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Protein Bioinformatics From Sequence to Function 1st
Edition M. Michael Gromiha Digital Instant Download
Author(s): M. Michael Gromiha
ISBN(s): 9788131222973, 8131222977
Edition: 1
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Year: 2010
Language: english

Protein Bioinformatics:
From Sequence to Function

Praise from the Field for
Protein Bioinformatics: From Sequence to Function
A protein structure oriented bioinformatics book has been long overdue and I would like to
congratulate Dr. Gromiha for his efforts to fill this gap.
Dr. Sandeep Kumar, Principal Scientist,
Pharmaceutical Sciences, Research and Development,
Global Biologics, Pfizer, Inc.
The book has excellent coverage for students wishing to practice bioinformatics.
Dr. Debnath Pal, Indian Institute of Science,
Bangalore, India
The author introduces the concepts in a simple manner so that even people with a
non-mathematical background will find it very useful. The literature covered is quite
extensive so that the beginner will find it as a very good platform to begin one’s work.
Dr. S. Selvaraj, Department of Bioinformatics,
Bharathidasan University, Tamil Nadu
To my knowledge, there is no source currently available that solidifies protein
bioinformatics the way this book does. The book tries to provide the broadest possible
coverage of protein bioinformatics; it has practice exercises, and it links and discusses
online databases/servers that the reader can actually use to analyze their own protein
datasets.
Dr. Eric Gaucher, Georgia Institute of Technology,
Atlanta, GA USA
This book is a “must have” for my Protein Engineering students who need to have easy
access to the most important bioinformatics tools for analyzing protein structures and
predicting how mutations will affect their structure and/or function.
Dr. Gerard Pujadas Departament de Bioquı́mica i
Biotecnologia, Universitat Rovira i Virgili, Catalonia

Protein Bioinformatics:
From Sequence to Function
M. Michael Gromiha
Computational Biology Research Center (CBRC),
National Institute of Advanced Industrial
Science and Technology (AIST), Tokyo, Japan
AMSTERDAM • BOSTON • HEIDELBERG • LONDON
NEW DELHI • NEW YORK • OXFORD • PARIS • SAN DIEGO
SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO
Academic Press is an imprint of Elsevier

Cover credits:
The figure is a portion of Color Insert 12 which was adapted from Gromiha MM, Pujadas G,
Magyar C, Selvaraj S, Simon I. “Locating the stabilizing residues in (alpha/beta) 8 barrel
proteins based on hydrophobicity, long-range interactions, and sequence conservation.”
Proteins: Structure, Function and Bioinformatics. 2004;55:316–329.
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Library of Congress Cataloging-in-Publication Data
Gromiha, M. Michael.
Protein bioinformatics : from sequence to function.
1. Proteins–Structure–Data processing. 2. Proteins–
Metabolism–Data processing. 3. Bioinformatics.
572.6
0285-dc22
British Library Cataloguing in Publication Data
A catalogue record for this book is available from the British Library
ISBN-13: 978-81-312-2297-3
Printed in India.
Published by Elsevier, A Division of Reed Elsevier India Pvt. Ltd.
10 11 12 5 4 3 2 1

Dedicated to the memory of my beloved father

Foreword
Bioinformatics is a broad field of research that escapes easy definition. As in all
natural sciences, progress in the life sciences depends on the interplay of empirical
research and data interpretation (theory building), as well as modeling (valida-
tion). Because of the vast improvements in data collection, we have to deal with
very large numbers of highly complex empirical data—in particular molecular and
genetic data—while our ability to understand the data in terms of new concepts
and theoretical frameworks often lags behind. The gap between data and scientific
understanding cannot be bridged without computational tools, and this is where
bioinformatics comes into the picture. Since all steps of data collection, preprocess-
ing, storage, and analysis have their particular computational tools, novices trying
to understand “bioinformatics” in general feel inevitably lost in the jungle of often
loosely defined terms and concepts.
Choosing protein bioinformatics as the subject of this book is highly justified
for two particular reasons. First, protein research is a well-defined subfield both
within the molecular sciences and within the industrial sector where protein prod-
ucts (pharmaceuticals, industrial enzymes, etc.) play dominant roles. Second, while
genome biologists deal with large amounts of lightly annotated data, protein re-
searchers often need to deal with a few, very well-characterized molecules, using a
variety of methods. The book seeks to offer an introduction of these methods and
tools.
Thepurposeofthebookistoprovidearesourcetohelpresearchersandstudents
to understand the structure, function, folding, stability, and interactions of proteins
through bioinformatics approaches. The book is meant for students and researchers
who need a first-time introduction to protein bioinformatics and is meant to serve
as a textbook. In addition, it will be an excellent reference book with up-to-date
information for protein researchers who want to apply computational tools in their
research projects.
The book is divided into seven chapters discussing protein fundamentals, se-
quence analysis, structure analysis, structure prediction, folding, stability, and pro-
tein interactions. Each chapter presents an overview of the main problem-solving
approaches and tools. All chapters provide an overview of the currently available
on-line tools, and many of them are illustrated with examples. The methods include
both basic and advanced methodologies, which help the reader become familiar
with the manifold approaches that characterize this varied and interdisciplinary
xv

xvi Foreword
field. At the end of each chapter, there is a list of exercises and problems that can
be directly used in teaching courses.
Bioinformatics is a fast-evolving field. The author, Michael Gromiha, chose a
well-defined target within this changing scenery and made an excellent summary
ofthecomputationaltoolsofproteinbioinformaticsasweseeittoday.Theapproach
is unique in the sense that it covers all aspects of proteins and their interactions in
a coherent manner, and it provides a concise but comprehensive overview of the
pertinent databases and Web servers. Students and researchers in protein research,
bioinformatics, biophysics, computational biology, molecular modeling, and drug
design will find this easy-to-understand book a ready reference for staying current
and productive in this changing, interdisciplinary field.
It is with these thoughts that I recommend this well-written book to the reader.
Sándor Pongor
International Centre for Genetic engineering and Biotechnology
Trieste, Italy

Preface
Proteins are one of the most versatile molecules in living organisms. The struc-
tural and functional roles of proteins have been investigated through several ap-
proaches and different points of view. It ranges from the experimental studies such
as the characterization of proteins using biochemical and molecular biological as-
pects, determination of protein structures, understanding the stability of proteins
at extreme conditions and mutations, and designing drugs using the concept of
protein–ligand interactions to numerous computational techniques for the analy-
sis of protein sequences and structures, folding behavior, stabilizing interactions,
prediction of protein structures, and docking studies.
The recent advances in information technology have enhanced the computa-
tional power as well as information exchange with rapid progress. This changed
the trend of developing simple methods to complicated machine learning tech-
niques to attain the highest accuracy between the data obtained from experiments
and computational models. The availability of high-speed computers with extreme
storage capacity has reduced the gap between experiment and theory.
The development of databases on various aspects and tools for different types
of analysis, and the availability of both of them on the Web considerably reduced
the work of experimentalists to search for the suitable sample and opened a way to
the bioinformatists to deepen the study. Based on the variety of investigations on
proteins and their importance on clinical aspects, it is necessary to compile most of
thebioinformatics-relatedworksonproteins,whichwillbeusefultoboththeexper-
imentalists as well as the bioinformatics/computational biology researchers. This
book will be of immense use and a valuable guide to the students and researchers
working on proteins and to those who have the interest to work on proteins. Fur-
thermore, it provides a comprehensive survey of literature and applications to the
students who are studying/interested on bioinformatics.
The main feature of the book is the coverage of all aspects of proteins, such as
sequence and structure analysis, prediction of protein structures, protein folding,
protein stability, and protein interactions. All the sections have the illustrations
and Web addresses of available databases and online tools, and the details to con-
struct the data and derive the important parameters. In addition, the literature has
been thoroughly surveyed from the beginning, and important methods have been
highlighted in detail.
xvii

xviii Preface
Chapter 1 provides the general introduction of proteins, structural organiza-
tions, databases for protein sequences and structures, and their applications. This
will give an overall idea about the availability of public domain resources to the
students and researchers.
Chapter 2 illustrates the works that can be done with protein sequences. This
includes the alignment of sequences, position specific scoring matrices, and the
delineation of several amino acid properties based on just sequence. Furthermore,
the available servers for analyzing protein sequences have been discussed.
Chapter 3 demonstrates the plenty of analyses carried out with known three-
dimensional structures of proteins. It includes the assignment of secondary struc-
tures, computation of solvent accessibility to obtain the information about the
residues that are in the interior of the protein and at the surface, contacts between
amino acid residues in protein structures, development of contact potentials, clus-
ters of amino acid residues in folded environment, and free energy calculations. In
addition, the comparison of protein structures using different methods has been
outlined with illustrated examples.
Chapter 4 deals with protein folding kinetics with the introduction of -value
analysis and the relationship between values and amino acid properties. In addi-
tion, the importance of protein folding rates and the parameters that are influencing
the folding rates of proteins have been surveyed. Furthermore, the relationship be-
tween values and folding rates has been outlined.
Chapter 5 focuses on the prediction of protein structures, that includes the
discrimination of different structural classes of globular and membrane proteins,
secondary structure content, secondary structural regions, contacts between amino
acidresidues,solventaccessibility,andthethree-dimensionalstructuresofproteins.
Chapter 6 is devoted to protein stability beginning with the determination of
protein stability using experimental techniques and the availability of databases for
protein stability. The relative contributions of noncovalent interactions to protein
stability and the factors influencing the stability of thermophilic proteins have been
surveyed. The development of different methods for discriminating the stabilizing
and destabilizing effect of proteins upon amino acid substitutions and predicting
the stability change upon mutations has been elaborated.
Chapter 7 enlightens the importance of protein interactions, such as protein–
protein, protein–nucleic acid, and protein–ligand interactions. The availability of
databases for the binding of proteins with other molecules has been listed, and the
analysis carried out to extract the principles governing the interactions has been
included. Furthermore, the development of Web servers for predicting the binding
sites in protein and their binding affinities has been explained.
Appendix A includes the collective lists of databases and Web servers, which
are important in the field of protein bioinformatics. The exercises given in each
chapter will be an added value to the book. In essence, this book would be a
valuable resource to students and researchers to deepen the knowledge about the
studies on proteins.
M. Michael Gromiha

Acknowledgments
I am deeply indebted to Professor P.K. Ponnuswamy, who introduced me to the
field of proteins, and encouraged to deepen the knowledge in protein research.
I am grateful to Dr. S. Pongor, Dr. A. Sarai, Dr. Y. Akiyama, Dr. R. Majumdar,
Dr. M. Lakshmanan, and Dr. K. Asai for their continuous support and advice.
My sincere thanks to my collaborators Dr. S. Ahmad, Dr. M. Babu, Dr. K. Fukui,
Dr. L-T. Huang, Dr. D. Jacobs, Dr. H. Kono, Dr. S. Kumar, Dr. T.S. Kumarevel, Dr. C.
Magyar, Dr. M. Oobatake, Dr. Y-Y. Ou, Dr. D.A.D. Parry, Dr. V. Parthiban, Dr. M.N.
Ponnuswamy, Dr. G. Pujadas, Dr. G.P.S. Raghava, Dr. S. Selvaraj, Dr. I. Simon, Dr.
M. Suwa, Dr. Y-h. Taguchi, Dr. H. Uedaira, and Dr. Y. Yabuki, who helped me in
several ways at different stages of the work.
I extend my gratitude to Dr. Dr. P. Chakrabarti, Dr. E. Gaucher, Dr. I. Ghosh,
Dr. E. Ortlund, Dr. Pal, Dr. K. Veluraja, and Dr. D. Velmurugan as well as my
collaborators for their comments on various chapters in the book.
It is my pleasure to acknowledge my wife A. Mary Thangakani, sons Michael
Mozim, and Michael Abejo, and daughter Angela Shalom, who have spared their
committed time and allowed me to work on the book. I also immensely acknowl-
edge my mother for her valuable guidance.
Finally, I thank all my well wishers and friends who encouraged me to write
this book.
xix

CHAPTER 1
Proteins
Proteins perform a variety of functions, including enzymatic catalysis, transport-
ing ions and molecules from one organ to another, nutrients, contractile system of
muscles, tendons, cartilage, antibodies, and regulating cellular and physiological
activities. The functional properties of proteins depend on their three-dimensional
structures.Thenativestructureofaproteincanbeexperimentallydeterminedusing
X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, electron
microscopy, etc. Over the past 40 years, the structures of more than 53,000 pro-
teins (as of May 12, 2009) have been determined. On the other hand, the amino
acid sequences are determined for more than eight million proteins (as of May 5,
2009). The specific sequence of amino acids in a polypeptide chain folds to generate
compact domains with a particular three-dimensional structure. Anfinsen (1973)
stated that the polypeptide chain itself contains all the information necessary to
specify its three-dimensional structure. Deciphering the three-dimensional struc-
ture of a protein from its amino acid sequence is a long-standing goal in molecular
and computational biology.
1.1 Building blocks
Protein sequences consist of 20 different kinds of chemical compounds, known
as amino acids, and they serve as building blocks of proteins. Amino acids con-
tain a central carbon atom (C), which is attached to a hydrogen atom, an amino
group (NH2), and a carboxyl group (COOH) as shown in Figure 1.1. The letter R in
Figure 1.1 indicates the presence of a side chain, which distinguishes each amino
acid.
1.1.1 Amino acids
Amino acids are naturally of 20 different types as specified by the genetic code
emerged from DNA sequences. Furthermore, nonnatural amino acids occur, in
rare cases, as the products of enzymatic modifications after translocation. The major
difference among the 20 amino acids is the side chain attached to the C through its
fourth valance. The variation of side chains in 20 amino acids is shown in Figure 1.2.
These residues are represented by conventional three- and one-letter codes. Most
of the databases use single-letter codes.
The amino acids are broadly divided into two groups, hydrophobic and hy-
drophilic, based on the tendency of their interactions in the presence of water
1

2 Chapter One
H2N Cα COOH
H
R
Amino group Carboxyl group
FIGURE 1.1 Representation of amino acids. R is the side chain that varies for the 20 amino acids.
molecule. The hydrophobic residues have the tendency of adhering to one another
in aqueous environment. Generally, amino acids, Ala (A), Cys (C), Phe (F), Gly (G),
Ile (I), Leu (L), Met (M), Val (V), Trp (W), and Tyr (Y), are considered as hydrophobic
residues. In this category, Ala, Ile, Leu, and Val contain aliphatic side chains; Phe,
Trp, and Tyr contain aromatic side chains; and Cys and Met contain sulfur atom. Gly
has no side chain, and it has hydrogen (H) at the fourth position. Two Cys residues
in different parts of the polypeptide chain but adjacent to each other in the three-
dimensional structure of a protein can be oxidized to form a disulfide bridge. The
Glycine
Gly, G
Alanine
Ala,A
Hydrophobic residues
Leucine
Leu, L
Isoleucine
Ile, I
Valine
Val,V
Aliphatic
H2N Cα COOH
H
H
H2N Cα COOH
H
CH3
H2N Cα COOH
H
CH2
CH
CH3
CH3
H2N Cα COOH
H
HC CH3
CH2
CH3
H2N Cα COOH
H
CH
CH3 CH3
Phenylalanine
Phe, F
H2N Cα COOH
H
CH2
H2N Cα COOH
H
CH2
OH
Tyrosine
Tyr,Y
H2N Cα COOH
H
Tryptophan
Trp, W
Methionine
Met, M
H2N Cα COOH
H
CH2
S
CH2
CH3
Cysteine
Cys, C
H2N Cα COOH
H
CH2
SH
Aromatic Sulfur containing
Hydrogen
CH2
FIGURE 1.2 The common 20 amino acids in proteins. The three- and one-letter codes for the
amino acids are also given. The amino acids are classified into hydrophobic (hydrogen, aliphatic,
aromatic, and sulfur containing) and hydrophilic (negatively charged, positively charged, and
polar). The side chains are marked with oval boxes.

Proteins 3
Aspartic acid
Asp, D
Glutamic acid
Glu, E
Hydrophilic residues
Histidine
His, H
Lysine
Lys, K
Positive charged
Cα COOH
H
CH2
Cα COOH
H
(CH2)4
+
NH3
Asparagine
Asn, N
Glutamine
Gln, Q
Serine
Ser, S
Threonine
Thr, T
Proline
Pro, P
Polar
Negative charged
H2N Cα
H2N Cα H2N Cα
COOH
H
CH2
COOH
H2N
H2N H2N H2N
Cα COOH
H
CH2
CH2
COOH
COOH
H
CH2
O
NH2
C
H2N Cα H2N Cα
COOH
H
CH2
OH
COOH
H
C
CH3
H OH
HN Cα COOH
H
CH2
H2C
CH2
COOH
H
CH2
CH2
O
NH2
C
Arginine
Arg, R
Cα COOH
H
NH
NH2
NH2
C
+
(CH2)3
N
HN
FIGURE 1.2 (Continued)
formation of disulfide bridges in protein structures stabilizes the protein, making
it less susceptible to degradation.
Amino acids, Asp (D), Glu (E), His (H), Lys (K), Asn (N), Pro (P), Gln (Q), Arg
(R), Ser (S), and Thr (T), are classified as hydrophilic residues. In this category,
Asp and Glu are negatively charged; His, Lys, and Arg are positively charged; and
others are polar and uncharged.
1.1.2 Formation of peptide bonds
The carboxyl group of one amino acid interacts with the amino group of another
to form a peptide bond by the elimination of water (Figure 1.3). Amino acids are
joined end-to-end during protein synthesis by the formation of such peptide bonds.
The peptide bond (C N) has a partial double-bond character due to resonance,
and hence there is no rotation about the peptide bond. In Figure 1.3, the peptide
is represented as a planar unit with the C O and N H groups positioning in
opposite directions in the plane. This is called trans-peptide. There is another form,
cis-peptide in which the C O and N H groups point in the same direction. To
avoid steric hindrance, the trans form is frequently presented in protein structures
for all amino acids except Pro, which has both trans and cis forms. The cis prolines
are found in bends of the polypeptide chains.

4 Chapter One
H2N Cα COOH
H
H2N Cα
H
Cα
H
Cα
H
R1
H N COOH
R2
N COOH
R2
H
+
C
R1 O
−H2O
H
Peptide bond
Carboxyl Amino
Peptide
FIGURE 1.3 Formation of a peptide bond by the elimination of a water molecule.
A protein chain is formed by several amino acids in which the amino group of
the first amino acid and the carboxyl group of the last amino acid remain intact,
and the chain is said to extend from the amino (N) to the carboxyl (C) terminus.
This chain of amino acids is called a polypeptide chain, main chain, or backbone.
Amino acids in a polypeptide chain lack a hydrogen atom at the amino terminal
and an OH group at the carboxyl terminal (except at the ends), and hence amino
acids are also called amino acid residues (simply residues). Nature selects the
combination of amino acid residues to form polypeptide chains for their function,
similar to the combination of alphabets to form meaningful words and sentences.
These polypeptide chains that have specific functions are called proteins.
1.2 Hierarchical representation of proteins
Depending on their complexity, protein molecules may be described by four lev-
els of structure (Nelson and Cox, 2005): primary, secondary, tertiary, and quater-
nary (Figure 1.4). Because of the advancements in the understanding of protein
structures, two additional levels such as supersecondary and domain have been
proposed between secondary and tertiary structures. A stable clustering of several
elements of secondary structures is referred to as a supersecondary structure. A
somewhat higher level of structure is the domain, which refers to a compact region
and distinct structural unit within a large polypeptide chain.
1.2.1 Primary structure
Primary structure describes the linear sequence of amino acid residues in a pro-
tein. It includes all the covalent bonds between amino acids. The relative spatial
arrangement of the linked amino acids is unspecified.

Proteins 5
Primary
structure
Secondary
structure
Tertiary
structure
Quaternary
structure
Met
Ile
Lys
Ala
Asn
Ile
Amino acid sequence α-helix, β-strand
3D structure with
atomic details
Assembled
subunits
FIGURE 1.4 Structural organization of proteins.
1.2.2 Secondary structure
Secondary structure refers to regular, recurring arrangements in space of adjacent
amino acid residues in a polypeptide chain. It is maintained by hydrogen bonds
between amide hydrogens and carbonyl oxygens of the peptide backbone. The
major secondary structures are -helices and -structures.
The -helical conformation was first proposed by Linus Pauling and co-workers
(1951), and a typical -helix is shown in Figure 1.5. In this structure, the polypeptide
backbone is tightly wound around the long axis of the molecule, and R groups of
the amino acid residues protrude outward from the helical backbone. The repeating
Hydrogen bond
Cα
N
C
R
O
H
H
FIGURE 1.5 Structure of a typical -helix. The hydrogen bonds between the residues n and n + 4
are shown as dotted lines. Figure was taken as a screenshot from the Web, http:/
/www.food-info.
net/uk/protein/structure.htm

6 Chapter One
(a) Antiparallel β-sheet
(b) Parallel β-sheet
Hydrogen bond
FIGURE 1.6 Structures of (a) antiparallel and (b) parallel -sheets. The dotted lines show the
hydrogen bonds between amino acid residues. The arrows indicate the directions of the
polypeptide chain, from N- to C-terminal. Figure was taken as a screenshot from the Web,
http:/
/www.food-info.net/uk/protein/structure.htm.
unit is a single turn of a helix, which extends about 0.54 nm along the axis, and
the number of amino acid residues required for one complete turn is 3.6. In an
-helix, each carbonyl oxygen (residue, n) of the polypeptide backbone is hydrogen
bonded to the backbone amide hydrogen of the fourth residue further toward the
C-terminus (residue, n + 4). The hydrogen bonds, which stabilize the helix, are
nearly parallel to the long axis of the helix.
The other common secondary structure is -structure that includes -strands
and -sheets. -strands are portions of the polypeptide chain that are almost fully
extended, and several -strands constitute -sheets. -sheets are stabilized by hy-
drogen bonds between carbonyl oxygens and amide hydrogens on adjacent -
strands (Figure 1.6). In -sheets, the hydrogen bonds are nearly perpendicular to
the extended polypeptide chains. The -strands may be either parallel (running in
the same N- to C-terminal) or antiparrallel (running in opposite N- to C-terminal
directions).
In a polypeptide chain, the -carbon atoms of adjacent amino acids are
separated by three covalent bonds arranged as C C N C. In these bonds,
rotation is permitted about the N C and C C bonds, and the torsional angles
are conventionally denoted as and , respectively. Every secondary structure
is described completely by these two torsional angles that are repeated at each
residue. The allowed values for and can be shown graphically by simply
plotting these values known as Ramachandran plot (Ramachandran et al. 1963).
Figure 1.7 shows the conformations that are permitted for most amino acid
residues in Ramachandran plot.

Proteins 7
FIGURE 1.7 Ramachandran plot showing the allowed regions of -helical and -strand
conformations. Figure was taken as a screenshot from the Web, http:/
/swissmodel.expasy.org/
course/text/chapter1.htm.
1.2.3 Tertiary structure
Tertiary structure refers to the spatial relationship among all amino acids in a
polypeptide; it is the complete three-dimensional structure of the polypeptide with
atomic details. Tertiary structures are stabilized by interactions of side chains of
nonneighboring amino acid residues and primarily by noncovalent interactions.
The formation of tertiary structure brings the amino acid residues that are far apart
in the primary structure close together.
1.2.4 Quaternary structure
Quaternary structure refers to the spatial relationship of the polypeptides or sub-
units within the protein. It is the association of two or more polypeptide chains
into a multisubunit or oligomeric protein. The polypeptide chains of an oligomeric
protein may be identical or different. The quaternary structure also includes the
cofactor and other metals, which form the catalytic unit and functional proteins.
1.3 Structural classification of proteins
Proteins are broadly classified into two major groups: fibrous proteins, having
polypeptide chains arranged in long strands, and globular proteins, with polypep-
tide chains folded into a spherical or globular shape.
1.3.1 Fibrous proteins
Fibrous proteins are usually static molecules and play important structural roles in
the anatomy and physiology of vertebrates, providing external protection, support,
shape, and form. They are water insoluble and are typically built upon a single,
repetitive structure assembled into cables or threads. Examples of fibrous proteins

8 Chapter One
(a) (b)
(c) (d)
FIGURE 1.8 Ribbon diagram for four typical protein structures in different structural classes (a)
all- (4MBN), (b) all- (3CNA), (c) + (4LYZ), and (d) / (1TIM). Figure was adapted from
Gromiha and Selvaraj (2004).
are-keratin,themajorcomponentofhairandnails,andcollagen,themajorprotein
component of tendons, skin, bones, and teeth.
1.3.2 Classification of globular proteins
Globular proteins are categorized into four structural classes: all-, all-, + ,
and / (Levitt and Chothia, 1976). The ribbon diagrams illustrating the structures
in each class are shown in Figure 1.8.
The all- and all- classes are dominated by -helices ( 40% and 5%) and
by -strands ( 40% and 5%), respectively (Figures 1.8a and b). The +
class contains both -helices (15%) and antiparallel -strands (10%) that do
not mix but tend to segregate along the polypeptide chain (Figure 1.8c). The /
class proteins (Figure 1.8d) have mixed or approximately alternating segments of
-helical (15%) and parallel -strands (10%).

Proteins 9
(a) (b)
M
e
m
b
r
a
n
e
Polar head
hydrophobic tail
FIGURE 1.9 Representation of (a) -helical and (b) -barrel membrane proteins. The membrane
spanning regions are shown within the disc. Protein structures were taken from Protein Data
Bank of Transmembrane Proteins (http:/
/pdbtm.enzim.hu/).
1.3.3 Membrane proteins
Membrane proteins, which require embedding into the lipid bilayers, have evolved
to have amino acid sequences that will fold with a hydrophobic surface in contact
with the alkane chains of the lipids and polar surface in contact with the aqueous
phases on both sides of the membrane and the polar head groups of the lipids
(Figure 1.9). In genomes, 30% of the proteins are suggested to be membrane pro-
teins, and most of the transmembrane helical and strand proteins are identified
as targets for drug design. Membrane proteins perform a variety of functions, in-
cluding cell–cell signaling and mediating the transport of ions and solutes across
the membrane. They are of two kinds: (i) transmembrane helical proteins in which
they span the cytoplasmic membrane with -helices (White and Wimley, 1999) and
(ii) transmembrane -barrel proteins that traverse the outer membranes of gram-
negative bacteria with -strands (Schulz, 2003). Figure 1.9 shows the structures of
membrane proteins with these two different motifs, -helices and -strands.
1.4 Databases for protein sequences
Recombinant DNA techniques have provided tools for the rapid determination of
DNA sequences and, by inference, the amino acid sequences of proteins from struc-
tural genes. The number of such sequences is increasing exponentially, and these
sequences have been deposited in the form of database, generally, known as protein
sequence databases. Specifically, Georgetown University, Washington, D.C., USA,
developed the database, Protein Information Resource (PIR). The Swiss Institute of
Bioinformatics and European Bioinformatics Institute developed SWISS-PROT and
TrEMBL databases. Recently, progress has been made to set up a single worldwide
database of protein sequence and function, UniProt, by unifying PIR, SWISS-PROT,
and TrEMBL database activities.
1.4.1 Protein Information Resource
PIR has evolved from the Atlas of Protein Sequence and Structure established
in the early 1960s by Margaret O. Dayhoff (Dayhoff et al. 1965). It produces the
largest, most comprehensive, annotated protein sequence database in the public

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What does that young boy Pao Yu do with himself
generally? asked the third Yu. Oh, it would be better to ask, what
does he not do! Certainly nothing very brilliant, replied Little Hsing
with a meaningful grin. He is utterly degenerate and has no interest
either in books or in arms. In the beginning his father, Mr. Cheng,
used to take some trouble with his upbringing, but he has given that
up long since. So the boy is left completely to his own devices.
Outwardly he is a very nice-looking, bright lad, and does not look at
all as if he is wanting in the top story, but when you have a good
look at him you can see that he's a weakwitted fool. He seems so
timid before strangers that he can hardly get out a sentence. He's a
real little sissy of a mother's darling and never feels at ease unless
he has a crowd of girls around him to fool about with and to pet and
cuddle. The poor soppy fellow lacks all seriousness and firmness of
character. With us servants, for instance, he's different every time,
according to his mood. If he's in a good humor he jokes with us as if
we were his equals and there was no such thing as difference of
class. If he's in a bad humor he retires into his shell and other
human beings simply do not exist for him any more. At such times
he doesn't even reprimand us if we behave improperly. Naturally, in
these circumstances it's impossible to have any respect for him.
You servants are certainly a difficult lot to handle, said the third
Yu, smiling. When you finally have a really goodhearted and
conscientious master or mistress you grumble and are discontented
with them. It's a pity about him, for he's a nice boy otherwise,
sighed the second Yu, compassionately. Oh, don't listen to all this
servants' gossip! continued the third Yu. Why, we have met him
once or twice ourselves, and have been able to form our own
judgment of him. There is certainly something effeminate in his
nature and behavior, but that is not surprising when a boy grows up
only among women and girls. But to call him weakminded and a fool
— no, that is quite ridiculous and unjust! Don't you remember when
we met him just recently at the funeral services at the bier of the

Prince Hermit? We girls were all standing together in a group. Then
when the monks filed in and took up their position all round the
coffin, Pao Yu came in and stood right in front of our group. That
was remarked upon unfavorably by everyone. Had he no eyes in his
head, and was it not most inconsiderate to stand so awkwardly right
in our way, blocking the view from us, people said. But afterwards
he told us the reason for his strange behavior. He knew perfectly
well what was correct, and he had very good eyes in his head, so he
said, but he had deliberately pushed himself in between us and the
monks so that we should not be treated too directly to their
unsavory odor. And 357

accurate
later, when you asked for a drink of tea and the serving
woman began to fill up the bowl for you which he had just drunk out
of, he stopped her and ordered her to rinse the bowl with water first.
From these two little incidents, which I observed dispassionately, I
could see that he is, in fact, a person of very fine feelings, and that
in the presence of young girls he has only eyes and thoughts for us
and forgets everything else on our account. Naturally, outsiders who
do not know his nature cannot understand his behavior. Hearing
you talk that way, it seems to me that you and he are already of one
mind, said the elder Yu gaily. How would it be if you and he were
to become betrothed? Because of the presence of the servant Little
Hsing, the third Yu chose to remain silent, and sat with bent head,
nibbling melon seeds. Yes, he would suit the young lady quite well,
interposed the presumptuous Little Hsing, but unfortunately he is
already disposed of. It's an open secret, after all, that Miss Ling is his
intended. Of course they're both very young yet, and besides, Miss
Ling has been very sickly of late. But in two or three years the old
Tai tai will no doubt open her mouth and settle the matter. While he
was saying this the servant Little Lung came back from the Yungkuo
palace. Prince Shieh is sending his son Chia Lien on an important
mission to Ping an Chow, he reported. He will set out in three or
four days and will be away for ,abouttwo weeks. He therefore asks
to be excused for today, as he is occupied with urgent preparations
for the journey, and he asks the Nai nai please to go ahead alone
with the matter she knows of. As soon as he comes back he will take
it up again. The two servants, Little Lung and Little Hsing, now
withdrew. The second Yu bolted the hall door behind them and she,
her mother, and her sister went early to bed, but before going to
sleep she questioned the younger sister persistently until she found
out from her the name of the man whom she wanted for her
bridegroom. When Chia Lien made a brief dash over early the next
day and wanted to know the name of the third Yu's chosen man as
quickly as possible, the second Yu replied with a laugh: There's no

hurry; the man in question is far away now, heaven knows where
and for how long. My sister declares that even if he stays away a
year, she will wait for him a year; if it is ten years until he returns,
she will wait patiently the ten years, but that if he dies in the
meantime she will have her hair cut off and go into a convent; but
she will never marry any other man, whatever happens. Who,
then, is this fellow who has taken her heart so completely? asked
Chia Lien, impatiently. Oh, that is a long story, but I will make it as
short as I can. It be358

accurate
gan five years ago in Grandmother's house on her birthday.
There was a theatrical performance in celebration of the day. The
performers were not professional actors, but all sons of good
families. Among them was one who played the part of the youthful
heroine. He is said to have fled from the town a long while ago on
account of some trouble or other, and to be travelling in distant
parts. His name is Liu Hsiang Lien. Oh, so that's the man! A fine,
handsome fellow. Your sister has good taste and g'ood eyes; one
must say that for her. But he's of a cold, proud nature. He has no
time for ordinary people, but he gets on splendidly with Pao Yu. Last
year he had a fight with our wild cousin Hsueh Pan, and to save our
family from further awkward incidents he simply disappeared from
the town immediately afterwards without leaving a trace. Perhaps
one could find out something about his whereabouts through Pao
Yu's servant, but he is certainly a drifting water plant. It may well be
years before it occurs to him to show his face here again, and your
sister may perhaps wait in vain. She would do much better to put
him right out of her mind. You seem not to know me yet, brother-
in-law, interrupted the third Yu, suddenly coming in from an
adjoining room. I'm in the habit of saying what I mean. It is either
Mr. Liu or no one, and that's that! From today on I will devote myself
to prayer and fasting and looking after my mother, while I wait for
him, even if I have to wait a hundred years. And if I do not mean
this sincerely, may my fate be the fate of this clasp! she continued
solemnly. Drawing a jade clasp from her hair, she broke it in two,
then disappeared into the adjacent room again. Chia Lien had to
resign himself to her decision. He tried to find out something about
the whereabouts of the Cold Knight through Pao Yu's personal
servant, Ming Yen, but Ming Yen knew nothing; and his inquiries in
the young man's former locality were equally fruitless. Shortly
afterwards Chia Lien set out on his journey to the prefecture of Ping
an Chow, after having spent two more nights secretly in the Lane of
the Flowering Branch. He had been three days on the way when a

trading caravan came towards him. As the two parties drew close he
perceived to his astonishment that his cousin Hsueh Pan was one of
the dozen men on horseback escorting the caravan. And what
astonished him still more as to see, riding peacefully by his side,
his former enemy, the Cold Knight. After mutual greetings, both
parties turned in to rest in a near-by inm How comes it that I see
you two former tilters and adversaries so peacefully united now?
asked Chia Lien. My caravan was attacked and looted by a band of
robbers in the neighborhood of Ping an Chow. Then just by chance
and in the nick 359

accurate
of time along came Brother Liu to our rescue with his party.
He put the robbers to flight, recovered the booty from them by force
of arms, and saved the lives of myself and my people. As he scorned
my thanks, I offered him blood brotherhood. And so we have
become true confederates and blood brothers forever and have been
travelling part of the way together. But our ways must soon part
again. I am going back to the capital, and he is going two hundred li
farther south to visit an aunt of his. As soon as I have finished my
business at home I intend to obtain a bride and a house for him and
to persuade him to settle down permanently in the capital. A
bride? Oh, that is splendid! I can suggest a suitable party straight
away, interposed Chia Lien eagerly. And he told of his secret
marriage to the second Yu sister and of the younger sister who was
still unwed. He refrained, however, from saying that the third Yu
sister had herself chosen the Cold Knight as her husband, and he
also prevailed upon his cousin not to breathe a word of the whole
story when he got home. You should have hit upon that sly plan of
yours long ago ! It serves my strait-laced cousin Phoenix quite
right! said Hsueh Pan maliciously; and gave his ready approval to
the new marriage plan too. But the Cold Knight declared: That is all
very well, but I have firmly resolved to marry only a really
outstanding beauty. I set less value upon money and lineage than
upon this. I shall have to make sure on this point before I can agree
to your suggestion. Oh, you can be quite easy on that score, Chia
Lien assured him zealously. But what are words? You must see her
for yourself, and then you will be convinced! It would be hard to find
another girl who could compare with her for beauty. Very well, I
agree; and I shall be in the capital in about two months' time, but I
want to visit my aunt first, declared the Cold Knight. I accept your
word, but you are a restless fellow, a drifting water plant. Would you
not prefer to back up your word in a visible way with some kind of
betrothal gift? suggested Chia Lien with a smile. Do not worry,
brother-in-law; you may rely upon my word. I am not in funds just

now, and besides I am travelling. Where would I get a suitable gift
right away? replied the Cold Knight. Let me procure the gift!
suggested Hsueh Pan. It need not be expensive gold and silver and
jewelry straight away. Any little trifle which you are wearing will do
just as well. Only it is best that it should be a personal souvenir of
yourself, for otherwise she may possibly not believe me, insisted
Chia Lien. Very well, then. She shall have my twin-blade sword. It is
an old 360

accurate
heirloom of my family which I have always treasured
faithfully, said the Cold Knight. They drank a few more glasses
together, then broke up and went their opposite ways. Chia Lien
carried out his mission to the Prefect of Ping an Chow and was back
again in the capital after two weeks. His first visit was to the Lane of
the Flowering Branch. Apart from two surreptitious and completely
fruitless visits from the princely brother-in-law, nothing of any
consequence had happened during his absence. The three ladies Yu
had remained quietly at home behind closed doors and passed the
time busy with their needles. Chia Lien proudly reported his
successful meeting with ttie Cold Knight and handed over to the
third Yu her betrothal gift, the ''duck couple sword with the twin
blades. She examined her strange betrothal gift more closely. One
single sheath studded with pearls and jewels concealed two
completely identical, coldly glistening, sharp-edged blades. The
symbol yuan, signifying little drake, was engraved on the one, and
the symbol yang, signifying little duck, on the other. The third Yu
was overjoyed. She took the twin sword and carried it into her
maiden chamber, where she hung it on the wall over her bed, for
she wished to have it continually before her eyes so that the sight of
it might strengthen and support her until the day when the beloved
himself would cpme to lead her to his home. Chia Lien remained for
two days with his Nai nai in the Lane of the Flowering Branch; only
then did he go to the western palace to present his report to his
father and to greet Phoenix. She had recovered meantime from her
long illness and was able to go out again, and to resume her
accustomed activities in the household. Finally he visited Prince Chen
and informed him of the successfully concluded betrothal of the third
Yu. The Prince listened to him rather indifferently and
unsympathetically. He was out of humor because of the failure of his
own efforts to win the favor of his beautiful sister-in-law. So he
confined himself to putting his hand in his pocket and taking out a
few dozen taels as his subscription towards the young lady's dowry,

leaving all the rest of the arrangements to his cousin Chia Lien. In
the eighth month the Cold Knight did in fact turn up once more in
the capital. On his first day there he visited Aunt Hsueh and his new
friend Hsueh Pan, who was in bed with a severe cold in the head.
Aunt Hsueh no longer bore him any grudge for his past quarrel with
her son, and now regarded him only as his friend and rescuer and
loaded him with thanks and attentions. In order to pay off some part
of their debt of gratitude, mother and son had undertaken to meet
all the ex361

accurate
penses of the coming marriage out of their own pockets, an
arrangement in which the Cold Knight gladly acquiesced. The next
day he paid a visit of friendship to Pao Yu and wanted to hear more
details about Chia Lien's secret marriage to the second Yu, but Pao
Yu too knew only a little about it. from hearsay through Ming Yen.
And in any case I should prefer to keep as clear of the delicate
matter as possible, said Pao Yu, parrying his questions. But tell me
about yourself. I hear that you met Cousin Chia Lien on the way to
Ping an Chow and had an important conversation with him. What
was it about, by the way? The Cold Knight told of his betrothal to
the third Yu, which had been agreed upon on the journey. Oh, you
are to be congratulated! Pao Yu assured him fervently. She is really
a ravishing beauty — a worthy partner for you. Oh, indeed? If she
is as beautiful as all that I cannot understand why a poor fellow like
me should fall to her lot. Besides, I am by no means on intimate
terms with her brother-in-law, said the Cold Knight thoughtfully.
The suspicion that a former light-of-love of Prince Chen's was being
foisted upon him suddenly crept into his mind. It really puzzles me
that I was pressed into this engagement in the course of our brief
chance meeting on the road. I cannot imagine a girl like that running
after a man. I cannot help feeling suspicious about the whole thing,
and now I am almost sorry that I handed over my sword as a
pledge. I should have preferred to make some inquiries myself first.
Your doubts are certainly quite unfounded. First you insist upon
getting an outstanding beauty, and now when you have got her you
begin to falter. Take her, and do not hesitate any longer ! Are you
so sure, then, that she is beautiful? For it seems to me that you do
not appear to know much more than I do about her person and her
family. She is a stepsister of Princess Chen, whose maiden name
was Yu. During the recent funeral solemnities in the eastern palace I
met her and her mother and sister I do not know how many times,
so I surely must know. The Cold Knight stamped his foot angrily.
Do not talk to me about your eastern palace! he cried. The only

creatures there that are not disreputable are the two marble lions in
front of the main gateway. No! The whole business seems most
shady to me. I will not go through with it! But he immediately
repented of his offensive outburst when he noticed the embarrassed
flush on his friend's cheeks. Bowing ceremoniously before him, he
hastened to apologize. I have 362

accurate
let my tongue run away with me, and I deserve death for
my bad manners, he said. But tell me at least something about her
character. Oh, please, why do you keep on asking me? Apparently
you are better informed than I am. Besides, I myself may perhaps
be disreputable and untrustworthy? Please do not be resentful. I
forgot myself. Very well. We shall say no more about it! The Cold
Knight perceived that he would get nothing more out of his offended
friend, so he bowed once more briefly and formally and took his
leave. He was determined to cancel the overhasty betrothal, which
seemed to him more and more questionable. He set out straight
away to report his decision to Chia Lien, who at this hour was with
the second Yu in the Lane of the Flowering Branch. The
unsuspecting Chia Lien received him in the most friendly and familiar
manner, and led him straight into the drawing room of the ladies of
the house, where 'he introduced him to his future mother-in-law. He
was not a little surprised at the stiff and formal manner in which 'his
presumptive brother-in-law bowed to Mother Yu, and still more
surprised when he heard him addressing her not as mother-in-law
but as old aunt, and referring to himself not as son-in-law but
simply as the later born one. But he was dumfounded when during
tea the Cold Knight suddenly said without any warning: Our recent
agreement was an overhasty, chance arrangement. My aunt, whom I
visited shortly afterwards, had already chosen my future bride and
she wishes me to marry her in the fourth month. Respect for my
aunt demands that I should fall in with her wishes. In, these
circumstances I must reluctantly renounce our agreement and
request the return of my pledge. If it had been any kind of ordinary
ornament, I would not insist upon its return; but that sword is
precious and sacred to me as a heritage handed down by my fathers
and ancestors. I must therefore reluctantly ask for its return. Chia
Lien could hardly believe his ears when he heard him speaking in
this way. My brother is mistaken: an agreement is an agreement,!'
he cried, aghast. That is just why I insisted upon a betrothal gift —

so that there would be a visible pledge if you should regret your
word. How would it be if every betrothed man could back out of his
betrothal whenever he liked? No! What you ask is impossible! I am
ready to make any satisfaction that my brother may stipulate, but I
must have my sword back, insisted the Cold Knight with a smile.
Chia Lien was about to make some reply, but the Cold Knight stood
up and asked him to step outside in order to avoid continuing the
dispute in the presence of Mother Yu. The two were just about to
leave 363

accurate
the room when the door leading to the adjacent room
suddenly opened and the third Yu walked in. She had been listening
to the whole argument from the next apartment. So she had been
waiting and waiting for the beloved, only to hear now that he wished
to have nothing to do with her! Perhaps he had heard an
unfavorable report of her in the Yungkuo palace? Perhaps she had
been described to him as a shame-less hussy, unworthy to become
his lawful wife? If she let him go now without further ado, there
would probably be a fierce quarrel outside on her account between
himself and Chia Lien. She did not want that. Suddenly resolved, she
tore the double sword down from the wall, drew out the female
blade, and, hiding it under her right arm, walked into the room,
carrying the male blade in the sheath. Your quarrel is
unnecessary; I return my betrothal gift voluntarily, she said in a
voice choked with tears, handing the Cold Knight the sheath with the
male blade. At the same time she drew out the hidden female blade
and, with a powerful thrust, pressed it into her delicate throat. The
others sprang forward aghast and tried to stop her, but it was
already too late. The petals of the broken peach blossom were
already falling in red splashes over the ground; the crumbling
nephrite h1* a would never rise again ! After the first shock the
mother of the dead girl raised a loud lamentation and broke out in
bitter curses and revilements of the Cold Knight. At the same time
Chia Lien took hold of him and ordered his servants to bind him with
ropes and drag him before the Court, but the second Yu dried her
tears in haste and made him desist from his intention. 'My sister did
what she did of her own free will; the gentleman has exercised
neither pressure nor force upon her, she said. To bring the matter
before the Court would not undo what is done, and would only bring
discredit on our house. Let the gentleman go his way! Chia Lien had
to admit that she was right, and he let the Cold Knight go. But the
latter remained standing where he was, as if stunned, wiping away
the tears which ran slowly down his cheeks. What a magnificent,

heroic girl! Who would have thought such a thing could happen? I
curse myself, bringer of woe! he murmured to himself, lost in
thought. And he remained there as if rooted to the spot until a coffin
was brought and the hapless girl was laid in it. Then, with his hand
on the coffin, he broke into loud lamentations, which continued for
some time. Having thus paid the dead girl her due tribute of
reverence, he took his leave and walked slowly away. Sunk in
thought and tortured with self-reproach, he walked on and on,
heedless of either road or goal. Suddenly he heard a gentle sound
like the rustling of silken garments 364

accurate
and the tinkling of golden bracelets and jade belt
ornaments, and lo and behold, there stood the third Yu before him.
In one hand she held his double sword, in the other a parchment
scroll, and she said to him in a tone of lamentation: In the vain folly
of my love I waited for you for five years. Fool that I was, I did not
know that your heart was as cold as your glance. I have had to pay
for my foolish passion with my life. I am now in the service of the
Fairy of Fearful Awakening, who reigns in the Realm of the Great
Void. It is my task to register, with my comments, in the archives of
unpaid love-debts, notable cases of unhappy love. I have come to
bid you a last farewell, for to see each other again is denied us.
With tears in her eyes, she bowed to him in salutation. He threw
himself upon her and tried to embrace her and hold her fast, but she
pushed his hands gently away from her and eluded him. Thereupon
he began to sob loudly, and the sound awoke him, -for he had only
been dreaming. When he raised his eyes he saw around him the
ancient, crumbling walls of a ruined temple. By his side sat a man
attired in the dirty robe of a Taoist priest. He was lame in one foot,
and was busily engaged catching fleas. Who are you, Master, and
where are we? he asked the peculiarlooking stranger. I do not
know that myself, and in any case it does not matter; I only know
that we have just been having a brief rest, came the strange reply.
Suddenly illuminated with inward light, the Cold Knight shuddered
with icy horror, and, drawing his sword, he raised it to his head and
cut off his hair. Then he followed the uncanny stranger, he knew not
whither. CHAPTER 34 Phoenix cross-examines the servant and so
finds out the master's deceits. The unhappy Yu girl allows herself to
be hired into a trap. 1 HE THIRD Yu WAS COFFINED ON THE VERY
DAY OF HER DEATH AND was buried very quietly outside the city
walls. The news of her sudden death was brought to Aunt Hsueh's
ears by a waiting maid. Aunt Hsueh was profoundly grieved and
dismayed. She had been delighted to help arrange the wedding with
the third Yu out of gratitude to the Cold Knight for having saved

Hsueh Pan's life on the highroad. She had intended, moreover, to
give him a complete house with furniture as a wedding present.
What do you think of this sad, puzzling occurrence? she asked her
366

accurate
daughter Precious Clasp with a sigh. The poor thing was
engaged to the Cold Knight, your brother's sworn friend. And now,
shortly before the marriage, she has taken her life! And her fiance
has since disappeared without leaving a trace! That confirms once
more the truth of the old saying that human fate is as incalculable as
the course of the wind and the form of the clouds. Between evening
and morning our fate can suddenly change for good or for ill. It was
her predetermined destiny, concluded Precious Clasp
philosophically. But what is the use of brooding and complaining?
She is dead and it cannot be helped, and we cannot bring her back
again. Get over it and turn your attention to our own lives and our
own affairs ! Brother Hsueh Pan has already been back from the
South some weeks and meantime has probably sold the goods he
brought back. It is high time for him to give a feast for his
associates, who spent months travelling with him, and shared his
hardships and dangers, to celebrate the happy return home. You
should speak to brother Hsueh Pan and urge him to comply at last
with this duty, proper to his position. Mother Hsueh was about to
reply when the subject of discussion himself appeared. He looked
quite distraught and cried excitedly to the two women: Have you
heard the sad news yet? Cousin Yu dead! Brother Liu disappeared!
We have just been speaking about it. What a sad case! sighed
Aunt Hsueh. They say he has gone off with a strange Taoist monk.
That makes the story all the more weird. How on earth could such a
sensible young man as that Liu get such a crazy idea? As he is your
friend and is all alone in the world, you must exert yourself for him
and have him searched for. He will scarcely go far with that wretched
begging monk. Presumably he is hiding in some temple in the
neighborhood. My men and I have already searched for him
everywhere inside and outside the town; but there was no trace of
him anywhere. Good, then you have already satisfied your
obligations as a friend. But do not neglect your own affairs because
of all this. You are bound in gratitude to prepare a worthy feast for

your men, who travelled three thousand li and shared all troubles
and dangers faithfully with you for four or five months. They well
deserve it. Hsueh Pan agreed with her, and that same day he sent
out invitations to a number of his business managers and travelling
companions summoning them to a banquet. When the guests were
assembled round the table, one of them called out during the
banquet: Two of our good companions are still missing from the
table. Who, then? they were asked. 367

accurate
Mr. Chia Lien and our host's sworn friend, the Cold Knight.
Why have they not been invited? Hsueh Pan's face darkened and he
sighed deeply. My cousin set out for Ping an Chow again a short
time ago, he said, and as for my friend Liu, the mere mention of
his name gives me pain. It is a very strange story. He suddenly
renounced the world and disappeared in the company of a stranger,
a Taoist priest. And he related all he knew of the tragic outcome of
the engagement of his friend, and of his sudden disappearance.
How strange! they all said, and one of the business managers
went on to tell: A similar case was spoken of recently by the
customers in the shop. The story was of a monk who with three or
four sentences bewitched somebody and made him invisible. I do
not know any more details; we were busy serving and had no time
to pay attention to the idle gossip of customers. Oh, what
nonsense! This is sure to be quite a different kind of case, said
another, skeptically. A courageous, sensible, strong fellow, used to
arms like Mr. Liu, would certainly never allow himself to be
bewitched by a wandering monk. Presumably he only pretended to
join him in order to get behind his magic tricks and hocus-pocus. He
will undoubtedly get the upper hand of the swindler in the end.
That is probably what has happened! agreed Hsueh Pan warmly.
Really, these mountebanks who bluff and dupe people with their
tricks should be put down with an iron hand. Have you not had a
search made for your lost friend? they asked him. Yes, of course, I
have had him searched for everywhere, both inside and outside the
city walls; but — and you may laugh at this — he cannot be traced
anywhere, declared Hsueh Pan, becoming despond.ent once more.
The company around the table fell into an embarrassed silence;
nothing could be heard but long and short sighs, no one could be
merry any more, and after just a few more glasses of wine the party
broke up in depressed mood. A few days afterwards Pearl went to
visit Phoenix in a free hour to inquire after her health, but she did
not stay long. She thought she perceived a certain uneasiness in the

air, she noticed excited, whispering waiting maids and caught
something about cross-examinations to which these porters or those
servants had been subjected by Phoenix. Therefore she did not want
to be in the way. She had hardly gone when Phoenix sent for Little
Wang, the gatekeeper of the second gate. Well, how was it? What
have you heard? she asked the waiting maid Little Ping before Little
Wang arrived. 368

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