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Protein
& Peptide Letters
ISSN: 0929-8665
Protein & Peptide Letters
Volume 12, Number 8, November 2005
Contents
Polypeptide Chemical Ligation Tools in Protein Engineering
Guest Editor: Julio A. Camarero
Editorial Pp. 721-722
Julio A. Camarero
Synthesis of Proteins by Native Chemical Ligation
Using Fmoc-Based Chemistry Pp. 723-728
Julio A. Camarero and Alexander R. Mitchell
[Abstract]
Chemical Synthesis of Proteins and Circular Peptides
Using Nα-(1-Phenyl-2-Mercaptoethyl) Auxiliaries
Pp. 729-735
Paolo Botti and Sylvie Tchertchian
[Abstract]
Chemical Synthesis Approaches to the Engineering
of Ion Channels Pp. 737-741
Gerd G. Kochendoerfer, Daniel Clayton and Christian
Becker
[Abstract]
Mimicking Reverse Protein Splicing by Three-Segment
Tandem Peptide Ligation Pp. 743-749
James P. Tam and Khee Dong Eom
[Abstract]
The Mechanism of Intein-Mediated Protein Splicing:
Variations on a Theme Pp. 751-755
Kenneth V. Mills and Francine B. Perler
[Abstract]
Incorporation of Selenocysteine into Proteins
Using Peptide Ligation Pp. 757-764
Robert J. Hondal
[Abstract]
Protein Chemical Ligation as an Invaluable Tool
for Structural NMR Pp. 765-768
Alexander Shekhtman
[Abstract]
Intein-Mediated, In Vitro and In
Vivo Protein Modifications with Small Molecules Pp.
769-775
Lay Pheng Tan and Shao Q. Yao
[Abstract]
Expressed Protein Ligation to Obtain Selectively
Modified Aldo/Keto Reductases Pp. 777-781
Michael P.O. Richter and Annette G. Beck-Sickinger
[Abstract]
Expressed Protein Ligation to Study Protein Interactions:
Semi-Synthesis of the G-Protein Alpha Subunit Pp.783-787
Lori L. Anderson, Garland R. Marshall and Thomas
J. Baranski
[Abstract]
Expressed Protein Ligation: A New Tool for the
Biosynthesis of Cyclic Polypeptides Pp. 789-794
Richard Kimura and Julio A. Camarero
[Abstract]
Using Siclopps for the Discovery of Novel Antimicrobial
Peptides and Their Targets Pp. 795-799
Lisa O. Nilsson, Mostafa Louassini and Ernesto
Abel-Santos
[Abstract]
General Articles
Regular Papers
Unusual Solute Protection Against Pressure Inactivation of
Yeast Alcohol Dehydrogenase Pp. 801-803
Gene Kidman and Dexter B. Northrop
[Abstract]
Prediction of Protein Secondary Structure Using
Improved Two-Level Neural Network Architecture Pp.
805-811
Xin Huang, De-Shuang Huang, Guang-Zheng Zhang,
Yun-Ping Zhu and Yi-Xue Li
[Abstract]
Proteome Analysis of Nelore Bull (Bos taurus indicus)
Seminal Plasma Pp. 813-817
T.I. Assumpção,W. Fontes, M.V. Sousa and
C.A.O. Ricart
[Abstract]
Crystallization Reports
Crystallization and Preliminary X-Ray Diffraction Studies
of Two Myotoxic Lys49-Phospholipases A2 Complexed
with α-Tocopherol Pp. 819-822
Juliana I. dos Santos, Agnes A.S. Takeda, Daniela
P. Marchi-Salvador, Andreimar M. Soares and Marcos R.M. Fontes
[Abstract]
Purification and Preliminary Crystallographic
Studies of CutC, a Novel Copper Homeostasis Protein from Shigella
flexneri Pp. 823-826
Yong-Qun Zhu, De-Yu Zhu, Hong-Xia Lu, Na Yang,
Gen-Pei Li, Da-Cheng Wang
[Abstract]
Abstracts
[Back to top]
Editorial
Julio A. Camarero
Chemistry has always played an important role in the study
of biological processes. Proteins have been a major focus
of biological chemistry research, both from the perspective
of fully understanding their innate biological functions and
from the perspective of harnessing those functions for a variety
of applications. Today, the study of proteins is even more
important because of the many successful genome-sequencing
projects that have revealed hundreds of thousands of new proteins
as predicted from sequence data. Numerous scientific studies
require access to chemically modified proteins. Some of these
molecules, however, are impossible to prepare using standard
DNA recombinant techniques. This is mostly due to the intrinsic
limitations of the genetic code, which only tolerates the
introduction of 20 naturally occurring amino acids. Chemical
synthesis, on the other hand, allows the incorporation of
non-natural amino acids in addition to posttranslational modifications.
During the last decade, peptide chemical ligation has emerged
as a promising method for the total synthesis and semisynthesis
of proteins. Chemical ligation uses efficient reactions between
unprotected peptides to form a stable peptide bond in a chemoselective
way between the a-carboxyl group of one of the peptide fragments
and the a-amino group of the other peptide fragment. This
method was developed to solve the intrinsic problems associated
with the historical approach for polypeptide synthesis employing
fragment condensation of large, protected polypeptide building
blocks. Early contributions to chemical ligation include the
thiol-capture procedure of Kemp [1], which relies on a proximity-based
entropic activation step, and the minimally protected peptide
condensation approach of Blake and Yamashiro [2], which employs
peptide thioacids and silver activation.
A defining point in chemical synthesis occurred when native
chemical ligation (NCL) was introduced by Kent and Tam in
1994 [3, 4]. In this reaction, two unprotected peptides, one
containing a C-terminal a-thioester group and the other an
N-terminal Cys, react chemoselectively under neutral conditions
to form a native peptide bond at the ligation site. This type
of thioester-based chemistry was first observed by Wieland
in the 1950s for the synthesis of small Cys-containing peptides
[5]. Since its introduction in 1994, NCL has been widely used
for the chemical synthesis of a multitude of chemically modified
and natural medium-sized proteins. Of particular interest
are the membrane proteins that are thought to form ˜30%
of the whole genome. In this special issue, Kochedoerfer and
coworkers describe the use of NCL for the chemical synthesis
and engineering of several bacterial membrane proteins.
The chemical synthesis of peptide thioesters using Fmoc-based
chemistry is a significant advance in the generic application
of NCL. C-terminal peptide thioesters are usually prepared
using standard Boc/benzyl-based solid-phase peptide synthesis.
Unfortunately this method requires the use of anhydrous hydrofluoric
acid, which is not very well suited for the synthesis of phospho-
and glycopeptides. Camarero and Mitchell review in this issue
the available methods for generating these important intermediates
using Fmoc-based chemistry.
NCL has undergone a number of improvements since it was first
introduced. One advance is the development of auxiliary groups
that in favorable cases overcome the requirement for an N-terminal
Cys. Botti and coworkers describe the use of Na-(1-phenyl-2-mercaptoethyl)-based
auxiliary groups for the synthesis of proteins lacking Cys
residues. This is illustrated in the total chemical synthesis
of cytochrome b562. Another improvement is the use of sequential
ligation strategies, which allows several peptides to be linked
together in series. Tam and coworkers exemplify this method
by sequentially combining NCL and Y-Pro ligation.
The NCL approach can also be used in the semisynthesis of
proteins from recombinant and synthetic fragments, which extends
the size and complexity of the protein targets available to
chemical engineering. As noted above, the NCL of two polypeptides
requires that one of the fragments possess an N-terminal Cys
and the other contain a a-thioester moiety. Polypeptides containing
N-terminal Cys residues can easily be obtained using standard
recombinant DNA expression methods. Methods for the biosynthesis
of recombinant polypeptide a-thioesters, on the other hand,
just recently have become available. The elucidation of the
protein-splicing mechanism by Perler and coworkers was key
for developing biosynthetic methods to produce these important
intermediates [6]. Perler and Mills review in this issue the
mechanism of protein splicing and how it can be modified to
provide new tools in protein engineering.
The NCL of recombinant polypeptide a-thioesters and synthetic
N-terminal Cys-containing polypeptides was first reported
in 1998 by the Muir and Xu groups, and it was named expressed
protein ligation (EPL) and intein-mediated protein ligation
(IPL), respectively [7, 8]. Since its introduction, EPL has
been applied to the engineering of many classes of protein
from both eukaryotic and prokaryotic organisms. These classes
include kinases, phosphatases, transcription factors, polymerases,
ion channels, cytoplasmic- and membrane-signaling proteins
as well as antibodies. A variety of chemical modifications
have been introduced into these proteins, and questions have
been addressed that would have been difficult to respond to
by other means.
Baranski and coworkers describe in this issue the use of
EPL to incorporate conformationally constrained nitroxide
spin labels and isotopically labeled amino acids into the
C-terminal tail of the subunit a of various G-proteins. These
site-specific labeled Ga-proteins are used to study the interaction
between the heterotrimeric G-protein and its membrane receptor
using electron paramagnetic resonance (EPR) and nuclear magnetic
resonance (NMR).
EPL has also been used to develop a segmental isotopic labeling
strategy designed to overcome the practical size limit for
protein structure determination using NMR. Shekhtman reviews
the combined use of EPL to generate segmental isotopically
labeled proteins with recently developed transverse relaxation
optimized–based NMR techniques. Combination of these
two techniques allows access to high-resolution structural
information in protein complexes larger than 50 kDa.
EPL, as previously mentioned, is an excellent tool for incorporating
unnatural amino acids into proteins. Hondal reviews the preparation
of N-terminal selenocysteine-containing peptides and their
use in EPL to generate selenocysteine-containing proteins.
A general method is not yet available for preparing recombinant
Selenocysteine-containing proteins. The ability to incorporate
this unnatural residue into proteins is exciting, especially
in those cases where the selenol group can be incorporated
into the catalytic group of an enzyme.
Several EPL-based strategies have been used to generate circular
proteins in vitro and in vivo. Circular proteins are of considerable
interest to researchers in the protein-engineering and protein-folding
communities. Kimura and Camarero describe the use of EPL for
the biosynthesis of cyclic polypeptides, which offers many
advantages over chemical synthetic methods. Large combinatorial
libraries of cyclic peptides may be generated and screened
in vivo using molecular biology tools. Abel-Santos and coworkers
describe the use of the naturally occurring Ssp DnaE split
intein for the generation of large libraries of cyclic polypeptides
inside E. coli, which are screened in vivo for their antimicrobial
properties.
EPL has also been used for the site-specific incorporation
of biophysical probes. Yao and coworkers describe the specific
biotinylation of proteins in vitro and in vivo using EPL.
These biotinylated proteins are later immobilized on avidin-coated
glass slides to produce protein microarrays. In a similar
way, Beck-Sickinger and coworkers describe the production
of site-specific biotinylated aldo/keto reductases for attachment
to avidin-coated surfaces. Using this approach ensures that
all the proteins will attach to the surface through the same
binding site and, thus, provide well-defined protein monolayers.
In summary, this special issue contains state-of-the art
reports on how NCL and EPL can be used for the chemical engineering
of proteins. I hope this will encourage others to continue
using these powerful tools for exploring the molecular basis
of protein function and the roles proteins play in complex
biochemical pathways within the cell.
REFERENCES
[1] Kemp, D. S., Leung, S. L. and Kerkman, D. J. (1981) Tetrahedron
Lett., 22, 181-184.
[2] Yamashiro, D. and Blake, J. (1981) Int. J. Pept. Protein
Res., 18, 383-392.
[3] Dawson, P. E., Muir, T. W., Clark-Lewis, I. and Kent,
S. B. H. (1994) Science, 266, 776-100%.
[4] Tam, J. P., Lu, Y. A., Liu, C. F. and Shao, J. (1995)
Proc. Natl. Acad. Sci. U.S.A., 92, 12485-12489.
[5] Wieland, T., Bokelmann, E., Bauer, L., Lang, H. U. and
Lau, H. (1953) Liebigs Ann. Chem., 583, 129.
[6] Xu, M.-Q. and Perler, F. B. (1996) EMBO J., 15, 5146-5153.
[7] Muir, T. W., Sondhi, D. and Cole, P. A. (1998) Proc.
Natl. Acad. Sci. U.S.A., 95, 6705-6710.
[8] Evans, T. C., Benner, J. and Xu, M.-Q. (1998) Protein
Sci., 7, 2256-2264.
[Back to top]
Synthesis of Proteins by Native Chemical Ligation Using
Fmoc-Based Chemistry
Julio A. Camarero and Alexander R. Mitchell
C-terminal peptide α-thioesters
are valuable intermediates in the synthesis/semisynthesis
of proteins by native chemical ligation. They are prepared
either by solid-phase peptide synthesis (SPPS) or biosynthetically
by protein splicing techniques. The present paper reviews
the different methods available for the chemical synthesis
of peptide α-thioesters
using Fmoc-based SPPS.
[Back to top]
Chemical Synthesis of Proteins and Circular Peptides Using
Nα-(1-Phenyl-2-Mercaptoethyl) Auxiliaries
Paolo Botti and Sylvie Tchertchian
An overview of the applications of Nα-(1-phenyl-2-mercaptoethyl)
auxiliary is presented. We describe the on resin preparation
(Cα-carboxy and thioester) of Nα-auxiliary
derivatives of glycine and the synthesis and incorporation
of preformed Nα-auxiliary derivatives of glycine
and alanine with the protection schemes, including the thiazolidine
strategy for SPPS. Such approaches allowed the synthesis of
the protein cytochrome b562 as well as native circular peptides
after successful removal of the auxiliary.
[Back to top]
Chemical Synthesis Approaches to the Engineering of Ion
Channels
Gerd G. Kochendoerfer, Daniel Clayton and Christian
Becker
Chemoselective ligation strategies have previously provided
synthetic access to water-soluble proteins with novel properties,
and more recently these strategies have been used to prepare
ion channels. Examples of ion channels prepared by total chemical
synthesis include bacterial mechanosensitive channels, and
viral ion channels. Chemical protein synthesis allows for
the generation of ion channel proteins with both native, and
engineered structural or conductance properties.
[Back to top]
Mimicking Reverse Protein Splicing by Three-Segment Tandem
Peptide Ligation
James P. Tam and Khee Dong Eom
Here we report a bi-directional and interchangeable three-segment
peptide ligation of N, M, and C-segments, mimicking the reverse
process of protein splicing to form, in tandem, a tripartite
NMC-peptide using a synthetic intein, a role served by the
M-segment with an N-terminal Ser or Thr and a C-terminal thioester.
[Back to top]
The Mechanism of Intein-Mediated Protein Splicing: Variations
on a Theme
Kenneth V. Mills and Francine B. Perler
Intein-mediated protein splicing is facilitated by four separate
but coordinated nucleophilic displacement reactions that result
in the excision of the intein and the ligation of the flanking
polypeptides, called the exteins. These reactions are catalyzed
by the intein plus the first downstream extein amino acid
without the assistance of cofactors or auxiliary enzymes.
Non-canonical inteins missing conserved nucleophilic residues
at the N- or C-terminus likely splice using variations of
the standard mechanism.
[Back to top]
Incorporation of Selenocysteine into Proteins Using Peptide
Ligation
Robert J. Hondal
Expressed protein ligation has become a frequently used technique
to insert non-standard amino acids into proteins. The technique
has been adapted to insert selenocysteine residues in place
of cysteine residue in proteins, taking advantage of the similarity
in the chemistries of sulfur and selenium. This replacement
can confer unique structural and catalytic properties to enzymes
and proteins. The development of this technique also allows
for naturally occurring selenoproteins to be produced semisynthetically.
[Back to top]
Protein Chemical Ligation as an Invaluable Tool for Structural
NMR
Alexander Shekhtman
This article reviews the methodology and recent applications
of expressed protein ligation for NMR structural studies of
proteins and protein complexes.
[Back to top]
Intein-Mediated, In Vitro and In Vivo
Protein Modifications with Small Molecules
Lay Pheng Tan and Shao Q. Yao
We review intein-mediated approaches for the site-specific
modifications of proteins and highlight their applications
in (1) the site-specific in vitro and in vivo
biotinylation of proteins for protein arrays and (2)
the site-specific in vivo labeling of proteins in
living cells.
[Back to top]
Expressed Protein Ligation to Obtain Selectively Modified
Aldo/Keto Reductases
Michael P.O. Richter and Annette G. Beck-Sickinger
Specific enzyme immobilization has moved into the focus for
many applications in biochemical research fields. Expressed
Protein Ligation (EPL) has been proven to be ideal to selectively
label proteins at single positions. Applying this technique
to enzymes of the aldo/keto reductase superfamily provides
a new approach to generate native or modified redox enzymes
for direct and indirect immobilization.
[Back to top]
Expressed Protein Ligation to Study Protein Interactions:
Semi-Synthesis of the G-Protein Alpha Subunit
Lori L. Anderson, Garland R. Marshall and Thomas J.
Baranski
Interactions between G proteins and GPCRs are fundamental
for transmitting signals for a multitude of physiologic responses.
Little is known regarding the protein-protein interface between
the G protein and the receptor, much less the mechanisms for
receptor activation of G proteins. Here, we will describe
how expressed protein ligation will aid in the study of protein-protein
interactions between semi-synthetic G alpha subunits and GPCRs.
[Back to top]
Expressed Protein Ligation: A New Tool for the Biosynthesis
of Cyclic Polypeptides
Richard Kimura and Julio A. Camarero
The present paper reviews the use of expressed protein ligation
for the biosynthesis of backbone cyclized polypeptides. This
general method allows the in vivo and in vitro
biosynthesis of cyclic polypeptides using recombinant DNA
expression techniques. Biosynthetic access to backbone cyclic
peptides opens the possibility to generate cell-based combinatorial
libraries that can be screened inside living cells for their
ability to attenuate or inhibit cellular processes.
[Back to top]
Using Siclopps for the Discovery of Novel Antimicrobial
Peptides and Their Targets
Lisa O. Nilsson, Mostafa Louassini and Ernesto Abel-Santos
High throughput screening of SICLOPPS libraries afforded
six distinct cyclic peptides that inhibit Escherichia
coli growth both in liquid and solid media. One of these
peptides (LN05) reduced both bacterial growth rate and caused
cell aggregation in liquid media. Mutant bacteria immune to
LN05 action were obtained at a frequency of 10-7. Over-expression
of an E. coli genomic library in the presence of
LN05 production resulted in enrichment of a single genomic
construct, a fragment of the NarZ gene.
[Back to top]
Unusual Solute Protection Against Pressure Inactivation
of Yeast Alcohol Dehydrogenase
Gene Kidman and Dexter B. Northrop
A 3:1 combination of sucrose and glycine provides significantly
greater protection against pressure-induced inactivation of
yeast alcohol dehydrogenase than either solute alone. Trehalose,
alone, gives much greater protection than sucrose alone, but
not so in combination with glycine. These are striking new
findings that cannot be accounted for by current theories
of protein stabilization.
[Back to top]
Prediction of Protein Secondary Structure Using Improved
Two-Level Neural Network Architecture
Xin Huang, De-Shuang Huang, Guang-Zheng Zhang, Yun-Ping
Zhu and Yi-Xue Li
In this paper we propose constructing an improved two-level
neural network to predict protein secondary structure. Firstly,
we code the whole protein composition information as the inputs
to the first-level network besides the evolutionary information.
Secondly, we calculate the reliability score for each residue
position based on the output of the first-level network, and
the role of the second-level network is to take full advantage
of the residues with a higher reliability score to impact
the neighboring residues with a lower one for improving the
whole prediction accuracy. Thirdly, considering it is indeed
a problem that the target protein can be lost in the multiple
sequence alignment we propose to code single sequence into
the second-level network. The experimental results show that
our proposed method can efficiently improve the prediction
accuracy.
[Back to top]
Proteome Analysis of Nelore Bull (Bos taurus indicus)
Seminal Plasma
T.I. Assumpção,W. Fontes, M.V. Sousa
and C.A.O. Ricart
The Nelore bull (Bos taurus indicus) seminal plasma
proteome was analyzed by MALDI-TOF MS and two-dimensional
gel electrophoresis. A total of 260 spots were visualized
in the 2-DE gel (pI range 3-10) and 13 spots could be identified
by peptide mass fingerprinting corresponding to 11 different
polypeptides. The results allowed the creation of the first
proteomic map of Bos taurus indicus seminal plasma. The roles of the
identified proteins in the bull seminal plasma are discussed.
[Back to top]
Crystallization and Preliminary X-Ray Diffraction Studies
of Two Myotoxic Lys49-Phospholipases A2 Complexed
with α-Tocopherol
Juliana I. dos Santos, Agnes A.S. Takeda, Daniela P.
Marchi-Salvador, Andreimar M. Soares and Marcos R.M. Fontes
BnSP-7 and BnSP-6, two Lys49-phospholipase A2
isolated from Bothrops neuwiedi pauloensis snake
venom, were co-crystallized with α-tocopherol and X-ray
diffraction data were collected for both complexes (2.2 and
2.6 Å). A new “alternative” quaternary conformation
for these two complexes compared with all other dimeric Lys49-PLA2
has been observed.
[Back to top]
Purification and Preliminary Crystallographic Studies of
CutC, a Novel Copper Homeostasis Protein from Shigella
flexneri
Yong-Qun Zhu, De-Yu Zhu, Hong-Xia Lu, Na Yang,
Gen-Pei Li, Da-Cheng Wang
CutC is a novel copper homeostasis protein containing 248
amino acids. Here we report the cloning, expression, purification,
crystallization and preliminary X-ray crystallographic studies
of CutC from Shigella flexneri 2a. Purification
of CutC and its selenomethionine (SeMet) derivate were done
using immobilized metal ion affinity chromatography, size-exclusion
and ion-exchange chromatography. The purified proteins were
crystallized using the hanging drop vapor diffusion method.
The diffraction data for the native and SeMet CutC, respectively,
have been collected with resolution of 1.7 Å and 2.1
Å. They belong to the space group C2221 and similar
cell dimension. The native protein crystals have cell parameters:
a=75.3267, b=97.6718, c=132.6910.
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