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Methods in
Molecular Biology 1860

Rutilio Fratti Editor

SNAREs
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY

Series Editor
John M. Walker
School of Life and Medical Sciences,
University of Hertfordshire,
Hatfield, Hertfordshire AL10 9AB, UK

For further volumes:

http://www.springer.com/series/7651
 SNAREs

Methods and Protocols

Edited by

Rutilio Fratti
Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, IL, USA
Editor
Rutilio Fratti
Department of Biochemistry
University of Illinois Urbana-Champaign
Urbana, IL, USA

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-4939-8759-7 ISBN 978-1-4939-8760-3 (eBook)
https://doi.org/10.1007/978-1-4939-8760-3
Library of Congress Control Number: 2018956143

© Springer Science+Business Media, LLC, part of Springer Nature 2019

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Preface

Eukaryotic cells are compartmentalized into membrane-bound organelles encased by a

plasma membrane. Vesicular content of membrane proteins and luminal constituents are
trafficked through cells using a set of highly regulated pathways that culminate in fusion with
a target membrane and transfer of products. The fusion of vesicles is critical for maintaining
cellular homeostasis and is essential for the release of neurotransmitters, hormones, anti-
bodies, as well as the turnover of receptors, the destruction of pathogens, and the generation
of antigens. The machinery that controls membrane fusion is conserved from yeast to
mammals, and mechanisms described in one system are applicable to most fusion models.
Although membrane trafficking requires numerous factors such as cargo receptors, coat
proteins, Rab GTPases, and tethering factors, the terminal catalysts of fusion are SNAREs
(soluble N-ethylmaleimide-sensitive factor attachment protein receptors). All SNAREs
contain a canonical heptad repeat, termed a “SNARE motif,” that is flanked by various
N-terminal domains and C-terminal membrane anchors. SNAREs form parallel four helical
bundles through their SNARE motifs. These regions are primarily composed of hydropho-
bic residues with the exception of a central polar glutamine (Q), or arginine (R) that interact
in the ionic zero layer. Each SNARE bundle is composed of 3 Q-SNAREs donated by one
membrane and 1 R-SNARE by its partner membrane.
This book covers many of the ways that SNAREs and their function are examined in the
laboratory. The methods described in each chapter are described in detail such that novice as
well as experienced researchers can explore the mechanisms of SNARE-mediated membrane
fusion. Therefore, I expect that this book will serve as a valuable tool for all investigators
interested in the field.
This volume of Methods in Molecular Biology contains 26 chapters that are grouped into
sections, starting with Biophysical and Computational Alaysis of SNAREs. Part II is dedi-
cated Biochemical methodologies for examining the interactions of SNAREs with proteins
and lipids. Part III includes Functional methods for measuring SNARE complex formation,
calcium transport and and membrane fusion. Part IV is comprised of advanced microscopy
methods to observe membrane fusion.

Urbana, IL, USA Rutilio Fratti

v
Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

PART I BIOPHYSICS AND COMPUTATIONAL ANALYSIS

1 Molecular Dynamics Simulations of the SNARE Complex . . . . . . . . . . . . . . . . . . . 3
Maria Bykhovskaia
2 Mesoscale Computational Modeling of Protein-Membrane
Interactions Based on Continuum Mean-Field Theory . . . . . . . . . . . . . . . . . . . . . . 15
George Khelashvili
3 EPR Lineshape Analysis to Investigate the SNARE Folding
Intermediates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Ryan Khounlo, Brenden J. D. Hawk, and Yeon-Kyun Shin
4 Dynamic Light Scattering Analysis to Dissect Intermediates
of SNARE-Mediated Membrane Fusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Byoungjae Kong, Yoosoo Yang, and Dae-Hyuk Kweon
5 SNAREpin Assembly: Kinetic and Thermodynamic Approaches . . . . . . . . . . . . . . 71
Feng Li and Frederic Pincet
6 Single-Molecule Optical Tweezers Study of Regulated SNARE
Assembly. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Lu Ma, Junyi Jiao, and Yongli Zhang
7 Studying Munc18:Syntaxin Interactions Using Small-Angle
Scattering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Andrew E. Whitten, Russell J. Jarrott, Shu-Hong Hu, Anthony P. Duff,
Gordon J. King, Jennifer L. Martin, and Michelle P. Christie
8 Using Force Spectroscopy to Probe Coiled-Coil Assembly
and Membrane Fusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Hannes Witt and Andreas Janshoff

PART II BIOCHEMISTRY

9 SNAP-25 S-Guanylation and SNARE Complex Formation. . . . . . . . . . . . . . . . . . . 163

Yusuke Kishimoto, Takaaki Akaike, and Hideshi Ihara
10 Analysis of the Role of Sec3 in SNARE Assembly
and Membrane Fusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Kunrong Mei, Peng Yue, and Wei Guo
11 Use of Microscale Thermophoresis (MST) to Measure Binding
Affinities of Components of the Fusion Machinery. . . . . . . . . . . . . . . . . . . . . . . . . . 191
Robert P. Sparks and Rutilio Fratti

vii
viii Contents

12 Use of Surface Plasmon Resonance (SPR) to Determine

Binding Affinities and Kinetic Parameters Between Components
Important in Fusion Machinery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
Robert P. Sparks, Jermaine L. Jenkins, and Rutilio Fratti
13 Determination of Sec18-Lipid Interactions by Liposome-Binding
Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
Matthew L. Starr and Rutilio Fratti
14 Using Nanodiscs to Probe Ca2+-Dependent Membrane Interaction
of Synaptotagmin-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
Ekaterina Stroeva and Shyam S. Krishnakumar
15 Functional Reconstitution of Intracellular Vesicle Fusion
Using Purified SNAREs and Sec1/Munc18 (SM) Proteins. . . . . . . . . . . . . . . . . . . 237
Haijia Yu, Lauren Crisman, Michael H.B. Stowell, and Jingshi Shen

PART III FUNCTIONAL ASSAYS

16 Assay of Lipid Mixing and Fusion Pore Formation in the Fusion

of Yeast Vacuoles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
Massimo D’Agostino and Andreas Mayer
17 A Nanodisc-Cell Fusion Assay with Single-Pore Sensitivity
and Sub-millisecond Time Resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
Natasha R. Dudzinski, Zhenyong Wu, and Erdem Karatekin
18 An In Vitro Assay of Trans-SNARE Complex Formation During Yeast
Vacuole Fusion Using Epitope Tag-Free SNAREs . . . . . . . . . . . . . . . . . . . . . . . . . . 277
Youngsoo Jun
19 A Cell-Free Content Mixing Assay for SNARE-Mediated
Multivesicular Body-Vacuole Membrane Fusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
Mahmoud Abdul Karim, Dieter Ronny Samyn,
and Christopher Leonard Brett
20 Reconstituted Proteoliposome Fusion Mediated by Yeast
SNARE-Family Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
Joji Mima
21 Real-Time Fluorescence Detection of Calcium Efflux
During Vacuolar Membrane Fusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323
Gregory E. Miner and Rutilio Fratti

PART IV MICROSCOPY

22 Single-Molecule Fluorescence Measurement of SNARE-Mediated

Vesicle Fusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335
Yachong Hu, Zhiqi Tian, and Jiajie Diao
23 Quantifying Intramolecular Protein Conformational Dynamics
Under Lipid Interaction Using smFRET and FCCS . . . . . . . . . . . . . . . . . . . . . . . . . 345
Pei Li, Yawei Dai, Markus Seeger, and Yan-Wen Tan
 Contents ix

24 Visualization of SNARE-Mediated Organelle Membrane

Hemifusion by Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361
Sevan Mattie, Tom Kazmirchuk, Jeannie Mui, Hojatollah Vali,
and Christopher Leonard Brett
25 Studies of the Secretory Machinery Dynamics by Total Internal
Reflection Fluorescence Microscopy in Bovine Adrenal Chromaffin
Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379
José Villanueva, Yolanda Gimenez-Molina, and Luis M. Gutiérrez
26 Imaging SNAP-29 in Drosophila . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391
Hao Xu and Bryan Stewart

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403
Contributors

TAKAAKI AKAIKE  Department of Environmental Medicine and Molecular Toxicology, Tohoku

University Graduate School of Medicine, Sendai, Japan
CHRISTOPHER LEONARD BRETT  Department of Biology, Concordia University, Montréal,
QC, Canada
MARIA BYKHOVSKAIA  Department of Neurology, Wayne State University School of Medicine,
Detroit, MI, USA; Department of Anatomy and Cell Biology, Wayne State University
School of Medicine, Detroit, MI, USA
MICHELLE P. CHRISTIE  Bio21 Molecular Science and Biotechnology Institute, The University
of Melbourne, Parkville, VIC, Australia
LAUREN CRISMAN  Department of Molecular, Cellular and Developmental Biology,
University of Colorado, Boulder, CO, USA
MASSIMO D’AGOSTINO  Département de Biochimie, Université de Lausanne, Epalinges,
Switzerland
YAWEI DAI  State Key Laboratory of Surface Physics and Department of Physics, Fudan
University, Shanghai, China; Department of Physics, The University of Hong Kong, Hong
Kong, China
JIAJIE DIAO  Department of Cancer Biology, University of Cincinnati College of Medicine,
Cincinnati, OH, USA
NATASHA R. DUDZINSKI  Interdepartmental Neuroscience Program, Yale University, New
Haven, CT, USA; Nanobiology Institute, Yale University, West Haven, CT, USA
ANTHONY P. DUFF  Australian Nuclear Science and Technology Organisation, Lucas
Heights, NSW, Australia
RUTILIO FRATTI  Department of Biochemistry, University of Illinois at Urbana-Champaign,
Urbana, IL, USA
YOLANDA GIMENEZ-MOLINA  Instituto de Neurociencias, Centro Mixto CSIC-Universidad
Miguel Hernández, Sant Joan d’Alacant, Alicante, Spain
WEI GUO  Department of Biology, University of Pennsylvania, Philadelphia, PA, USA
LUIS M. GUTIÉRREZ  Instituto de Neurociencias, Centro Mixto CSIC-Universidad Miguel
Hernández, Sant Joan d’Alacant, Alicante, Spain
BRENDEN J. D. HAWK  Roy J. Carver Department of Biochemistry, Biophysics and Molecular
Biology, Iowa State University, Ames, IA, USA
SHU-HONG HU  Griffith Institute for Drug Discovery, Griffith University, Nathan, QLD,
Australia
YACHONG HU  Department of Cancer Biology, University of Cincinnati College of Medicine,
Cincinnati, OH, USA; School of Life Science and Technology, Xi’an Jiaotong University,
Xi’an, China
HIDESHI IHARA  Department of Biological Science, Graduate School of Science, Osaka
Prefecture University, Sakai, Japan
ANDREAS JANSHOFF  Institute of Physical Chemistry, University of Goettingen, Göttingen,
Germany
RUSSELL J. JARROTT  Griffith Institute for Drug Discovery, Griffith University, Nathan,
QLD, Australia

xi
xii Contributors

JERMAINE L. JENKINS  Structural Biology and Biophysics Facility, University of Rochester,

Rochester, NY, USA
JUNYI JIAO  Department of Cell Biology, Yale University School of Medicine, New Haven,
CT, USA; Integrated Graduate Program in Physical and Engineering Biology, New
Haven, CT, USA
YOUNGSOO JUN  School of Life Sciences, Cell Logistics Research Center, and Silver Health Bio
Research Center, Gwangju Institute of Science and Technology, Gwangju, Republic of
Korea
ERDEM KARATEKIN  Nanobiology Institute, Yale University, West Haven, CT, USA;
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New
Haven, CT, USA; Department of Molecular Biophysics and Biochemistry, Yale University,
New Haven, CT, USA; Centre National de la Recherche Scientifique (CNRS), Paris,
France
MAHMOUD ABDUL KARIM  Department of Biology, Concordia University, Montréal, QC,
Canada; Department of Cell Biology, University of Alberta, Edmonton, AB, Canada
TOM KAZMIRCHUK  Department of Biology, Concordia University, Montréal, QC, Canada
GEORGE KHELASHVILI  Department of Physiology and Biophysics, Weill Cornell Medical
College of Cornell University, New York, NY, USA; Institute for Computational
Biomedicine, Weill Cornell Medical College of Cornell University, New York, NY, USA
RYAN KHOUNLO  Roy J. Carver Department of Biochemistry, Biophysics and Molecular
Biology, Iowa State University, Ames, IA, USA
GORDON J. KING  Centre for Microscopy and Microanalysis, The University of Queensland, St
Lucia, QLD, Australia
YUSUKE KISHIMOTO  Department of Biological Science, Graduate School of Science, Osaka
Prefecture University, Sakai, Japan
BYOUNGJAE KONG  Department of Integrative Biotechnology, College of Biotechnology and
Bioengineering, Sungkyunkwan University, Suwon, Republic of Korea
SHYAM S. KRISHNAKUMAR  Department of Cell Biology, Yale University School of Medicine,
New Haven, CT, USA; Department of Clinical and Experimental Epilepsy, Institute of
Neurology, University College London, London, UK
DAE-HYUK KWEON  Department of Integrative Biotechnology, College of Biotechnology and
Bioengineering, Sungkyunkwan University, Suwon, Republic of Korea; Biomedical
Institute for Convergence, Sungkyunkwan University, Suwon, Republic of Korea
FENG LI  Department of Cell Biology and Nanobiology Institute, School of Medicine, Yale
University, New Haven, CT, USA
PEI LI  State Key Laboratory of Surface Physics and Department of Physics, Fudan
University, Shanghai, China
LU MA  Beijing National Laboratory for Condensed Matter Physics and CAS Key
Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing,
China
JENNIFER L. MARTIN  Griffith Institute for Drug Discovery, Griffith University, Nathan,
QLD, Australia
SEVAN MATTIE  Department of Biology, Concordia University, Montréal, QC, Canada;
Montreal Neurological Hospital and Institute, McGill University, Montréal, QC, Canada
ANDREAS MAYER  Département de Biochimie, Université de Lausanne, Epalinges,
Switzerland
KUNRONG MEI  Department of Biology, University of Pennsylvania, Philadelphia, PA, USA
JOJI MIMA  Institute for Protein Research, Osaka University, Osaka, Japan
 Contributors xiii

GREGORY E. MINER  Department of Biochemistry, University of Illinois at Urbana-

Champaign, Urbana, IL, USA
JEANNIE MUI  Facility for Electron Microscopy Research, Department of Anatomy and Cell
Biology, McGill University, Montréal, QC, Canada
FREDERIC PINCET  Department of Cell Biology and Nanobiology Institute, School of
Medicine, Yale University, New Haven, CT, USA; Laboratoire de Physique Statistique,
Ecole Normale Supérieure, PSL Research University, Université Paris Diderot Sorbonne
Paris Cité, Sorbonne Universités UPMC Univ Paris 06, CNRS, Paris, France
DIETER RONNY SAMYN  Department of Biology, Concordia University, Montréal, QC,
Canada
MARKUS SEEGER  Biological Imaging Center, Technische Universit€ at München, München,
Germany
JINGSHI SHEN  Department of Molecular, Cellular and Developmental Biology, University of
Colorado, Boulder, CO, USA
YEON-KYUN SHIN  Roy J. Carver Department of Biochemistry, Biophysics and Molecular
Biology, Iowa State University, Ames, IA, USA
ROBERT P. SPARKS  Department of Biochemistry, University of Illinois at Urbana-
Champaign, Urbana, IL, USA
MATTHEW L. STARR  Department of Biochemistry, University of Illinois at Urbana-
Champaign, Urbana, IL, USA
BRYAN STEWART  Department of Biology, University of Toronto Mississauga, Mississauga,
ON, Canada
MICHAEL H. B. STOWELL  Department of Molecular, Cellular and Developmental Biology,
University of Colorado, Boulder, CO, USA
EKATERINA STROEVA  Department of Cell Biology, Yale University School of Medicine, New
Haven, CT, USA
YAN-WEN TAN  State Key Laboratory of Surface Physics and Department of Physics, Fudan
University, Shanghai, China
ZHIQI TIAN  Department of Cancer Biology, University of Cincinnati College of Medicine,
Cincinnati, OH, USA; School of Life Science and Technology, Xi’an Jiaotong University,
Xi’an, China
HOJATOLLAH VALI  Facility for Electron Microscopy Research, Department of Anatomy and
Cell Biology, McGill University, Montréal, QC, Canada
JOSÉ VILLANUEVA  Instituto de Neurociencias, Centro Mixto CSIC-Universidad Miguel
Hernández, Sant Joan d’Alacant, Alicante, Spain
ANDREW E. WHITTEN  Australian Nuclear Science and Technology Organisation, Lucas
Heights, NSW, Australia
HANNES WITT  Institute of Physical Chemistry, University of Goettingen, Göttingen,
Germany
ZHENYONG WU  Nanobiology Institute, Yale University, West Haven, CT, USA;
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New
Haven, CT, USA
HAO XU  School of Biological, Environmental, and Earth Sciences, University of Southern
Mississippi, Hattiesburg, MS, USA
YOOSOO YANG  Biomedical Research Institute, Korea Institute of Science and Technology
(KIST), Seoul, Republic of Korea; Division for Bio-Medical Science and Technology, KIST
School, Korea University of Science and Technology, Seoul, Republic of Korea
PENG YUE  Department of Biology, University of Pennsylvania, Philadelphia, PA, USA
xiv Contributors

HAIJIA YU  Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life
Sciences, Nanjing Normal University, Nanjing, China; Department of Molecular,
Cellular and Developmental Biology, University of Colorado, Boulder, CO, USA
YONGLI ZHANG  Department of Cell Biology, Yale University School of Medicine, New
Haven, CT, USA
 Part I

Biophysics and Computational Analysis

 Chapter 1

Molecular Dynamics Simulations of the SNARE Complex

Maria Bykhovskaia

Abstract
Molecular dynamics (MD) simulations enable in silico investigations of the dynamic behavior of proteins
and protein complexes. Here, we describe MD simulations of the SNARE complex and its interactions with
the neuronal protein complexin. Complexin is an effector of neuronal secretion that inhibits spontaneous
fusion and is thought to clamp the fusion process via the interactions with the SNARE complex. We
describe MD simulations of the SNARE complex alone and bound to complexin. The MD simulations
under external forces imitating the repulsion between lipid bilayers enabled us to investigate unraveling and
assembly of the SNARE complex.

Key words Synaptic transmission, Exocytosis, Synaptobrevin, Syntaxin, SNAP25, Forces, Assembly

1 Introduction

Molecular dynamics (MD) is a powerful computational approach

that enabled investigations of protein dynamics at the atomic level
[1]. This approach has proved to be instrumental in guiding site-
directed mutagenesis of receptors, ion channels, and enzymes
[2–4]. Until recently, a critical limitation of MD simulations was
the inability to simulate protein dynamics at timescales beyond a
few nanoseconds. However, recent advances in supercomputing,
such as MD software packages developed for parallel platforms [5],
a new generation of supercomputers, and specialized supercompu-
ters designed for MD simulations [6], have enabled a break-
through. Currently, the dynamics of protein complexes can be
investigated employing MD simulations at sub-microsecond or
even microsecond range, approaching the timescales required to
detect major conformational transitions in proteins and protein
complexes.
We employed MD simulations [7, 8] to develop dynamic mod-
els of the final steps of SNARE unraveling and assembly and their
regulation by the protein complexin (Cpx). Cpx is a SNARE-

Rutilio Fratti (ed.), SNAREs: Methods and Protocols, Methods in Molecular Biology, vol. 1860,
https://doi.org/10.1007/978-1-4939-8760-3_1, © Springer Science+Business Media, LLC, part of Springer Nature 2019

3
4 Maria Bykhovskaia

interacting protein which is thought to prevent full SNARE assem-

bly and clamp spontaneous fusion [9–12]. Initial MD studies of the
SNARE complex enabled assessing overall flexibility of the SNARE
bundle [13, 14] and also investigating it in the presence of Cpx
[15]. We performed MD simulations of the SNARE complex under
external forces to imitate the influence of lipid bilayers, and inves-
tigated how Cpx affects the separation and assembly of membrane-
proximal layers of the SNARE bundle. Combined with targeted
mutagenesis in Drosophila [8], these simulations enabled us to
develop a model for the dynamic interaction of Cpx with the
SNARE bundle and its role in neuronal section.

2 Materials

1. ZMM software package (www. zmmsoft.com [16, 17])

enabling initial optimization and homology modeling was
used for the system setup.
2. VMD (Visual Molecular Dynamics, NIH Center for Macro-
molecular Modeling and Bioinformatics, University of Illinois
at Urbana-Champaign) software (http://www.ks.uiuc.edu/
Research/vmd/) was used for the system setup and data analy-
sis. We used the versions 1.8 and 1.9 for Windows.
3. NAMD Scalable Molecular Dynamics [5] (NIH Center for
Macromolecular Modeling and Bioinformatics, University of
Illinois at Urbana-Champaign, versions 2.8–2.10) were used
for MD computations. The heating phase was performed
under Windows platform at PC computers, while production
runs were performed at Stampede and Ranger supercomputers
through XSEDE (eXtreme Science and Engineering Discovery
Environment, www.xsede.org) network.
4. The software package Vega ZZ (Drug design Laboratory,
http://nova.disfarm.unimi.it/cms/index.php?Software_pro
jects:VEGA_ZZ) under Windows platform was used for the data
analysis. The software package PyMol (www.PyMol.org) was
used to create illustrations.
5. MatLab software (MathWorks) was used for the system setup
and data analysis.
6. All the X-ray structures used for the initial system setup were
downloaded from the protein data bank (http://www.rcsb.
org).
7. Drosophila protein sequences were downloaded from NCBI
(National Center for Biotechnology Information) database.
 MD Simulations of the SNARE Complex 5

3 Methods

3.1 System Setup Use the high-resolution (1.4A) X-ray structure 1N7S for the initial
topology of the SNARE complex [18]. Optimize the structure
3.1.1 SNARE-Cpx
employing the Monte-Carlo Minimization (MCM) method [19]
Complex
with ZMM software package.
Construct the initial topology of the SNARE-Cpx complex out
of two X-ray structures: 1N7S, the high-resolution structure of the
SNARE complex, and 1KIL [20], the structure of the SNARE-Cpx
complex obtained by a combination of crystallography and NMR
approaches with 2.3 A resolution. Construct the SNARE-Cpx
model using the ZMM/MVM package in the following way:
1. The SNARE bundle structure (from 1N7S) is kept rigid, and
Cpx (from 1KIL) is docked to the bundle by imposing har-
monic distance constraints obtained from 1KIL SNARE-Cpx
structure. The constraints are imposed on all the atoms of the
SNARE bundle and Cpx that are within van der Waals (VdW)
distances.
2. Optimize the resulting structure employing the MCM algo-
rithm with ZMM/MVM package and imposing constraints on
all the C-alpha atoms, which are rigidly pinned.
3. Remove all the constraints and optimize the structure of the
SNARE-Cpx complex employing MCM with no constraints.

3.1.2 Homology Perform sequence alignments between the Drosophila and mamma-
Modeling of the Drosophila lian protein fragments using the BLAST algorithm in NCBI
Complex (National Center for Biotechnology Information) database. Derive
the 3D model of the Drosophila SNARE-Cpx complex from the
model of the mammalian complex (described in the previous sec-
tion) employing ZMM package. Perform the residue substitutions
on one helix at a time (overall five rounds for five helixes comprising
the complex), and optimize the structure after each round with
MCM employing ZMM package. Manually perform the substitu-
tions within the *.pep file generated by ZI module of ZMM pack-
ager (see Note 1). After each round of substitutions, optimize the
structure with Cɑ atoms being rigidly pinned, and then optimize
again with no constraints. The resulting 3D structure of the Dro-
sophila complex was similar to the mammalian complex (Fig. 1).

3.1.3 Molecular Perform operations using VMD software. Build the molecular
Topology, Single-Point topology file *.psf employing the Automatic Psf Generator (see
Mutations, Water Box, Note 2). Introduce single-point mutations in Syx, Syb, and Cpx
and Ionization employing VMD Mutator. Add the water box using Add Solvation
Box function. The size of the box is 150
70
70 Å (Fig. 2). Add
ions employing the Add Ions function. Add K+ and Cl+ ions to
neutralize the negative charge of the protein complex and to yield
150 mM concentration of KCl (see Note 3).
6 Maria Bykhovskaia

Fig. 1 Two views of superimposed mammalian (blue) and Drosophila (green)

SNARE-Cpx complexes after the optimization show almost perfect overlay of the
backbone (top) and moderate deviations of side chains (bottom)

Fig. 2 The periodic cell containing water molecules, potassium ions (blue
spheres), and chlorine ions (green spheres). SNARE-Cpx complex is shown in
the cartoon representation (blue)

3.2 Molecular Perform MD simulations employing NAMD package and

Dynamics CHARMM22 force field [21–23] (see Note 4) with periodic
boundary conditions and Ewald electrostatics at NTP ensemble.
Keep bond lengths of water molecules fixed (see Note 5). Regulate
the pressure with Berendsen barostat.
 MD Simulations of the SNARE Complex 7

3.2.1 Energy Perform the energy minimization for 100 iterations (see Note 6),
Minimization and Heating followed by the heating phase. At the heating phase, set the time
Phase step to 1 fs. Set the initial temperature to 200 K and adjust every
500 steps in increments of 50 K by velocity rescaling (see Note 7).
Set the Berendsen barostat parameters to:

useGroupPressure Yes
BerendsenPressureTarget 1.01325
BerendsenPressureCompressibility 4.57E-5
BerendsenPressureRelaxationTime 40
BerendsenPressureFreq 2

Set the heating phase to last 10 ps (see Note 8). Use a scaling of
2 for electrostatic interactions: fullElectFrequency ¼ 2.

3.2.2 MD Perform production runs with a step of 2 fs. Use a scaling of 2 and
Production Runs 4 for non-bonded and electrostatic interactions, respectively:

timestep 2.0
nonbondedFreq 2
fullElectFrequency 4

Control the temperature with Langevin thermostat with the

damping factor langevinDamping ¼ 5.0. Set the parameters of
Berendsen barostat to optimize the speed (see Note 9):

BerendsenPressureRelaxationTime 160
BerendsenPressureFreq 8

Set the production runs to last 200–300 ns (see Note 10).

To avoid a rotation of the molecular complex in the prolonged
rectangular water box, apply harmonic constraints to Cɑ atoms of
the C-terminal residue (K256) of syntaxin (Syx), the C-terminal
residue (K83) of SN1 unit of SNAP25, and the N-terminal residue
(G139) of SN2 unit of SNAP 25 (Fig. 3a, see Note 11).

3.2.3 SNARE Separation Apply external forces to the Cɑ atom of the C-terminal residue
Under External Forces (W89) of synaptobrevin (Syb). Compute the direction of the force
as a vector perpendicular to the plain defined by the constrained
atoms of the t-SNARE bundle (Fig. 3a, see Note 12). Perform
these calculations employing MatLab (see Note 13). Vary the mag-
nitude of the force within the limits predicted by the computations
of the membrane electrostatic potential ([7, 24], 2–4 kcal/mol/Å,
see Note 14). The force of 2 kcal/mol/Å produced unraveling of
the bundle within 100–200 ns (Fig. 3b).
8 Maria Bykhovskaia

Fig. 3 Simulations of SNARE unraveling under external forces. (a) The positions
of the C-terminal residue of Syx, C-terminal residue of SN1 subunit of SNAP25,
and N-terminal residue of SN2 subunit of SNAP25 are constraint (black circles).
The external force (arrow) is directed perpendicular to the plain defined by the
constraint atoms. (b) Unraveling of Syb under the external force of 2 kcal/mol/Å
at different time points of the trajectory. The structures are shown in two
representations: left—backbone; right—all-atom as VdW spheres

Use a time step of 1 fs for simulations under external forces and

maintain the temperature by velocity rescaling (as during the heat-
ing phase, see Note 15).

3.2.4 SNARE Assembly For the simulations of the SNARE assembly, use the initial states
obtained by the simulations under pulling forces (Fig. 3b), as
described in the previous section. Perform the simulations of the
SNARE assembly in two different ways [7]: (1) with no external
force applied, and (2) with a weak external force applied (see Note
16). We found that the second paradigm produced a faster zipper-
ing (see Note 17), since in the absence of constraints the unstruc-
tured C-terminus Syb tended to interact with distal layers of the
bundle.
The parameters of the simulation were identical to those
described in Subheading 3.2.2 for the paradigm 1, and to the
parameters described in Subheading 3.2.3 for the paradigm 2.
 MD Simulations of the SNARE Complex 9

3.3 Trajectory 1. Set the interval between trajectory points to 10 ps.

Analysis 2. Evaluate the separation of Syb and Cpx from the SNARE
bundle by computing the distance between Cɑ atoms of several
key residues. Compute the distance using Vega ZZ software
(Analysis/Measure function).
3. Check the periodic images of the complex at several trajectory
points employing VMD software (see Note 18).
4. Compute the interaction energies of different parts of the
complex employing VMD/namd2 interface (NAMD Energy
function within the VMD package).
5. Generate images of the models employing PyMol and VMD
packages.

4 Notes

1. This approach was practical, since homology was relatively high

(>70%). With lower homology, automated approaches should
be used, such as Swiss-Model (https://swissmodel.expasy.org/)
or similar software.
2. It is critical to ensure that the topology parameter file used to
build the molecular structure (*.psf) matches the force-field
parameter file used for MD computations. Current CHARMM
topology and force-field parameter files can be obtained from
the MacKerell Lab, www site (http://mackerell.umaryland.
edu/charmm_ff.shtml).
3. Potassium ions were added, since we aimed to simulate the
SNARE complex inside the cell. If the goal is to reproduce the
results of in vitro experiments, then the ion concentration and
composition should closely match the buffer solution.
4. Currently, the latest version of the CHARMM force field is
CHARMM36 [21] (http://mackerell.umaryland.edu/
charmm_ff.shtml), and earlier versions can be considered obso-
lete. Importantly, the major changes in force-field parameters
between the versions CHARMM22, 27, and 36 involved
nucleic acids, lipids, and carbohydrates, but not proteins.
Since we have a molecular system composed out of proteins
in water and ion environment, it is unlikely that the behavior of
this system would be altered when switching from
CHARMM22 to CHARMM36. However, the CHARMM36
version is presently considered the most accurate.
5. Sometimes the system becomes unstable during the heating
phase or at the initial stages of the production run, and in such
cases it may be helpful to equilibrate the system with all the
bond length being flexible. However, this would drastically
10 Maria Bykhovskaia

increase the simulation time, and this is not recommended for

production runs.
6. If the minimization length is not sufficient, then the system
would become unstable at the beginning of the heating phase.
The most common error in these cases would be velocities of
selected atoms exceeding the maximally allowed values. In such
cases, additional minimization should be performed. However,
the minimization algorithms employed within NAMD are very
powerful, and after 400–500 steps of minimization the system
can considerably deviate from its initial state. Thus, if 200–300
minimization steps were not sufficient, the system setup should
be checked thoroughly.
7. If the system becomes unstable during the heating phase, it
may be helpful to start from lower temperatures (50–100 K)
and proceed in smaller increments (10–20 K).
8. The pressure should be checked in the end of the heating
phase. The value of Group Pressure Average should fluctuate
within 5 to 5 bar range. If the values are consistently higher,
additional equilibration may be needed.
9. If the values of Group Pressure Average (see above) become too
high, both parameters should be reduced.
10. Supercomputers of the latest generation enable MD simula-
tions of longer trajectories (at a microsecond range) for this
system. It would be of interest to investigate how the accessory
helix of Cpx interacts with the SNARE bundle at longer time-
scales. Our simulations at the 200–300 ns range demonstrated
that these interactions are dynamic and that Cpx may tightly
interact with the bundle, or it may deviate from the bundle.
Simulations at a microsecond scale would be of interest for
performing a statistical analysis of the Cpx states.
11. In vivo, the residue K256 of Syx would be attached to the
plasma membrane, and the palmitoylated loop of SNAP25,
which spans between the C-terminus of its SN1 unit and the
N-terminus of its SN2 unit, would be attached to the mem-
brane as well. These interactions would restrict movements of
both proteins. Thus, our constraints imitate the attachment of
the t-SNARE (Syx and SNAP25 complex) to the plasma mem-
brane via the transmembrane domain of Syx and the palmitoy-
lated loop of SNAP25.
12. An alternative design would be applying opposing forces to
C-termini of Syb and Syx. This paradigm usually produces
unraveling of both Syb and Syx from the bundle. Such simula-
tions would be appropriate to imitate unraveling of the SNARE
complex in vitro. In contrast, applying constraints to Syx and
SNAP25 and applying a force to Syb, as described here, pro-
duce unraveling of Syb from the core t-SNARE bundle. Such
 MD Simulations of the SNARE Complex 11

scenario appears most appropriate to imitate SNARE unravel-

ing in vivo, with the core t-SNARE bundle being attached to
the membrane.
13. The following MatLab code computes the force vector:
a=[]
b=[]
c=[]
d=a-b
e=c-a
k=cross(d,e)
lk=sqrt(sum(k.*k))
nk=k./lk

Coordinates of the three constrained atoms should be entered

for a, b, and c, and the unitary vector perpendicular to the plain
defined by these three atoms will be computed. This vector
should be multiplied by the value of the force.
14. The computations of the electrostatic repulsion predict that the
force applied to the SNARE bundle would be 2–4 kcal/Mol/Å
when the complex is fully or nearly fully zippered, and decrease
exponentially as the distance between Syx and Syb C-termini
increases, becoming negligibly small at a separation of 5–6 nm
[7, 24]. However, various forces have been used in biophysical
in vitro experiments to investigate SNARE unraveling. When
the force is below 1.5 kcal/mol/Å, the complex usually does
not unravel within 100–200 ns, and therefore we employed the
forces of 2 kcal/Mol/Å or higher. However, as longer MD
simulations become realistic, it would be of interest to explore
SNARE unraveling under weaker forces and to relate this
dynamics to single-molecule experiments.
15. We found that in this system external forces are not compatible
with Langevin thermostat, since this combination destabilized
the system. Similarly, increasing the time step compromised the
stability.
16. We used the forces of 0.5 or 0.25 kcal/Mol/Å. These forces
would imitate the inertia imposed on Syb by the attached
vesicle. This force should not be applied when simulating the
dynamics of the isolated SNARE complex.
17. Within the simulation times employed in our studies
(200–400 ns), we never observed full SNARE assembly if
more than one C-terminal helical turn of Syb was initially
unraveled. Zippering of a single C-terminal helical turn of
Syb (Layer 8) was usually observed within 20–100 ns of the
simulation.
12 Maria Bykhovskaia

18. One needs to ensure that the unraveled terminus of Syb does
not contact with the periodic image of the SNARE complex.
The cell size we chose was sufficient as long as Syb was unra-
veled up to the layer 6. If a more radical separation is simulated,
the size of the periodic cell has to be increased.

References
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dynamics simulations of biomolecules. Nat hon HT, Sudhof TC, Brose N, Rosenmund C
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10.1038/nsb0902-646 +dependent neurotransmitter release. Cell
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studies of membrane channels. Structure 12 12. Kummel D, Krishnakumar SS, Radoff DT,
(8):1343–1351. https://doi.org/10.1016/j. Li F, Giraudo CG, Pincet F, Rothman JE,
str.2004.06.013 Reinisch KM (2011) Complexin cross-links
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35(3):179–185. https://doi.org/10.1016/j. 13. Durrieu MP, Lavery R, Baaden M (2008)
tibs.2009.10.007 Interactions between neuronal fusion proteins
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41:83–89. https://doi.org/10.1016/j.sbi. bury DJ (2010) Chemomechanical regulation
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https://doi.org/10.1002/jcc.21367
 Chapter 2

Mesoscale Computational Modeling of Protein-Membrane

Interactions Based on Continuum Mean-Field Theory
George Khelashvili

Abstract
Quantitative computational modeling of protein-membrane interactions is of great importance as it aids in
the interpretation of experimental results and enables design and exploration of new experimental systems.
This review describes one such computational approach conceived specifically to treat electrostatically
driven interactions between a lipid membrane and a protein (or protein domains) adsorbing onto the
membrane. The methodology is based on self-consistent minimization of the governing free energy
functional which is expressed in the mean-field approximation and has contributions from electrostatic
interactions as well as from mixing entropy of lipids in the membrane and ions in the solution. The method
enables calculation of the free energy of the binding process and quantification of the steady-state lipid
distribution around the adsorbing protein. The extension of the method to include membrane deformation
degrees of freedom further allows calculation of the equilibrium bilayer shape upon the protein binding.

Key words Poisson-Boltzmann theory, Cahn-Hilliard equation, Coarse-grained theory, Lipid

mixing, Electrostatic interactions, PIP2 lipids, Lipid diffusion and segregation, Cell signaling

1 Introduction

To perform their physiological function, signaling proteins and

other macromolecules are often required to associate with the cell
plasma membrane by means of nonspecific electrostatic interactions
between anionic lipids residing in the intracellular leaflet of the
plasma membrane and clusters of basic residues on the adsorbing
protein [1–3]. This process is not only energetically preferred, but
also driven by entropic factors responsible for the mobility of ions
in the bulk solution. Indeed, when in isolation, a protein and a lipid
membrane maintain charge neutrality by sequestering on or near to
their surfaces the so-called counterions from the solution
[4–7]. Upon protein-membrane binding, these counterions can
be released to the bulk solution to produce a gain in translational
entropy, while the protein and membrane electro-neutralize each
other [5, 8–10]. Thus, the stronger the extent of the aggregation of

Rutilio Fratti (ed.), SNAREs: Methods and Protocols, Methods in Molecular Biology, vol. 1860,
https://doi.org/10.1007/978-1-4939-8760-3_2, © Springer Science+Business Media, LLC, part of Springer Nature 2019

15
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Aurelia, and other poems. *$2 Dutton 821

20–15351

“The sequence of ‘Sonnets to Aurelia’ gives the story of a

disappointed lover with his mistress whose falseness, though ugly,
intensifies the helpless passion of the man. The form of the sonnet in
which the poet tells his story is Shakspearean.”—Boston Transcript

“Mr Nichols, like many of the minor Elizabethan lyrists, uses the
fourteen lines of the sonnet simply for the sake of their sound, their
rich baroque handsomeness of appearance. That is the principal and,
to our mind, damning defect of his sonnets. They have no substance.
The fountains are dry, the parched stone faces open their mouths to
no purpose; we are at a loss to see why the monument was built.”

− Ath p765 Je 11 ’20 1200w

“Among Mr Nichols’s most potent qualities the quality of vision is

the steadiest and strongest. Among the most recent English poets he
is the richest in this endowment.” W. S. B.

+ Boston Transcript p6 S 8 ’20 1200w

“The result is, to my taste, like a dish flavoured with nutmeg and
cinnamon to which has been added a dash of tabasco sauce.” J: G.
Fletcher

− Freeman 2:331 D 15 ’20 600w

“With some of the faults of youth, Mr Nichols has all of its virtues.
He is adaptable, he is resourceful, he is restlessly eager to try new
methods, to pour his soul into an unaccustomed vessel. He has force,
eloquence, fire, and passion.”

+ − Spec 124:22 Jl 3 ’20 850w

“The conspicuous fault of ‘Aurelia’ is the insecurity

of its style. Here is a series of quasi-Shakespearian
sonnets, in which we have conceits without
gracefulness, artifices aimed at intensifying rather
than at easing the situations they describe—in brief a
conscious author, magnifying an experience the
content of which was meagre at the best in imitation
of a spontaneous one the experience of which is too
full to be contained.”

− + The Times [London] Lit Sup p418 Jl 1

’20 1000w

NICHOLSON, MEREDITH. Blacksheep!

blacksheep! il *$1.75 Scribner

20–7287
 “At a dinner in Washington the hero, one Archibald Bennett,
whose income encourages his neurasthenia, sits next to a girl who
tells him that no man whose life motto is ‘Safety first!’ is likely to
have a very good time or escape a bored anaemia. Several days later
the same Archie goes to Maine to look at a house for his sister, and
the next thing he knows he has shot a man and is a fugitive from
justice in the stolen car driven by the ‘governor’! After that you,
together with the police forces of most of the states in the Union, are
completely in the ‘governor’s’ power.”—N Y Times

Booklist 16:314 Je ’20

Cleveland p72 Ag ’20 60w

“It is as breathlessly contrived and as diverting to follow as a

crooked street in a mediaeval town, along which anything might
happen.”

+ N Y Times 25:220 My 2 ’20 550w

“The tale furnishes pleasant diversion.”

+ Springf’d Republican p11a Je 6 ’20

480w

NICHOLSON, WATSON. Anthony Aston,

stroller and adventurer. *$1.25 The author, South
Haven, Mich.
 20–19783

This brief monograph forms a footnote to stage history. Little has

been known of Tony Aston, the author says, “save that he was a
strolling player for many years, the author of an unsuccessful play
and the much more important Brief supplement to Colley Cibber’s
apology.” An autobiographical sketch which he happened upon in the
British museum in 1914 has been made the basis of Mr Nicholson’s
account. This sketch is appended, as is the “Brief supplement.”

“The reprint is welcome and every student interested in ancient

Bohemias will be delighted to hear Aston tell, with complete
disregard for syntax and in the authentic pot-house style of Ned
Ward and the other blackguard wits, of his amazingly varied career.”

+ Nation 112:sup246 F 9 ’21 450w

“The student of the stage and society will find his career
interesting for the light it throws upon the provincial and illegitimate
stage of the time, concerning which practically nothing is known.” J.
W. Krutch

+ N Y Evening Post p8 N 27 ’20 380w

NICHOLSON, WATSON. Historical sources of

DeFoe’s Journal of the plague year. $2 Stratford co.
942.06

DeFoe’s “Journal of the plague year” which has hitherto been

classified as fiction and has been accounted as a “masterpiece of the
imagination” is here proven, by the aid of extracts from original
documents in the Burney collection and manuscript room of the
British museum, to be “a faithful record of historical facts, that it was
so intended by the author and is as nearly correct as it was humanly
possible to make it from the sources and time at his command.” The
contents are: Originals and parallels of the stories in DeFoe’s
Journal; The historical sources of the Journal; Errors in the Journal;
Summary. The appendices consist of excerpts from the original
sources of the Journal and from hitherto unpublished documents
illustrative of the plague. There is a bibliography.

Outlook 125:281 Je 9 ’20 90w

Spec 124:834 Je 19 ’20 400w
Springf’d Republican p8 Jl 23 ’20 240w

“Dr Watson Nicholson’s book suffers a little from the researcher’s

usual impatience with those who preceded him; a little more from his
sometimes odd and slack English; more still from careless proof-
reading. But those who are interested in DeFoe should read the book,
because the author does more than work his case out closely.”

+ − The Times [London] Lit Sup p418 Jl 1

’20 800w

NICOLAY, HELEN. Boys’ life of Lafayette. il

*$1.60 (2½c) Harper
 20–16920

The author writes of Lafayette as “a very gallant, inspiring figure

uniting the old world with the new.” She tells her young readers in
the preface: “This is no work of fiction. It is sober history; yet if the
bare facts it tells were set forth without the connecting links, its
preface might be made to look like the plot of a dime novel.” The
book is illustrated and has an index.

+ Booklist 17:123 D ’20

“Even tho this is pure history, as the author declares, there is a

deal of romance in the life of Lafayette to fascinate the young
reader.”

+ Lit D p96 D 4 ’20 40w

NIETZSCHE, FRIEDRICH WILHELM.

Antichrist. (Free lance books) *$1.75 (5c) Knopf 230

20–4092

Mr Mencken has made a new translation of “The antichrist,”

Nietzsche’s last work with the exception of his “Ecce homo.” The
introduction states: “The present translation of ‘The antichrist’ is
published by agreement with Dr Oscar Levy, editor of the English
edition of Nietzsche. There are two earlier translations, one by
Thomas Common and the other by Anthony M. Ludovici.... I began
this new Englishing of the book, not in any hope of supplanting
them, and surely not with any notion of meeting a great public need,
but simply as a private amusement in troubled days. But as I got on
with it I began to see ways of putting some flavour of Nietzsche’s
peculiar style into the English, and so amusement turned into a more
or less serious labour.” Mr Mencken’s introduction offers a critical
interpretation of Nietzsche.

“Mr Mencken’s translation of Nietzsche’s last considerable work is

lively and energetic, and his introduction is a happy example of his
critical writing.”

+ Ath p557 Ap 23 ’20 130w

Booklist 17:85 N ’20

Reviewed by Preserved Smith

Nation 110:sup483 Ap 10 ’20 250w

The Times [London] Lit Sup p243 Ap
15 ’20 60w

NIVEN, FREDERICK JOHN. Tale that is told.

*$1.90 (1½c) Doran

20–17825

The simple uneventful chronicle of a Scotch clergyman’s family,

told in a leisurely manner. The father is a genial egotist who had
preached to Queen Victoria at Balmoral and who never lets this fact
be lost sight of. The story follows the course of the six children’s lives
after his death, telling of their worldly success, business affairs, love
affairs and marriages. For a time three of the brothers conduct a
book store and circulating library, of which an amusing account is
given.

+ − Ath p439 O 1 ’20 820w

“He takes such a ‘slice of life’ as might delight Mr Hugh Walpole,

and he treats it quite in the manner of Mr Walpole, only—and it is an
important difference—he lacks something of his vitality. The
substance is more level, a level quality not due to restraint but to
quality of vision.” D. L. M.

+ − Boston Transcript p4 O 20 ’20 700w

“The scenes in the library are especially good.”

+ Cleveland p105 D ’20 40w

“The novel as a whole reflects the commonplace lives of the vast

majority of us, ‘such poor little figures struggling along in the jungle’
with considerable accuracy.”

+ N Y Times p18 N 14 ’20 850w

“I wish I could feel the glow that so many writing people seem to
be feeling about Frederick Niven’s ‘A tale that is told.’ It is pleasant
enough, human enough in its somewhat lacklustre fashion; but in the
end not much more than ‘a long preparation for something that
never happens.’”

+ − Review 4:57 Ja 19 ’21 640w

“The characters are unusually alive; it is a pity that they all lack
charm. The book is well constructed; the author has distinct ability.”

+ − Spec 126:56 Ja 8 ’21 40w

“It is refreshing to come upon a man who can write both lightly
and profoundly and who can mingle tenderness and humor without
losing the force of either.”

+ Springf’d Republican p9a N 14 ’20 380w

“It is not a story with a pattern, but there is a frame to it that gives
it bounds and a focus that gives it coherence; there is sunlight in it—
the pale northern sunlight of Scotland. The characterization is clear
and the more pungent for its tolerance.”

+ The Times [London] Lit Sup p633 S 30

’20 470w

NOGUCHI, YONÉ (MISS MORNING

GLORY, pseud.). Japanese hokkus. *$2 Four seas
co. 895

20–20445
 The hokku is the seventeen syllable poem of Japan which the
author describes at some length in the preface. This preface is in
itself a prose poem in its quaint English and with the vista it opens
into the Japanese mind. The real value of the hokku, we are told, is
not in what it expresses but how it expresses itself spiritually: not in
its physical directness but in its psychological indirectness. It is “like
a spider-thread laden with the white summer dews, swaying among
the branches of a tree; ... that sway indeed, not the thread itself, is
the beauty of our seventeen syllable poem.” Of the translating of the
hokku the author says, it is like the attempt to bring down the spider-
net and hang it up in another place. The epilogue is a reflection on
the introduction of western civilization into Japan.

“‘Japanese hokkus’ is remarkable for at least two reasons; one,

because its poems are of that sensitive and illusive loveliness that is
rare in the realism of contemporary publications, and another
because the book links the literature of the Orient and the Occident
rather more than any other poet whom we recall.” K. B.

+ Boston Transcript p7 O 2 ’20 1100w

“Whether it is because he is writing in a foreign language, or

because English cannot have packed into it the associations of
thousands of years and the treasure of half-forgotten philosophies,
the Japanese poet fails to produce the effect achieved by Waley in his
translations.” Babette Deutsch

+ − Dial 70:206 F ’21 230w

“To enjoy this present volume and to be deaf to Mr Walter de la

Mare—or to Shakespeare’s songs, for that matter—is to enjoy the art
page of the newspaper more than a visit to the originals in the art
gallery.” Llewellyn Jones
 − + Freeman 2:260 N 24 ’20 600w
+ N Y Evening Post p18 O 23 ’20 110w

NOLEN, JOHN. New ideals in the planning of

cities, towns and villages. il $1 (3c) Am. city bureau,
Tribune bldg., N.Y. 710

20–211

“The cities of the United States have not yet made many of those
public improvements that are so essential to modern life, especially
for the new era.... They have not yet applied in a businesslike and
economical manner the methods characteristic of the modern city
planning movement. Therefore the American city still suffers in
many ways from haphazard, piecemeal and shortsighted procedure.”
(Part 1) To show how these shortcomings are to be remedied, how
the new civic spirit is growing, what has already been done and what
is the promise of the future is the object of the book. Among the
topics discussed in the first part are: Two main divisions of city
planning; Specific needs of the smaller city; How to replan a city;
How to get a city plan into action. Part 2 contains in part: The city
planning movement; Local data as basis of city plan; Types of city
plans; Elements of city plans; Professional training and experience;
New towns and new standards; Public opinion and city planning
progress.

+ Booklist 17:20 O ’20

 + Springf’d Republican p11a Jl 11 ’20
220w

“We have never seen within such small compass a clearer

description of the processes of town planning or of the principles that
underlie good planning. A special merit of the book is that it reckons
with the limitations and difficulties of the small town where at the
present time such leadership as this is most needed and where
examples taken from the costly improvement schemes of large cities
are not helpful.” B. L.

+ Survey 43:592 F 14 ’20 320w

NORRIS, KATHLEEN (THOMPSON) (MRS

CHARLES GILMAN NORRIS). Harriet and the
piper. il *$1.90 (2c) Doubleday

20–13977

Harriet Fields had an emotional adventure when she was

seventeen, and her romantic fancy was captured by Royal Blondin’s
talk on Yogi philosophy, oriental religion and poetry. She even went
through a bogus marriage ceremony with him when her youthful
timorousness saved her from further disaster. Ten years later, when
she is filling a position of trust, as companion to the wife of the rich
Richard Carter and governess to his daughter Nina, Blondin crosses
her path again holding their former relationship over her as a sword,
to enlist her aid in the furtherance of his new schemes, i. e., marrying
young Nina Carter and possessing himself of her fortune. It involves
Harriet in many temptations. Twixt the overcoming of and yielding
to them her character is clarified. After Mrs Carter’s elopement and
sudden death, Harriet enters a marriage of convenience with Richard
Carter, whom she secretly loves and admires and the wooing by the
husband of his new wife forms part of the interest of the book.

“A lively and interesting story.”

+ − Ath p813 D 10 ’20 60w

Booklist 17:35 O ’20

“Well worn, even threadbare as her material is, Kathleen Norris

has contrived to concoct from it a very pleasant little story, and one
which holds the reader’s attention until the last half dozen chapters,
when it begins to drag badly. It is smoothly written, and agreeable to
read.”

+ − N Y Times p24 Ag 1 ’20 950w

“Readers who can put aside the insubstantial theme and the
artificial dilemmas attributed to the principals, will find some
entertainment in the flow of life and color through their vaguely
troubled days.”

− + Springf’d Republican p11a S 12 ’20

200w
The Times [London] Lit Sup p801 D 2
’20 100w