Protein and Peptide Letters - Volume 21, Issue 3, 2014

Most proteinaceous pores are characterized as ionic channels. However, some are also involved in protein translocation through phospholipidic membranes. This concept has evolved slowly in cell biology and in biophysics, requiring the development of adapted electrical and biochemical methods. Protein translocation in mitochondria biogenesis, secretion by endoplasmic reticulum or bacteria, and bacterial toxins internalization are the main fields where proteinconducting pores have been described. The concept is now well established and progress at the molecular and atomic levels have shown how different this paradigm is from ionic channels involved in neurobiology. Protein-conducting pores are often parts of large complexes and electrical analysis gives on-line information at the single-molecule level. They have a large conductance that, in certain membranes, should be highly regulated to prevent ionic leaking through the membrane. Finally, they are involved not only in protein translocation, but also in membrane protein insertion (α-helix and β-barrel types).

When macromolecules such as proteins are forced to translocate through a narrow pore, their conformational entropy is reduced, resulting in a free energy barrier. This free energy barrier is additionally modulated by protein-pore interactions. Furthermore, the driving force of the translocation such as the electrochemical potential gradient and electroosmotic flow navigates the transport of the protein through the free energy landscape. Depending on the specifics of the protein-pore system and the driving force, the details of the translocation process and their statistical properties such as the average translocation time can vary significantly. Nevertheless, there are a few fundamental physical concepts that underly the ubiquitous phenomenon of polymer translocation, which are reviewed here.

Biological processes such as protein degradation and mitochondrial protein import require protein passage, or translocation, across narrow pores. In addition to its biological significance, protein translocation through biological or engineered nanopores offers a powerful analytic tool for biophysics and nanotechnology. This mini-review discusses the physical mechanisms of protein translocation, as revealed by computational and theoretical studies. A simple, simulationbased model of translocation is presented, which provides a comprehensive description of this process and allows one to estimate experimentally observable quantities such as the dwell time of a protein inside the pore and the frequency of translocation events. Limitations of this model are further described and possible strategies to overcome them are outlined. Recent simulation studies are beginning to provide insights into the physical mechanisms that drive protein translocation in living systems, which are also discussed here.

We survey the transport of proteins across nanopores in the framework of coarse-grained modeling. The advantage of a reduced complexity with respect to full-atomistic techniques lies in the possibility of massive sampling of events, thus allowing a statistical mechanical description of translocation in terms of ensemble averages. Often, protein transport through narrow channels tightly couples with unfolding pathways causing a richer phenomenology compared to unstructured polymer translocation. This reflects into a process controlled by the presence of protein-specific free-energy barriers which can be conveniently estimated by statistical mechanical methods implemented in coarse-grained simulations. We illustrate how protein transport dynamics can be characterized by the statistical properties of trajectories and sometimes interpreted as driven diffusion of a single collective coordinate over a free-energy landscape. We also discuss, through selected examples, the connection between reduced-model simulations and recent experimental results.

Recent studies in the area of single-molecule detection of proteins with nanopores show a great promise in fundamental science, bionanotechnology and proteomics. In this mini-review, I discuss a comprehensive array of examinations of protein detection and characterization using protein and solid-state nanopores. These investigations demonstrate the power of the single-molecule nanopore measurements to reveal a broad range of functional, structural, biochemical and biophysical features of proteins, such as their backbone flexibility, enzymatic activity, binding affinity as well as their concentration, size and folding state. Engineered nanopores in organic materials and in inorganic membranes coupled with surface modification and protein engineering might provide a new generation of sensing devices for molecular biomedical diagnostics.

In this minireview, the nanopore analysis of peptides and proteins in the presence of divalent metal ions will be surveyed. In all cases the binding of the metal ions causes the peptide or protein to adopt a more compact conformation which can no longer enter the α-hemolysin pore. In the absence of Zn(II) the 30-amino acid Zn-finger peptide can readily translocate the pore; but upon addition of Zn(II) the peptide folds and only bumping events are observed. Similarly, the octapeptide repeat from the N-terminus of the prion protein binds Cu(II), which prevents it from translocating. The fulllength prion protein also undergoes conformational changes upon binding Cu(II), which results in an increase in the proportion of bumping events. Myelin basic protein of 170 residues is intrinsically disordered and, perhaps surprisingly, for a basic protein of this size, can translocate against the electric field based on the observation that the event time increases with increasing voltage. It, too, folds into a more compact conformation upon binding Cu(II) and Zn(II), which prevents translocation. Finally even proteins such as maltose binding protein which does not contain a formal binding site for metal ions undergoes conformational changes in the presence of the metal chelator, EDTA. Thus, contamination of proteins with trace metal ions should be considered when studying proteins and peptides by nanopore analysis.

In this work, we review the process of protein unfolding characterized by a solid-state nanopore based device. The occupied or excluded volume of a protein molecule in a nanopore depends on the protein’s conformation or shape. A folded protein has a larger excluded volume in a nanopore thus it blocks more ionic current flow than its unfolded form and produces a greater current blockage amplitude. The time duration a protein stays in a pore also depends on the protein’s folding state. We use Bovine Serum Albumin (BSA) as a model protein to discuss this current blockage amplitude and the time duration associated with the protein unfolding process. BSA molecules were measured in folded, partially unfolded, and completely unfolded conformations in solid-state nanopores. We discuss experimental results, data analysis, and theoretical considerations of BSA protein unfolding measured with silicon nitride nanopores. We show this nanopore method is capable of characterizing a protein’s unfolding process at single molecule level. Problems and future studies in characterization of protein unfolding using a solid-state nanopore device will also be discussed.

In this mini-review we introduce and discuss a new method, at single molecule level, to study the protein folding and protein stability, with a nanopore coupled to an electric detection. Proteins unfolded or partially folded passing through one channel submitted to an electric field, in the presence of salt solution, induce different detectable blockades of ionic current. Their duration depends on protein conformation. For different studies proteins through nanopores, completely unfolded proteins induce only short current blockades. Their frequency increases as the concentration of denaturing agent or temperature increases, following a sigmoidal denaturation curve. The geometry or the net charge of the nanopores does not alter the unfolding transition, sigmoidal unfolding curve and half denaturing concentration or half temperature denaturation. A destabilized protein induces a shift of the unfolding curve towards the lower values of the denaturant agent compared to the wild type protein.Partially folded proteins exhibit very long blockades in nanopores. The blockade duration decreases when the concentration of denaturing agent increases. The variation of these blockades could be associated to a possible glassy behaviour.

In order to predict enzyme subclasses, this paper builds a new enzyme database in term of previous ideas and methods. Based on protein sequence, by selecting increment of diversity value, low-frequency of power spectral density, matrix scoring values and motif frequency as characteristic parameters to describe the sequence information, a Random Forest algorithm for predicting enzyme subclass is proposed. Using the Jack-knife test, the overall success rate identifying the 18 subclasses of oxidoreductases, the 8 subclasses of transferases, the 5 subclasses of hydrolases, the 6 subclasses of lyases, the 6 subclasses of isomerases, and the 6 subclasses of ligases are 90.86%, 95.24%, 96.42%, 98.60%, 97.53% and 98.03%. Furthermore, the same way is used to the previous database, the better results are obtained.

Ribosome recycling factor (RRF) and elongation factor-G catalyze disassembly of the post-termination complex and recycling of the ribosomal subunits back to a new round of initiation. Thermoanaerobacter tengcongensis survive high temperatures that Escherichia coli cannot, partly due to the higher thermal stability of T. tengcongensis ribosome recycling factor (tteRRF). Here we compared the structural stability of tteRRF and E. coli RRF (ecoRRF) and explore the reasons for the differences. We obtained the values of the thermodynamic parameters. Salt could reduce the thermal stability of tteRRF, which suggested that ion pairing was an important stabilizing factor in the case of tteRRF. The value of the heat capacity change of tteRRF unfolding, δCp, is significantly smaller than that of ecoRRF. A consequence of the small δCp value is that the change in free energy upon unfolding (δG) of tteRRF is larger than that of ecoRRF, while the values of the enthalpy change (δH) and the entropy change multiplied by temperature (T*δS) are smaller. The small δCp of tteRRF appears to be the main stabilizing factor for tteRRF.

Leucine rich repeats (LRRs) are present in over 20,000 proteins from viruses to eukaryotes. Two to sixty-two LRRs occur in tandem. Each repeat is typically 20-30 residues long and can be divided into an HCS (Highly conserved segment) and a VS (Variable segment). The HCS part consists of an eleven or a twelve residue stretch, LxxLxLxxNx(x/- )L, in which “L” is Leu, Ile, Val, or Phe, “N” is Asn, Thr, Ser, or Cys, “x” is a non-conserved residue, and “-” is a possible deletion site. Eight classes have been recognized. However, there are many unclassified or unrecognized LRRs. Here we performed to search novel LRRs using protein sequence database. The novel LRR domains are present over three hundred proteins, which include fungal ECM33 protein and Monosiga brevicollis LRR receptor kinase, from unicellular eukaryotes and bacteria. The HCS part is clearly different from that of the known LRRs and consists of a twelve or a thirteen residue stretch, VxGx(L/F)x(L/C)xxNx(x/-)L, that is characterized by the addition of Gly between the first conserved Val and the second conserved Leu. The novel LRRs identified here form a new family. The novel LRR domains were classified into four classes. The VS parts of the two classes are consistent with those of known, normal “SDS22-like” and “IRREKO” classes, while the other two classes have unique VS parts. The structures, functions, and evolution of the novel LRR domains and their proteins are described. The present results should stimulate various experimental studies.

KGF (Keratinocyte Growth Factor), also known as FGF7, is a potent mitogen for different types of epithelial cells, which regulates migration and differentiation of these cells and protects them from various insults under stress conditions. KGF is produced by mesenchymal cells and exerts its biological effects via binding to its high-affinity receptor, a splice variant of FGF receptor 2 (FGFR2-IIIb), which is expressed by various types of epithelial cells, including epidermal keratinocytes, intestinal epithelial cells, and hepatocytes. This expression pattern of KGF and its receptor suggests that KGF acts predominantly in a paracrine manner. After acute injury, in various tissues - including the skin, the bladder as well as in chronically injured tissue - KGF expression is strongly up-regulated. This up-regulation is likely to be important for the healing of injured epithelia. In addition, KGF could also exert a protective effect on these cells. There are many researches have been underway to identify clinical applications for KGF. Specifically, KGF is currently being evaluated in clinical trials sponsored by Amgen (Thousand Oaks, CA) to test its ability to ameliorate severe oral mucositis (OM) that results from cancer chemoradiotherapy. In this paper, we provide an overview of the knowledge on molecular properties, biological functions and the recent findings on clinical application of KGF.