the
backbone of Arg33 The hydrophobic residues are marked as green
balls.
Trang 6transition curves were recorded (Fig 3) The values for
[D]50%and mDGas well as the change in free energy by the
mutation at the transition midpoint of wild-type RNase A
DDG[D]50%were determined (Table 3)
All mutations in the Ala20 loop as well as the control
N34D did not significantly affect the GdnHCl-induced
unfolding so that the transition curves of those
RNase A variants resemble that of wild-type RNase A
(group I; Fig 3) with a mean value for [D]50% of
2.85 ± 0.10M (Table 3) In contrast, all other mutations
in the proposed unfolding region resulted in a considerable
decrease in the transition midpoint (group II) Interestingly,
the variants K31A/R33S, L35S, L35A, and F46Y show
a remarkably coincident decrease in the thermodynamic
stability ([D]50%¼ 1.85 ± 0.10M; Table 3, Fig 3) whereas
the variants obtained by the combination of destabilizing
mutations in the unfolding region (K31A/R33S/F46Y, L35S/F46Y, and L35A/F46Y) are characterized by a further slight but uniform decrease in stability ([D]50%¼ 1.59 ± 0.10M; Table 3, Fig 3)
A similar destabilizing effect by the mutations was observed in thermal transition curves determined for wild-type RNase A and the variants F46Y and L35S/F46Y (not shown) Values of Tmwere 62.0 ± 0.1C, 53.0 ± 0.1 C, and 48.0 ± 0.1C, respectively, corresponding to values
of DDG25C of 10.4 ± 1.5 kJÆmol)1 and 23.6 ± 1.4 kJÆ mol)1 caused by the mutations F46Y and L35A/F36Y (calculated with DCp¼ 9.4 kJÆK)1Æmol)1 for wild-type RNase A [27] and the DHmvalues of 544 ± 14 kJÆmol)1,
481 ± 8 kJÆmol)1, and 341 ± 5 kJÆmol)1 for wild-type RNase A and the variants F46Y and L35S/F46Y, respect-ively, obtained from the van’t Hoff plot)
Kinetic stability The decreased thermodynamic stability of the RNase A variants with mutations in the proposed unfolding region could arise from faster unfolding or slower refolding (or both) To dissect the effect of the mutations, rate constants
of unfolding of wild-type RNase A and its variants were determined Owing to the isomerization of natively cis proline peptide bonds in the unfolded state [34,35] refolding
of RNase A is known to be rather complex [36] and the introduction of further proline residues at positions 20 and
21 is expected to further increase this complexity, as indicated by the decreased mDG values for the A20P and A20P/S21P variants (Table 3) Hence, we refrained from refolding experiments
Unfolding rate constants were determined by limited proteolysis with thermolysin at three different temperatures between 35.0C and 57.5 C (Fig 4) By linear extrapola-tion of the Eyring plots [29], values of kUat 25C were obtained which were used to calculate values of DDG#Uat
25C (Table 3) Wild-type RNase A and all variants of group I with respect to their thermodynamic stability unfold with the same rate constants at 47.5–57.5C (Fig 4),
Table 3 Activity and thermodynamic and kinetic parameters of wild-type RNase A and its variants at 25 C Values of k cat /K m were determined as described in Experimental Procedures with 6-FAM-dArU(dA) 2 -6-TAMRA as substrate in 100 m M Mes/NaOH, pH 6.0, containing 100 m M NaCl The thermodynamic parameters were determined from the GdnHCl-induced transition curves at 25 C as described in Experimental Procedures Values of DDG#U (25 C) were calculated from parameters obtained from the Eyring plot (Fig 4) as described in Experimental Procedures RNase A
variant
10)7· k cat /K m
(s)1Æ M )1 )
[D] 50%
( M )
m DG
(kJÆmol)1Æ M )1 )
DDG [D]50%
(kJÆmol)1)
DDG #
U 25 C (kJÆmol)1)
[GdnHCl] (M)
0.0
0.5
1.0
Fig 3 GdnHCl-induced transition curves The transition curves of
wild-type RNase A (teal) and its variants A20P (black), S21P (grey),
S21L (bright green), A20P/S21P (blue), N34D (red), L35A (cyan),
L35S (green), F46Y (dark red), K31A/R33S (pink), L35A/F46Y (dark
yellow), L35S/F46Y (dark blue), and K31A/R33S/F46Y (violet) were
determined by fluorescence spectroscopy in 50 m M Tris/HCl, pH 8.0,
at 25 C.
Trang 7indicating that the kinetic stability is also not affected by
these mutations All the thermodynamically less stable
RNase A variants also show a large increase in kU Even
though the effects are not as uniform as for the
thermo-dynamic stability, the comparison of DDG[D]50% and
DDG#U25
C shows that the decrease in the thermodynamic
stability is mainly caused by an increase in the unfolding rate
constant, i.e a decrease in the kinetic stability Whereas the
introduction of a cleavage site for thermolysin in the
control variant S21L-RNase A facilitated degradation of
the RNase A molecule under native conditions (not
shown), this variant was degraded like wild-type RNase A
under denaturing conditions
Discussion
As in the folding of proteins, confined regions of the protein
structure have a crucial role in the unfolding process and
are, thus, particularly important for kinetic stability [8,9,12]
These regions are mostly located on the surface of the
protein molecule, and loops in particular often represent
critical spots [8,11,17]
RNase A possesses two structural sections that might
function as such a critical region (Fig 1): (a) the loop region
around Ala20, which is highly flexible under native
condi-tions [14,15] as reflected in efficient proteolytic attack by
nonspecific proteases such as proteinase K and subtilisin
Carlsberg [37–39]; (b) the region from the end of helix II to
the adjacent a-sheet (Lys31–Phe46), which becomes
access-ible to an H–D exchange [40] and to the proteases
thermolysin and trypsin when the molecule starts to unfold
[17] Furthermore, this region (residues 31–39) is the last one
that becomes protected against tryptic attack during
RNase A folding [31]
RNase A variants with amino-acid substitutions in the
two regions fell into two classes with respect to
thermo-dynamic stability (Fig 3) The RNase A variants with
similar unfolding transition curves to wild-type RNase A (group I) are obtained by mutations in the loop region around Ala20 or by the control mutation N34D These amino-acid residues are not involved in interactions like hydrogen bonds, salt bridges or hydrophobic clusters, as reflected in great flexibility of the loop region around Ala20 [15,16] So even the replacement of two adjoined residues in this region by proline (A20P/S21P) was tolerated On the other hand, the introduction of the proline residues, i.e the decrease in loop flexibility, did not increase the global stability of the RNase A molecule
By introducing a cleavage site for thermolysin (S21L-RNase A), the flexibility of the Ala20 loop became traceable for this protease Nevertheless, the unfolding rate constants
of this RNase A variant correspond to those of wild-type RNase A (Fig 4), indicating that the local unfolding of the Ala20 loop is independent of the global unfolding of the RNase A molecule In the control variant Asn34-RNase A,
a solvent-exposed residue that belongs to the unfolding region (Lys31–Phe46) and serves as anchor for the stabil-izing carbohydrate moiety in the related RNase B [41], was replaced As expected, the mimicked deamidation does not affect interactions essential for stability
In contrast, the less stable RNase A variants of group II (L35S, L35A, F46Y, K31A/R33S, L35S/F46Y, L35A/ F46Y, and K31A/R33S/F46Y) all of which were obtained
by mutations in the region Lys31–Phe46 indicate a consid-erable contribution of this region to the thermodynamic stability of the entire RNase A molecule The coincidence of the degree of destabilization in these variants points to an effect on the stability of the entire region rather than on a particular interaction A similar destabilization was also found by Chatani et al [42] and Kadonosono et al [43] by replacement of Phe46 with Leu, Val, Ala, Lys, or Glu The authors concluded that Phe46 has an important role in the folding reaction through hydrophobic interactions and by the correct packing of the amino-acid side chains between two structural domains [42] However, from the rate of oxidative protein folding, they concluded that there was a decreased kU, i.e kinetic stabilization of the F46L, F46V, and F46A variants In contrast, we found an acceleration
of the unfolding reaction, i.e kinetic destabilization, for the variants and the similarity of DDG[D]50%and DDG#U indicates that the decrease in the thermodynamic stability is mainly caused by an increase in kU Furthermore, our results suggest that Leu35, the side chain of which is buried
in the interior of the molecule like that of Phe46 (Table 2),
is involved in the formation of a hydrophobic cluster with Phe46, Met29 and Met30 (Fig 2A) and consequently plays a similar role to Phe46 Molecular modeling revealed that any mutation in position 35 destabilizes the entire molecule by disturbing these complex hydrophobic inter-actions (G Vriend, University of Nijmegen, personal communication)
In addition to these hydrophobic interactions, this region
is stabilized by a network of hydrogen bonds between the side chain of Arg33 and the backbone of Met13 and Arg10 (three hydrogen bonds) and between the side chain of Arg10 and the backbone of Arg33 (one hydrogen bond; Fig 2B) Because no hydrogen bonds were identified for the side chain of Lys31 of RNase A (analysis using the program
[32]) and its exchange with Cys results in only a
-14
-12
-10
Fig 4 Eyring plot for the unfolding of wild-type RNase A and its
vari-ants Values for k U of wild-type RNase A (teal) and its variants A20P
(black), S21P (grey), S21L (bright green), A20P/S21P (blue), N34D
red), L35A (cyan), L35S (green), F46Y (dark red), K31A/R33S (pink),
L35A/F46Y (dark yellow), L35S/F46Y (dark blue), and K31A/R33S/
F46Y (violet) were determined by limited proteolysis with thermolysin
in 50 m M Tris/HCl, pH 8.0, at 35.0–57.5 C as described in
Experi-mental procedures.
Trang 8slight decrease in the stability [44], the destabilizing effect
of the mutation K31A/R33S is probably caused by the
mutation of Arg33
Generally, changes in the thermodynamic stability by
mutations can be caused by effects on the native and/or the
unfolded state, whereas changes in the kinetic stability are
due to a change in the native and/or transition state The
determination of the unfolding rate constants of wild-type
RNase A and its variants (Fig 4) allowed differentiation
between the several possibilities The RNase A variants
with GdnHCl-induced transition curves similar to that of
wild-type RNase A, i.e the members of group I, also show
thermal unfolding rate constants and consequently DG#U
values comparable to that of wild-type RNase A (Table 3),
indicating that the native state, relative to the transition
state, is not affected by the mutations The labile RNase A
variants show a large increase in the unfolding rate
constants For the variants K31A/R33S and K31A/R33S/
F46Y, a value of DDG#U was obtained that corresponds
very well to that of DDG[D]50%(Table 3), indicating that the
decrease in the thermodynamic stability is caused by
destabilization of the native state relative to the unfolded
state The decrease in the thermodynamic stability of the
other less stable variants, all of which were exclusively
obtained by exchanges of the hydrophobic residues Leu35
and/or Phe46, is not solely attributable to faster unfolding
The differences between DDG[D]50%and DDG#Ualso point
to slower refolding, e.g by disturbance of the formation of a
hydrophobic cluster [42] Nevertheless, the decrease in the
thermodynamic stability is mainly caused by the faster
unfolding resulting from destabilization of the native state
relative to the transition state, underlining the predominant
importance of this region for maintaining the natively
folded structure of the RNase A molecule
Interestingly, the hydrophobic nature of residues 29, 30,
35, and 46 is conserved throughout the members of the
ribonuclease A superfamily (Fig 5) While Phe46 and
Met30 (numbered by the RNase A sequence) are found in
all members, Met29 and Leu35 can be occupied by Met, Ile,
or Ala and Leu, Met, or Ile, respectively (Fig 5, cf [45])
Furthermore, with the exception of mammalian
ribonuc-leases 2 and frog ribonucribonuc-leases, the charged residue Arg33 is
conserved (Fig 5)
Altogether, whereas the loop region between helices I and
II, i.e around Ala20, does not contribute to the stability of
the RNase A molecule and local flexibility does not lead to
global unfolding, the interface between helix II and the
adjacent a-sheet is stabilized by a multitude of interactions and is very sensitive to mutations Connecting regions between different folding motifs have also been found to be crucial for the stability of other proteins [46–49] Despite a vast number of RNase A variants produced by protein engineering (for a review, see [50]), only two variants concerning this region are more stable than wild-type RNase A: the naturally occurring glycosylated RNase B (at Asn34 [41]) and the chemically cross-linked RNase A (Lys7–Lys41) [51] For both variants, the thermodynamic stabilization is comparable to the kinetic stabilization [27,51] Also the effect of the mutations reported here on the thermodynamic stability can mainly be attributed to effects on the kinetic stability of the protein, providing further evidence for the validity of the concept of the unfolding region
Acknowledgements
We are grateful to Professor R T Raines (University of Wisconsin, Madison, WI, USA) for the gift of the plasmid pBXR, to Professor
G Vriend (University of Nijmegen, the Netherlands) for molecular modeling, and to Y Markert for providing the plasmids for the variants A20P, S21P, and A20P/S21P J K was supported by a grant from the Max-Buchner-Forschungsstiftung, Frankfurt, Germany.
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