Preface
Molecular complexes of interacting proteins govern virtually all biological processes such as
metabolism, cell signaling, DNA repair, or gene expression. Macromolecular assemblies are
also of great biomedical relevance as factors that perturb biomolecular interaction networks
underlie a number of diseases, and deliberate inhibition of protein–protein interactions is an
increasingly common strategy in drug discovery initiatives. Unraveling their functions and
mechanisms of action is often only potentially accessible through a detailed structural
description and the integration of dynamic information. This volume of the Methods in
Molecular Biology series aims to provide the scientific community with strategies and detailed
protocols for the preparation of macromolecular complexes and their characterization in
view of structural analysis.
Protein engineering and production are essential tools for structural, biophysical, and
functional studies as well as for biotechnology and medical applications. Strategies to
prepare proteins and protein complexes have tremendously been improved in part thanks
to recent structural genomic programs. Yet, no universal solution has been implemented,
and the production and/or reconstitution of protein complexes remains a major bottleneck.
This is in particular the case for complexes composed of many subunits which are often
incompletely characterized. Additional difficulties result from their low natural abundance
or their versatile nature, in part because regulation often involves the formation of transient
complexes with low binding constants and in part because their composition varies with the
physiological context.
The first section of this book focuses on sample preparation. While Chapters 1 and
2 concentrate on strategies for recombinant expression of multiprotein complexes in prokaryotic and eukaryotic hosts, Chapter 3 illustrates how genome editing with the CRISPRCas9 system can be used to precisely modify protein coding genes in mammalian cells; this
offers the possibility to replace any coding gene by a reshaped version fused to an affinity tag
protein or to a fluorescent reporter, enabling the characterization of endogenous macromolecular complexes expressed under near physiological conditions. In the first section, the
production of recombinant antibodies and artificial binding proteins which emerge as key
reagents in multiprotein complex research, as inhibitors of protein–protein interactions to
modulate activity, as probes for cellular imaging or to facilitate structural determination is
also discussed. Chapter 4 details the production of recombinant antibodies in different
formats, Chapter 5 the intervening removable affinity tag (iRAT) system for the production
of recombinant antibody fragments, and Chapter 6 the isolation of artificial binding proteins
(Affimer reagents) based on a non-antibody scaffold.
The second section of the book details a set of biophysical methods that can provide
useful indicators for sample optimization and often complement structural information
obtained with core technologies for structure determination (X-ray crystallography, nuclear
magnetic resonance, and cryo-electron microscopy) by quantitative solution data, helping to
understand how biological systems function. Three chapters focus on techniques for the
biophysical characterization of biological macromolecules and their complexes. Chapter 7
details interaction measurements of protein–DNA complexes by isothermal titration calorimetry (ITC) and microscale thermophoresis (MST), Chapter 8 the use of the switchSENSE technology for the analysis of enzyme kinetics, and Chapter 9 the sedimentation
v
Molecular complexes of interacting proteins govern virtually all biological processes such as
metabolism, cell signaling, DNA repair, or gene expression. Macromolecular assemblies are
also of great biomedical relevance as factors that perturb biomolecular interaction networks
underlie a number of diseases, and deliberate inhibition of protein–protein interactions is an
increasingly common strategy in drug discovery initiatives. Unraveling their functions and
mechanisms of action is often only potentially accessible through a detailed structural
description and the integration of dynamic information. This volume of the Methods in
Molecular Biology series aims to provide the scientific community with strategies and detailed
protocols for the preparation of macromolecular complexes and their characterization in
view of structural analysis.
Protein engineering and production are essential tools for structural, biophysical, and
functional studies as well as for biotechnology and medical applications. Strategies to
prepare proteins and protein complexes have tremendously been improved in part thanks
to recent structural genomic programs. Yet, no universal solution has been implemented,
and the production and/or reconstitution of protein complexes remains a major bottleneck.
This is in particular the case for complexes composed of many subunits which are often
incompletely characterized. Additional difficulties result from their low natural abundance
or their versatile nature, in part because regulation often involves the formation of transient
complexes with low binding constants and in part because their composition varies with the
physiological context.
The first section of this book focuses on sample preparation. While Chapters 1 and
2 concentrate on strategies for recombinant expression of multiprotein complexes in prokaryotic and eukaryotic hosts, Chapter 3 illustrates how genome editing with the CRISPRCas9 system can be used to precisely modify protein coding genes in mammalian cells; this
offers the possibility to replace any coding gene by a reshaped version fused to an affinity tag
protein or to a fluorescent reporter, enabling the characterization of endogenous macromolecular complexes expressed under near physiological conditions. In the first section, the
production of recombinant antibodies and artificial binding proteins which emerge as key
reagents in multiprotein complex research, as inhibitors of protein–protein interactions to
modulate activity, as probes for cellular imaging or to facilitate structural determination is
also discussed. Chapter 4 details the production of recombinant antibodies in different
formats, Chapter 5 the intervening removable affinity tag (iRAT) system for the production
of recombinant antibody fragments, and Chapter 6 the isolation of artificial binding proteins
(Affimer reagents) based on a non-antibody scaffold.
The second section of the book details a set of biophysical methods that can provide
useful indicators for sample optimization and often complement structural information
obtained with core technologies for structure determination (X-ray crystallography, nuclear
magnetic resonance, and cryo-electron microscopy) by quantitative solution data, helping to
understand how biological systems function. Three chapters focus on techniques for the
biophysical characterization of biological macromolecules and their complexes. Chapter 7
details interaction measurements of protein–DNA complexes by isothermal titration calorimetry (ITC) and microscale thermophoresis (MST), Chapter 8 the use of the switchSENSE technology for the analysis of enzyme kinetics, and Chapter 9 the sedimentation
v