Protein & Peptide Letters, Volume 12, No. 2, 2005
Contents
Protein and Peptide Folding
Guest Editor: Masa Cemazar
Editorial
Masa Cemazar
Letters:
The
Designability Hypothesis and Protein Evolution
Pp.111-116
Edo
Kussell
The
Protein Folding Transition State: What Are f-Values Really Telling Us? Pp.117-122
Daniel
P. Raleigh and Kevin W. Plaxco
Model
Study:
HPr
as a Model Protein in Structure, Interaction, Folding and Stability Studies Pp.123-137
A.I.
Azuaga, J.L. Neira and N.A.J. van Nuland
Techniques:
Millisecond
Protein Folding Studied by NMR Spectroscopy Pp.139-146
Markus
Zeeb and Jochen Balbach
Protein
Folding Coupled with Disulfied Formation:
Oxidative
Folding of the Cystine Knot Motif in Cyclotide Proteins Pp.147-152
David.
J. Craik and Norelle. L. Daly
Prosequence-Mediated
Disulfide Coupled Folding of the Peptide Hormones Guanylin and Uroguanylin Pp.153-158
Thomas
Lauber and Ute C. Marx
Reviews:
Understanding
Protein Folding Through Peptide Models Pp.159-164
John
J. Osterhout
How
Do Cofactors Modulate Protein Folding? Pp.165-170
Catherine
L. Higgins, B.K. Muralidhara and Pernilla Wittung-Stafshede
Protein
Folding In Vivo and Aggregation:
Polypeptide
Models to Understand Misfolding and Amyloidogenesis and Their Relevance in
Protein Design and Therapeutics Pp.171-187
Jesus
Zurdo
Conformations
of Co-Translational Folding Intermediates Pp.189-195
Michael
S. Evans, Thomas F. Clarke IV and Patricia L. Clark
General Articles
Modulatory
Effects of pH, Cu+2 and Sheet Breakers on Aggregation of Amyloid
Peptides Pp.197-202
Kris
Beking, Xiaolei Hao, Sarmistha Basak and Ajoy Basak
Effect
of Metal Ions and EGTA on the Optical Properties of Concanavalin A at Alkaline
pH Pp.203-206
Mohammad
Tashfeen Ashraf and Rizwan Hasan Khan
Efficient
Immobilization of Enzymes on Microchannel Surface Through His-Tag and
Application for Microreactor Pp.207-210
Masaya
Miyazaki, Jun Kaneno, Susumu Yamaori, Takeshi Honda, Maria Portia P. Briones,
Masato Uehara, Kazunari Arima, Kenichi Kanno, Kenichi Yamashita, Yoshiko
Yamaguchi, Hiroyuki Nakamura, Hiroo Yonezawa, Masayuki Fujii and Hideaki Maeda
Abstracts
[Back to top] Editorial
Masa
Cemazar
This special issue of Protein and Peptide Letters focuses on protein and peptide folding in order to bring to the general reader an idea of the current state of this field. It brings together ten articles which expose various themes and range from letters discussing the folding theory to reviews of specific topics, a case study and developments of novel techniques.
Evolution has selected protein
sequences which can efficiently fold after their synthesis on the ribosome to
give a specific function. Protein folding is the underlying process that guides
the protein from its synthesis to its globular native form and remains a
challenge of modern science with two parts to its problem. First, it needs to
be deciphered how a specific sequence encodes a specific structure, which
inevitably defines the function for which the protein has evolved. Second, it
is important to understand how the sequence achieves the three-dimensional
array for which it was designed in terms of the mechanism and proceedings. All
pathways of the protein folding studies started with Levinthal, who pointed out
that a random unbiased search for the correct structure is impossible. Most
studies thereafter have sought for laws which govern the fact that only a
relatively small number of possible conformations need to be accessed by any
given protein on its transition from a random coil to the native structure. The
understanding of this complex process has gone a long way. Theories and models
justifying and explaining these phenomena have been proposed, and some of the
most prominent and successful ones include the framework model, hydrophobic
collapse theory and the nucleation-condensation mechanism. The combination of
experiment and molecular dynamics simulation has been able to predict protein
folding on atomic resolution. In parallel, the biophysical measurements with
increased time-resolution up to milli and microsecond scale have revealed the
rapid formation of some secondary structural elements. Another important
advance is the measurement of protein folding kinetics on the level of a single
molecule. There are still limitations of technology, especially computation,
which await novel advances before new discoveries can be made in protein
folding. A challenging future task will be to extend the research into the
faster and bigger. The studies of folding of large multi-domain proteins at
even faster time-scales will put the principles deduced for small proteins to a
test. In the last few years, an increasing number of intriguing protein
misfolding and aggregation studies show the importance of understanding protein
fold stability and raise the question of folding beyond an academic challenge.
Only correctly folded proteins are stable in vivo in the crowded
cellular environment and any protein that is slightly unstable or unable to
fold correctly could be the beginning of a debilitating pathological condition.
This adds additional motivation to produce a special issue that could serve as
a reference to studies involved in the research of protein folding diseases.
This issue starts with two letters that present different topic on protein
folding. Kussell writes about the designability hypothesis, which states that a
successful fold has an exponentially larger number of compatible sequences.
This part of protein evolution is probably a component of fold fitness, but
most probably not a dominant one. Designability might have an initial function
when the discovery of fold is done, but features less prominently later in the
evolution process. On the other hand, in the letter by Raleigh and Plaxco, the
credibility of the phi-value analysis in protein folding is discussed. The fact
that the structural interpretation of these values can be ambiguous and maybe
provides little insight into backbone participation is considered, and the
relationship between folding rates and phi-defined transition state structure
is evaluated.
The model study of Azuaga et al. reviews the studies on HPr protein, which combined detailed studies of protein folding and stability with structural and interaction studies. Emphasis is placed on the need for an integrated multi-techniques approach in order to understand the details of the folding reactions of even simple proteins.
The next section includes two articles, all of which focus on protein folding coupled with disulfide formation, the so-called oxidative folding. The First article, by Daly and Craik, speaks about the oxidative folding of a specific group of proteins, the cyclotides. These are cyclic peptides with a cystine knot motif. A detailed account is given of how the conformational and oxidative folding is coupled in these peptides and how they compare to other cystine knots. The second article in this section, by Marx and Lauber, discusses how certain hormones require their prosequences to be able to fold correctly in vitro. The prosequences play a crucial role in the oxidative folding of guanylin and pro-guanylin and their structures have a direct effect on the hormone topology.
The article by Zeeb et al. presents current developments in the field of real time NMR spectroscopy to follow protein folding, which allows protein folding studies to be followed on a microsecond to millisecond time scale. The manuscript also includes the millisecond protein folding studies by NMR on the cold shock protein CspB from Bacillus subtilis, for which it is possible to extract the folding and refolding rates.
Further, two inspiring reviews are presented. Osterhout reviews how peptide models have been used to describe the factors that stabilise elements of secondary structure. His article begins with a short review of the folding theories and then describes in detail a variety of peptide models and theories which have helped us in the understanding of the stability of secondary structures and kinetics of protein folding. Higgins et al., on the other hand, review the folding process of cofactor containing proteins. Not only are cofactors essential for biological activity, but it has also been shown that their interaction with unfolded proteins may affect both the mechanism and the speed of folding. Three proteins with their different cofactors, copper, flavin, and an iron-sulfur cluster, are contrasted in terms of the mechanism of their protein folding processes.
Last but not least, two articles focus on the protein folding in vivo, protein aggregation and its consequences. Patricia Clark’s group presents an article which addresses the subject of in vivo protein folding and provides a review of the known experimental results in this field. They examine the fact that macromolecular crowding in the cell promotes both the folding and aggregation and discuss the studies which have attempted to describe the conformations of the co-translational folding intermediates. Jesús Zurdo, on the other hand, presents an extensive review of the consequences of protein misfolding and aggregation. He first illustrates the protein folding in vivo in the crowded environment of the cell and then examines the phenomena of protein aggregation and amyloid formation. Amyloid models which are necessary to understand diseases and mechanisms of amyloid fibril formation are mentioned. Most importantly, the links between protein misfolding, aggregation process and human diseases are reviewed in detail.
Altogether, this issue gives a perspective of the same problem from different experimental techniques, theoretical backgrounds and different, although similar, biological systems. The importance of a multi-angle approach in the understanding of the complex folding process has been underlined for several years now, and I hope that the same thing will be shown with this issue. It was put together in the hope that the increasing number of enthusiastic protein folders from around the globe will persist in their research in order to help solve one of the remaining challenges of modern bioscience.
[Back to top]
The Designability
Hypothesis and Protein Evolution
Edo
Kussell
The usage of protein folds in nature is known to be non-uniform: a few folds are used often, while most others are used relatively rarely. What makes one fold more successful than another? The designability explanation, which posits that successful folds have an exponentially larger number of compatible sequences, is critically reviewed, and compared with other structural and functional explanations. It is argued that designability is one component of fold fitness, but most likely not a dominant one.
[Back to top]
The Protein Folding
Transition State: What Are f-Values Really Telling Us?
Daniel
P. Raleigh and Kevin W. Plaxco
Protein engineering-based studies of the folding transition state have accelerated significantly in the last decade, and more than a half dozen proteins have been subjected to extensive f-value analysis. A general picture is emerging from these studies of a transition state in which the large majority of experimentally characterized side chains participate in relatively homogeneous and energetically weak interactions playing only a relatively small role in defining relative folding rates.
[Back to top] HPr as a Model Protein in Structure, Interaction, Folding
and Stability Studies
A.I.
Azuaga, J.L. Neira and N.A.J. van Nuland
The small size and lack of disulphide bonds or cofactors in the Histidine-containing phosphocarrier protein (HPr) makes it an attractive system with which to study structure, interaction to its enzymatic partners, and its stability and folding. Here we give an overview on the immense work that has been performed on this protein and we will show that HPr has been widely used as a model protein to study important aspects in modern Structural Biology.
[Back
to top] Millisecond Protein Folding
Studied by NMR Spectroscopy
Markus
Zeeb and Jochen Balbach
Proteins are involved in virtually every biological process and in order to function, it is necessary for these polypeptide chains to fold into the unique, native conformation. This folding process can take place rapidly. NMR line shape analyses and transverse relaxation measurements allow protein folding studies on a microsecondto- millisecond time scale. Together with an overview of current achievements within this field, we present millisecond protein folding studies by NMR of the cold shock protein CspB from Bacillus subtilis.
[Back to top] Oxidative Folding of the Cystine
Knot Motif in Cyclotide Proteins
David.
J. Craik and Norelle. L. Daly
The cyclotides are a large family of plant proteins that have a cyclic backbone and a knotted arrangement of three conserved disulfide bonds. Despite the apparent complexity of their cystine knot motif it is possible to efficiently fold these proteins, as exemplified by oxidative folding studies on the prototypic cyclotide, kalata B1. This mini-review reports on the current understanding of the folding process in cyclotides. The synthesis and folding of these molecules paves the way for their application as stable molecular templates.
[Back to top] Prosequence-Mediated Disulfide
Coupled Folding of the Peptide Hormones Guanylin and Uroguanylin
Thomas
Lauber and Ute C. Marx
In contrast to their prohormones the mature peptide hormones guanylin and uroguanylin are not able to fold to their native disulfide connectivities upon oxidative folding. Structural properties of both peptide hormones and their precursor proteins as well as the role of their prosequences in proper disulfide coupled folding are reviewed. In addition, the structural behavior of a proguanylin mutant that closely resembles prouroguanylin has been investigated to gain further insight into structural properties of this homologous precursor protein.
[Back to top] Understanding Protein Folding
Through Peptide Models
John
J. Osterhout
From the time it was recognized that proteins are made up primarily of secondary structures, theories of protein folding have used secondary structural elements as important building blocks. Peptides have played a central role in elucidating the factors that stabilize individual elements of secondary structure and are now being employed to study higher levels of organization. The control of conformation in peptides has taken on new relevance with the realization that protein folding plays a central role in many disease states.
[Back to top] How Do Cofactors Modulate Protein
Folding?
Catherine
L. Higgins, B.K. Muralidhara and Pernilla Wittung-Stafshede
Cofactors are essential components of many proteins for biological activity. Characterization of several cofactor-binding proteins has shown that cofactors often have the ability to interact specifically with the unfolded polypeptides. This suggests that cofactor-coordination prior to polypeptide folding may be a relevant path in vivo. By binding before folding, the cofactor may affect both the mechanism and speed of folding. Here, we discuss three different cofactors that modulate protein-folding processes in vitro.
[Back to top] Polypeptide
Models to Understand Misfolding and Amyloidogenesis and Their Relevance in
Protein Design and Therapeutics
Jesus
Zurdo
The study of amyloid polypeptide models (polypeptides able to generate amyloid structures not necessarily connected with any pathology) provides an excellent tool to increase the understanding of the generic aspects of misfolding and aggregation as well as the details of the mechanism of polypeptide deposition in disease. This knowledge can be integrated and applied to different problems in therapy and biotechnology, and in particular to re-designing bioactive polypeptides (biopharmaceuticals) with improved properties.
[Back to top] Conformations
of Co-Translational Folding Intermediates
While in vitro experiments have contributed much to our understanding of protein folding, we know much less about how proteins fold in the more complex environment of the cell. This review summarizes our current knowledge of the earliest in vivo folding intermediates: the conformations adopted by nascent polypeptides during synthesis by the ribosome. The challenges related to successful folding in the cellular environment, including off-pathway aggregation and macromolecular crowding, are also discussed.
[Back to top] Modulatory
Effects of pH, Cu+2 and Sheet Breakers on Aggregation of Amyloid
Peptides
Kris
Beking, Xiaolei Hao, Sarmistha Basak and Ajoy Basak
The study explores in vitro by circular dichroism and mass spectrometry the effects of pH, Cu+2 ions and sheetbreakers on the secondary structures and self-aggregation of b-amyloid peptides [Ab43, Ab42 and Ab40] of Alzheimer's disease. Within pH 5.4-7.3, more sheet structures and aggregates containing up to 11 peptide units were observed. Cu+2 ions led to oxidative degradation or aggregation depending on its concentration and time of incubation. b-sheet breakers can reverse the self-aggregation process, suggesting their potential therapeutic use.
[Back to top] Effect
of Metal Ions and EGTA on the Optical Properties of Concanavalin A at Alkaline
pH
Mohammad
Tashfeen Ashraf and Rizwan Hasan Khan
In our earlier communications, we reported the effect of salts and alcohols on a-chymotrypsinogen [1] and the existence of stable intermediates at low pH in bromelain [2] and glucose oxidase [3]. In the present study, the role of metal ions and EGTA on the conformation of concanavalin A at alkaline pH was studied by near- and far-UV circular dichroism, fluorescence emission spectroscopy and binding of a hydrophobic dye, 1-anilino-8-naphthalene sulfonate (ANS). Far-UV CD spectra showed the transition from an ordered secondary structure at pH 7 with a trough at 223 nm to a relatively unordered state at pH 12. Near-UV CD spectra showed the loss of signal at 290 nm, thereby indicating the disruption of native three dimensional structure. Maximum ANS binding occurred at pH 12 suggesting the presence of an intermediate or molten globule-like state at alkaline pH.
[Back to top] Efficient
Immobilization of Enzymes on Microchannel Surface Through His-Tag and
Application for Microreactor
Masaya
Miyazaki, Jun Kaneno, Susumu Yamaori, Takeshi Honda, Maria Portia P. Briones,
Masato Uehara, Kazunari Arima, Kenichi Kanno, Kenichi Yamashita, Yoshiko
Yamaguchi, Hiroyuki Nakamura, Hiroo Yonezawa, Masayuki Fujii and Hideaki Maeda
We developed a simple immobilisation method for His-tagged enzymes on a microchannel surface. It facilitates immobilisation of protein molecule on microchannel surface through Ni-complex, using crude or purified protein solutions. By this method, we could immobilize proteins on microcapillary constantly. This method might be useful for further development of microreactor with reversibly immobilized enzymes.