Biochemical and Biophysical Research Communications 325 (2004) 1082–1089
BBRC
[Link]/locate/ybbrc
Role of weak interactions in thermal stability of proteins
B. Syed Ibrahim, Vasantha Pattabhi*
Department of Crystallography and Biophysics, University of Madras, Guindy campus, Chennai 600 025, India
Received 20 October 2004
Available online 6 November 2004
Abstract
A database analysis was done to study the role of weak interactions such as CH O, CH PIm and NH PIm in the thermal
stability of proteins. The CH O and CH PIm interactions are more in the case of thermophilic proteins as compared to meso-
philes. Amino acid analysis showed that hydrophobic amino acids like Val and Ile, and Cys contribute more to CH O hydrogen
bonds where as Pro and Gly contribute more to CH PIm interactions. Though NH PIm interactions are dominated by Lys and
Arg in thermophiles and mesophiles, the Arg contribution is significantly higher in thermophiles. Interestingly, Glycine is a predom-
inant contributor to all the weak interactions. The number of aromatic amino acids in the thermophiles is more and hence a large
number of aromatic clusters were observed in this class. Thus, a cumulative effect of weak interactions seems to be important in
thermal stability of proteins. The study also shows that introduction of Gly, Arg, Phe, Pro, and Tyr may enhance the thermal
stability.
Ó 2004 Elsevier Inc. All rights reserved.
Keywords: Weak interactions; Thermal stability; Aromatic cluster; Thermophiles
Microorganisms have been classified in to three stability of thermophilic proteins as compared to its
broad categories according to the temperature in which mesophilic pair. Such an analysis is essential for not only
they live. Mesophilic, living at normal temperature, understanding the factors behind the stability of pro-
thermophilic, living at higher temperature and psychro- teins but also for designing efficient enzymes that can
philic, living at low temperature are the three main cat- work at higher temperatures. Such enzymes may be use-
egories. Some of these organisms not only live at ful for several industrial applications, such as detergent
extreme temperatures but can also survive high pH, high manufacturing, food and starch processing, production
salt concentration, acidic condition, etc. For such an of high fructose corn syrup, and PCR [4]. It has also
organism a basic question arises about the stability of been reported that thermophilic enzymes are more resis-
their proteins in those environments. One may be able tant to proteolysis than their mesophilic homologues [5].
to address this question by investigating the structure Some of the important factors attributed to the great-
of the same protein in two or three different er stability of the thermophilic proteins include greater
environments. hydrophobicity [6], better packing, deletion or shorten-
Many investigations have been carried out to achieve ing of loops [7], smaller and less number of cavities, in-
this goal and many theories have been proposed for pro- creased surface area buried upon oligomerisation [8],
tein stability [1–3]. Several groups have carried out data amino acid substitutions within and outside the second-
base analysis to establish the reason for the enhanced ary structures [9,6,10], increased occurrence of proline
residues [6,11,12], decreased occurrence of thermolabile
*
Corresponding author. Fax: +91 044 223 00 122.
residues [7], increased helical content, increased polar
E-mail addresses: vasantha@[Link], pvasantha@[Link] surface area [6,13,14], increased hydrogen bonding
(V. Pattabhi). [13,14] and salt bridges [15–18].
0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved.
doi:10.1016/[Link].2004.10.128
B.S. Ibrahim, V. Pattabhi / Biochemical and Biophysical Research Communications 325 (2004) 1082–1089 1083
Table 1
Thermophilic and mesophilic proteins used for the analysis
Protein family name Thermophilic organism PDB entry, Mesophilic PDB entry, % identitya rmsdb (Å)
resolution (Å) organism resolution (Å)
oligomeric state, oligomeric state,
No. of residues No. of residues
Ferredoxin Bacillus thermoproteolyticus 1IQZ Clostridium 2FDN 16.4 1.11
0.92 Acidi-urici 0.94
Mononer, 81 Monomer, 55
Glutamate Pyrococcus furiosus 1GTM Clostridium 1BGV 27 1.84
dehydrogenase 2.2 symbiosum 1.9
Trimer, 1257 Monomer, 449
Thermolysin Bacillus thermoproteolyticus 1LNF Bacillus cereus 1NPC 72 0.9
and neutral protease 1.7 2.0
Monomer, 316 Monomer, 317
CheYk Thermotoga maritima 1TMY Escherichia coli 3CHY 28 1.8
1.9 1.66
Monomer, 118 Monomer, 128
Endo-1,4-b xylanase Thermomyces lanuginosus 1YNA Bacillus circulans 1XNB 43.8 0.9
1.55 1.49
Monomer, 193 Monomer, 185
Methionine Pyrococcus furiosus 1XGS Escherichia coli 2MAT 20.2 1.2
aminopeptidase 1.75 1.9
Dimer, 590 Monomer, 263
Adenylate kinase Bacillus 1ZIN Saccharomyces 1AKY 41.5 1.5
Stearothermophilus cerevisae 1.65 1.63
Monomer, 217 Monomer, 218
Thermitase Thermoactenomyces vulgaris 1THM Bacillus lentus 1ST3 41.6 1.6
1.4 1.4
Monomer, 279 Monomer, 269
Rubredoxin Pyrococcus furiosus 1BRF Escherichia coli 1RB9 65.4 0.5
0.95 0.92
Monomer, 53 Monomer, 53
b-Glycanesus Clostridum thermocellum 1XYZ Cellulomonas fimi 2EXO 41.3 1.4
1.4 1.8
Dimer, 694 Monomer, 312
Superoxide dismutase Thermus thermophilus 3MDS Escherichia coli 1D5N 37.9 0.9
1.8 1.55
Monomer, 203 Tetramer, 820
Spermidine synthase Thermotoga maritima 1INL Bacillus subtilis 1IY9 42 2.1
1.5 2.3
Tetramer, 1184 Tetramer, 1100
Rnase H Thermus thermophilus 1JL2 Escherichia coli 1JL1 74.3 0.9
1.76 1.3
Tetramer, 624 Monomer, 155
Aspartatetrans Pyrococcus abyssi 1ML4 Escherichia coli 3CSU 50.0 0.9
carbamylase 1.8 1.88
Monomer, 308 Trimer, 930
Malate dehydrogenase Thermus flavus 1BMD Aquaspirillum arcticum 1B8P 61 1.7
1.9 1.9
Dimer, 754 Monomer, 329
Tryptophan synthase Thermus thermophilus 1UJP Salmonella typhimurium 1QOP 25 2.1
1.34 1.40
Monomer, 271 Dimer, 664
Citrate synthase Pyrococcus furiosus 1AJ8 Chicken heart 1CSH 26.2 1.6
1.9 1.6
Dimer, 742 Monomer, 435
a
Identity between the sequence of thermophilic and mesophilic pair.
b
r.m.s. deviation obtained when Ca atoms of thermophilic and mesophilic pair were superimposed.
The importance of hydrogen bonds in the stabiliza- OAH PIm, CAH O, and CAH PIm also play a role
tion of secondary structures [19], protein folding and in protein stability [25,26]. Though CAH O is consid-
stability [20,21] is well established [22–24]. Recent stud- ered to be a weaker interaction as compared to
ies showed that weaker interactions such as NAH PIm, NAH O and OAH O hydrogen bonds it constitutes
1084 B.S. Ibrahim, V. Pattabhi / Biochemical and Biophysical Research Communications 325 (2004) 1082–1089
20–25% of the total number of hydrogen bonds in pro- [26,28,29]. The above mentioned studies have been car-
teins [27] and the importance of CAH O hydrogen ried out for protein stability in general. We have now ex-
bonds in protein folding and stability is well recognized plored the role of weak interactions and aromatic
clusters in the thermal stability of proteins from thermo-
philic organisms.
Table 2
CaAH O interaction (M, monomer; D, dimer; T, tetramer; I, trimer;
the values given in the parentheses are for complete oligomer; this
Methods
notation is common for all the tables)
PDB ID No. of contacts Selection of PDB structures. High-resolution X-ray crystal struc-
Thermophilic proteins tures from the December 2003 update of the PDB database [30] were
1IQZ-M 10 selected for the analysis. Seventeen pairs of non-redundant families of
1UJP-M 19 thermophilic and mesophilic proteins upto 2.3 Å resolution were se-
1BMD-D (59) 29 lected and the selection criterion used was similar to that of Kumar
1JL2-T (90) 22 et al. [31]. The selected PDBÕs are listed in Table 1 with its property,
1INL-T (164) 43 PDBID, resolution, oligomeric state, etc.
1AJ8-D (31) 16
1GTM-I (113) 39
1LNF-M 31
Results and discussion
1TMY-M 9
1XGS-D (81) 44
1YNA-M 59 CAH O interactions
1ZIN-M 11
1THM-M 27 CaAH O interactions and other weak interactions
1BRF-M 4
were calculated using the program available for this pur-
1XYZ-D (50) 26
3MDS-D (17) 10 pose in the NCI server [32]. The hydrogen atoms were
1ML4-M 31 fixed by the program based on the geometry. The geom-
etry criterion used was H O < 2.5 Å, Ca O < 3.9 Å
Mesophilic proteins
2FDN-M 3 and CaAH O > 90°. Only Ca atoms were considered
1QOP-D (58) 17 for the calculation of CAH O interactions as Ca atoms
1B8P-M 26 are considered to be more acidic than the others [33].
1JL1-M 23 Very often the thermophilic proteins exist in oligomeric
1IY9-T (152) 38
state, in which case the interactions were checked for
1CSH-M 6
1BGV-M 33 monomers as well as oligomers.
1NPC-M 27 The results listed in Table 2 show that in general the
3CHY-M 18 thermophilic proteins have significantly more number of
2MAT-M 38 interactions both in monomeric as well as oligomeric
1XNB-M 45
states. Amino acid analysis showed that the influence
1AKY-M 13
1ST3-M 27 of most of the amino acids in making CaAH O inter-
1RB9-M 4 actions is higher in thermophiles than in mesophiles. In
2EXO-M 19 order to remove the bias due to amino acid distribution
1D5N-T (31) 9 in these proteins, percentage (P) of each of the 20 amino
3CSU-I (85) 30
acids involved in interactions was calculated.
Fig. 1. Percentage of each amino acid (P) involved in CaH O interactions.
B.S. Ibrahim, V. Pattabhi / Biochemical and Biophysical Research Communications 325 (2004) 1082–1089 1085
P¼
Number of residues of a particular type involved in interactions total number of interactions involving all the amino
Total number present in the data set acids was calculated, and was denoted as S
100:
The values obtained for both the classes of proteins were Interactions involving a particular amino acid
S¼ 100:
plotted (Fig. 1). The plot shows that interactions involv- Total number of interactions
ing hydrophobic residues are predominant. In particular
Val, and Trp have more contacts than others in both the The values of S obtained for all the amino acids were
classes of proteins, while the contribution of Ile and Cys plotted (Fig. 3). The plot shows that Ile and Ala contrib-
in thermophilic proteins is nearly double that of meso- ute more in thermophilic proteins, while Leu and Val
philic proteins. Though the total number of Cys residue contribute more in mesophilic proteins. Glycine contrib-
is significantly low (Fig. 2), its contribution to the Ca– utes equally to both the classes of proteins.
H O interaction is significant. The difference in the total
number of Phe, Asp, Gln, and Ala present in the two clas- PIm interaction
ses of proteins is less, but their percentage contribution in
thermophilic proteins is more. On the other hand Met, Hydrogen bonds between XAH donors and the PIm-
Asn and His contributes more to mesophilic proteins. electron cloud of an aromatic moiety (called XAH PIm
In order to identify the percentage contribution by an hydrogen bonds) was first proposed by Wulf et al. [34]. In
amino acid to the stability, the ratio between the number this type of interaction, the donor group XAH is placed
of interactions involving a particular amino acid to the roughly above the center of an aromatic ring and the
Fig. 2. Amino acid distribution in thermophiles and mesophiles.
Fig. 3. Percentage contribution (S) by an amino acid to CaH O interactions.
1086 B.S. Ibrahim, V. Pattabhi / Biochemical and Biophysical Research Communications 325 (2004) 1082–1089
XAH vector points at it. The large surface of a PIm sys- M are 3.2–3.8 Å, but the long-distance end is diffuse as
tem makes PIm—acceptors a ‘‘target that is easy to hit’’ is usual for hydrogen bonding in general, and shorter dis-
[35]. Normal distances of X to the aromatic midpoint tances than 3.2 Å are occasionally found [36]. The
parameters used in the calculation were default values gi-
ven in the NCI server viz. C PIm 6 4 Å and
Table 3 CaAH PIm P 120° and H PIm 6 3.8 Å.
CaAH PIm interactions In the present analysis CaAH PIm, CdAH PIm,
PDB ID No. of contacts OAH PIm, and NAH PIm interactions were consid-
Thermophilic proteins ered (Table 3). In some cases either thermophilic or mes-
1IQZ-M — ophilic protein does not have any PIm interaction but on
1UJP-M 4 an average these interactions are found more often in
1BMD-D (6) 3
1JL2-T (8) 3
thermophilic proteins than in its mesophilic counter
1INL-T (17) 5 part. From Table 3 it can be seen that excepting in three
1AJ8-D (9) 5 cases the thermophilic proteins have more CaAH PIm
1GTM-I (11) 3 interaction than its mesophilic pair.
1LNF-M 4 Amino acid analysis for proton donors in CaAH PIm
1TMY-M —
1XGS-D (5) 2
interactions (Fig. 4) and the percentage (P) of amino acids
1YNA-M 7 involved in CH PIm interaction showed that Gly and
1ZIN-M 1 Pro are favourably poised to form CH PIm interaction.
1THM-M 4 This is in agreement with the results obtained for the en-
1BRF-M 1 tire protein database by [33]. In general hydrophobic res-
1XYZ-D (13) 7
3MDS-D (16) 8
idues are less frequently involved in CH PIm
1ML4-M 2 interaction. The aromatic residues, Phe and Tyr act as
both donor and acceptor in this interaction but Trp does
Mesophilic proteins
2FDN-M 2 not participate in this type of interaction. The difference in
1QOP-D (6) 3 contribution by other amino acids is not significant.
1B8P-M 2 The percentage ratio S versus amino acid plot (Fig. 5)
1JL1-M 1 shows that glycine is the maximum contributor to this
1IY9-T (12) 3
interaction as well. This can be attributed to the pres-
1CSH-M 4
1BGV-M 2 ence of two free hydrogens at the Ca of glycine.
1NPC-M 3 The CdAH in proline also contributes to PIm interac-
3CHY-M — tion. Hence the analysis was extended to Cd atoms of
2MAT-M — prolines and a total of 16 interactions (in monomer
1XNB-M 4
only) in thermophilic and 11 in mesophilic proteins were
1AKY-M 1
1ST3-M 3 observed. The number of contacts observed though
1RB9-M 2 small, suggests that these interactions are also more pre-
2EXO-M 2 valent in thermophilic proteins.
1D5N-T (25) 7 The role of cation-PIm interaction in protein stability
3CSU-I (11) 4
has been shown by Gallivan and Dougherty [37] and
Fig. 4. Percentage of each amino acid involved (P) in CH PIm interaction.
B.S. Ibrahim, V. Pattabhi / Biochemical and Biophysical Research Communications 325 (2004) 1082–1089 1087
Fig. 5. Percentage contribution (S) by an amino acid to CH PIm interaction.
Gromiha et al. [38]. An analysis of NAH PIm interac- make the maximum contribution to this interaction.
tions involving backbone nitrogen only gave very few Though Lys–Tyr pair has more interactions in thermo-
contacts for our data set. Hence, NAH PIm (cat- philes (Table 4), Arg contribution to thermal stability
ion PIm) interactions involving backbone and side- is higher than that of Lys (Fig. 6).
chain nitrogens of lysine and arginine with the Extension of the analysis to OAH PIm from Ser,
aromatic residues, phenylalanine, tyrosin and trypto- Thr, and Tyr and SAH PIm from cysteine showed that
phan were considered for the analysis. The interactions there are very few interactions of this type. This is not
were calculated for two radii cut off viz 4 and 6 Å using
the program NCI server [32] and CAPTURE [37],
respectively. Very few contacts were obtained within
4 Å radius and hence the calculations were repeated with
6 Å cut off using the program CAPTURE [37]. NCI ser-
ver calculations are based on geometry criteria whereas
the program CAPTURE [37] uses an energy criterion.
Default parameters given in the programs were used.
The results (Table 4) show that Tyr interacts with Lys
more often in thermophiles while Trp–Lys interactions
predominate in mesophiles. This result agrees with that
of Gromiha et al. for thermophiles but for mesophiles
they have implicated Phe instead of Trp.
Amino acid analysis shows that, very few amino acids
are involved in this type of interaction. The percentage
(P) calculated for NH PIm interaction is shown in Fig. 6. Percentage of amino acids involved (P) in NH PIm
(Fig. 6). The plot shows that Arg is preferred for this interaction.
type of interaction in thermophilic proteins, while Lys
is preferred in mesophiles. Similarly the percentage ratio
(S) calculated (Fig. 7) shows that Arg, Gly, and Lys
Table 4
(NAH PIm) interactions involving lysin and arginine
Distance 4 Å 6 Å
Meso Thermo Meso Thermo
Arg–Phe 2 4 14 13
Arg–Tyr 2 6 11 14
Arg–Trp 2 6 4 19
Lys–Phe 3 2 9 14
Lys–Tyr 1 2 7 28
Fig. 7. Percentage contribution (S) by an amino acid to NH PIm
Lys–Trp 3 1 17 12
interaction.
1088 B.S. Ibrahim, V. Pattabhi / Biochemical and Biophysical Research Communications 325 (2004) 1082–1089
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