Single Protein Level Research Articles

MMDB and VAST+: tracking structural similarities between macromolecular complexes

The computational detection of similarities between protein 3D structures has become an indispensable tool for the detection of homologous relationships, the classification of protein families and functional inference. Consequently, numerous algorithms have been developed that facilitate structure comparison, including rapid searches against a steadily growing collection of protein structures. To this end, NCBI’s Molecular Modeling Database (MMDB), which is based on the Protein Data Bank (PDB), maintains a comprehensive and up-to-date archive of protein structure similarities computed with the Vector Alignment Search Tool (VAST). These similarities have been recorded on the level of single proteins and protein domains, comprising in excess of 1.5 billion pairwise alignments. Here we present VAST+, an extension to the existing VAST service, which summarizes and presents structural similarity on the level of biological assemblies or macromolecular complexes. VAST+ simplifies structure neighboring results and shows, for macromolecular complexes tracked in MMDB, lists of similar complexes ranked by the extent of similarity. VAST+ replaces the previous VAST service as the default presentation of structure neighboring data in NCBI’s Entrez query and retrieval system. MMDB and VAST+ can be accessed via http://www.ncbi.nlm.nih.gov/Structure.

Single-molecule spectroscopy reveals photosynthetic LH2 complexes switch between emissive states

Photosynthetic organisms flourish under low light intensities by converting photoenergy to chemical energy with near unity quantum efficiency and under high light intensities by safely dissipating excess photoenergy and deleterious photoproducts. The molecular mechanisms balancing these two functions remain incompletely described. One critical barrier to characterizing the mechanisms responsible for these processes is that they occur within proteins whose excited-state properties vary drastically among individual proteins and even within a single protein over time. In ensemble measurements, these excited-state properties appear only as the average value. To overcome this averaging, we investigate the purple bacterial antenna protein light harvesting complex 2 (LH2) from Rhodopseudomonas acidophila at the single-protein level. We use a room-temperature, single-molecule technique, the anti-Brownian electrokinetic trap, to study LH2 in a solution-phase (nonperturbative) environment. By performing simultaneous measurements of fluorescence intensity, lifetime, and spectra of single LH2 complexes, we identify three distinct states and observe transitions occurring among them on a timescale of seconds. Our results reveal that LH2 complexes undergo photoactivated switching to a quenched state, likely by a conformational change, and thermally revert to the ground state. This is a previously unobserved, reversible quenching pathway, and is one mechanism through which photosynthetic organisms can adapt to changes in light intensities.

Use of proteomics to understand seed development in rice

Rice is an important cereal crop and has become a model monocot for research into crop biology. Rice seeds currently feed more than half of the world's population and the demand for rice seeds is rapidly increasing because of the fast-growing world population. However, the molecular mechanisms underlying rice seed development is incompletely understood. Genetic and molecular studies have developed our understanding of substantial proteins related to rice seed development. Recent advancements in proteomics have revolutionized the research on seed development at the single gene or protein level. Proteomic studies in rice seeds have provided the molecular explanation for cellular and metabolic events as well as environmental stress responses that occur during embryo and endosperm development. They have also led to the new identification of a large number of proteins associated with regulating seed development such as those involved in stress tolerance and RNA metabolism. In the future, proteomics, combined with genetic, cytological, and molecular tools, will help to elucidate the molecular pathways underlying seed development control and help in the development of valuable and potential strategies for improving yield, quality, and stress tolerance in rice and other cereals. Here, we reviewed recent progress in understanding the mechanisms of seed development in rice with the use of proteomics.

High-throughput gene expression analysis at the level of single proteins using a microfluidic turbidostat and automated cell tracking

We have developed a method combining microfluidics, time-lapsed single-molecule microscopy and automated image analysis allowing for the observation of an excess of 3000 complete cell cycles of exponentially growing Escherichia coli cells per experiment. The method makes it possible to analyse the rate of gene expression at the level of single proteins over the bacterial cell cycle. We also demonstrate that it is possible to count the number of non-specifically DNA binding LacI-Venus molecules using short excitation light pulses. The transcription factors are localized on the nucleoids in the cell and appear to be uniformly distributed on chromosomal DNA. An increase in the expression of LacI is observed at the beginning of the cell cycle, possibly because some gene copies are de-repressed as a result of partitioning inequalities at cell division. Finally, a size-growth rate uncertainty relation is observed where cells living in rich media vary more in the length at birth than in generation time, and the opposite is true for cells living in poorer media.

Graded Tuning of Phosphatase Activity of VSP Coupled with the Intermediate State of the Voltage Sensor

Voltage-sensing phosphatase (VSP) consists of the voltage sensor domain and the cytoplasmic phosphatase region. We have shown that the movement of the voltage sensor domain (VSD) is coupled to the phosphatase activity over a wide range of membrane potential (Sakata et al., 2011). However, it still remains elusive how the phosphatase activity is regulated by the movement VSD at the level of single molecule. One possible idea is that constant level of phosphatase activities is attained when VSD is in a fully activated state, and the number of molecules in the active state increases as the membrane potential becomes more positive. The other probability is that the enzymatic activity of single VSP proteins can be graded upon distinct activated states of VSD. In this case, partial activation of VSD leads to certain level of the phosphatase activity. To distinguish between two possibilities, we studied Dr-VSP with mutations in VSD, Dr-VSP(T156R/I165R), whose Q-V plot was able to be fitted by the sum of two Boltzmann equations. Analyses of ‘gating’ current and voltage clamp fluorometry showed that VSD of this mutant moves in two steps. The enzyme-defective mutant or the protein with mutation in the linker between VSD and the phosphatase domain also showed two-step movements of the VSD. Measurements of the phosphatase activity of Dr-VSP(T156R/I165R) revealed that both transitions of VSD activation increase the phosphatase activity. These suggest that the full activation of the voltage sensor is not necessary to exhibit the phosphatase activity, and the phosphatase activity at the single protein level could be graded dependent upon the magnitude of the movement of the VSD.

Protein Logic: A Statistical Mechanical Study of Signal Integration at the Single-Molecule Level

Information processing and decision-making is based upon logic operations, which in cellular networks has been well characterized at the level of transcription. In recent years, however, both experimentalists and theorists have begun to appreciate that cellular decision-making can also be performed at the level of a single protein, giving rise to the notion of protein logic. Here we systematically explore protein logic using a well-known statistical mechanical model. As an example system, we focus on receptors that bind either one or two ligands, and their associated dimers. Notably, we find that a single heterodimer can realize any of the 16 possible logic gates, including the XOR gate, by variation of biochemical parameters. We then introduce what to our knowledge is a novel idea: that a set of receptors with fixed parameters can encode functionally unique logic gates simply by forming different dimeric combinations. An exhaustive search reveals that the simplest set of receptors (two single-ligand receptors and one double-ligand receptor) can realize several different groups of three unique gates, a result for which the parametric analysis of single receptors and dimers provides a clear interpretation. Both results underscore the surprising functional freedom readily available to cells at the single-protein level.

Cohesive behavior of soft biological adhesives: Experiments and modeling

Extracellular proteins play a key role in generating and maintaining cohesion and adhesion in biological tissues. These “natural glues” are involved in vital biological processes such as blood clotting, wound healing and maintaining the structural integrity of tissues. Macromolecular assemblies of proteins can be functionally stabilized in a variety of ways in situ that include ionic interactions as well as covalent crosslinking to form protein networks that can extend both within and between tissues. Within tissues, myriad cohesive forces are required to preserve tissue integrity and function, as are additional appropriate adhesive forces at interfaces both within and between tissues of differing composition. While the mechanics of some key structural adhesive proteins have been characterized in tensile experiments at both the macroscopic and single protein levels, the fracture toughness of thin proteinaceous interfaces has never been directly measured. Here, we describe a novel and simple approach to measure the cohesive behavior and toughness of thin layers of proteinaceous adhesives. The test is based on the standard double-cantilever beam test used for engineering adhesives, which was adapted to take into account the high compliance of the interface compared with the beams. This new “rigid double-cantilever beam” method enables stable crack propagation through an interfacial protein layer, and provides a direct way to measure its full traction–separation curve. The method does not require any assumption of the shape of the cohesive law, and the results provide abundant information contributing to understanding the structural, chemical and molecular mechanisms acting in biological adhesion. As an example, results are presented using this method for thin films of fibrin—a protein involved in blood clotting and used clinically as a tissue bio-adhesive after surgery—with the effects of calcium and crosslinking by Factor XIII being examined. Finally, a simple model is proposed, demonstrating how a bell-shaped cohesive law forms during the failure of the fibrin interface based on an eight-chain model whose structure degrades and changes configuration with stress.

Pengaruh Level Subtitusi Protein Sel Tunggal (Cj Prosin) Pada Pakan Komersial Terhadap Performan Ayam Broiler

The effect of level substitution of single cell protein (CJ Prosin) by commercial feed on broiler performanceABSTRACT. A research to find out the effect of the substitution level of single cell protein in commercial feed on Broiler’s performance was conducted from June to December 2011. The purpose of this experiment is to find out alternative protein sources for animal feedstuff that can be used as protein substitution in animal feed on body weight, feed consumption and feed conversion efficiency. The experiment is considered as preliminary experiment to observe the response of single cell protein on broiler performance. This research was carried out at experimental farm, Animal Husbandry Department, Syiah Kuala University. Totally, 250 DOC of Cobb breed with the average body weight 36,78±2,32 gr. used in this experiment. Completely Randomized Design was applied in this experiment with 5 treatments; P0 (commercial feed), P1(commercial feed substituted with 6% SCP), P2 (commercial feed substituted with 12% SCP) dan P3 (commercial feed substituted with 18%CSP). Each treatment consist of 5 replications with total of 20 treatment’s units. Feed was formulated with isoprotein and isoenergy based on NRC requirement ( 23% protein ; 3200 kcal ME/kg at the age of 1-2 week and 20 % protein ; 3200 kcal ME/kg at the age of 4-7 week). Data was analyzed by using SPSS program SPSS (Window 2008). Differences between treatment were stated by Duncan’s multiple range test. The results of the experiment showed that commercial feed substituted with the level of SCP was not significantly difference (P0.05) on body weight gain, feed consumption, and feed conversion ratio. On the other hand, administration of SCP as substituted feed commercial on the level of 12 % and 16 % influenced significantly (P0.05) on body weight gain and feed conversion ratio, but not on feed consumption at the level of 12% SCP. As a conclusion, the use of SCP as animal feed sources in commercial feed only could be tolerated at the level of 6%.

Handling of artificial membranes using electrowetting-actuated droplets on a microfluidic device combined with integrated pA-measurements.

Artificial membranes, as a controllable environment, are an essential tool to study membrane proteins. Electrophysiology provides information about the ion transport mechanism across a membrane at the single-protein level. Unfortunately, high-throughput studies and screening are not accessible to electrophysiology because it is a set of not automated and technically delicate methods. Therefore, it is necessary to automate and parallelize electrophysiology measurement in artificial membranes. Here, we present a first step toward this goal: the fabrication and characterization of a microfluidic device integrating electrophysiology measurements and the handling of an artificial membrane which includes its formation, its displacement and the separation of its leaflets using electrowetting actuation of sub-μL droplets. To validate this device, we recorded the insertion of a model porin, α-hemolysin.

Direct Quantification of the Effects of Osmolytes on Protein Unfolding and Refolding Pathways using AFM Techniques

There is considerable interest in developing new effective therapies based on small molecules for the treatment of devastating pathologies, such as Alzheimer, Huntington and Parkinson diseases. Osmolytes are a class of small organic molecules found in all taxa that can profoundly affect protein stability and aggregation. Osmolytes such as sorbitol or trimethylamine-N- oxide can act as “chemical chaperones” by increasing the stability of native proteins, assisting refolding of unfolded polypeptides, inhibiting protein aggregation. However, the mechanism of protecting osmolytes action on protein folding and stability remains controversial, where the protein backbone seems to play a key role. Here we used single-molecule AFM to systematically analyze the effect of several naturally occurring osmolytes (sorbitol, sarcosine, TMAO, inositol, trehalose, proline, glycerol, and taurine) on the stability and folding kinetics on PKD domains which are normally exposed to high urea concentrations in the kidney. Our approach enables us to directly quantify the effects of osmolytes on the folded state and on the exposed protein backbone in the absence of chemical denaturants. Here we show that a mixture of different protecting osmolytes (e.g. 0.5M sorbitol + 0.5M sarcosine) counteracts the effects of 1M urea equally well as 1M sorbitol on the unfolding/refolding rates. These results indicate an additive effect of these protecting osmolytes on domain stability. However, we also found osmolytes mixtures that work synergistically on protein stability. Our results demonstrate a robust approach to study the mechanism of action of osmolytes at the single protein level.

Systematic control of protein interactions for systems biology

Since its birth, systems biology has gained a great deal from the protocols devised to study phenomena at the level of single proteins and nucleic acids. Such protocols find broad markets and utility at higher levels of biological organization, from next-generation sequencing, which uses modified nucloeotides and fluorescent identifiers (1), to ChIP-seq analysis, which identifies histone modifications and binding sites in protein–DNA interactions (2). In PNAS, Sivaramakrishnan and Spudich introduce a system for interrogating interactions between pairs of proteins, domains, and peptides (3), and it is very possible that their invention will find applicability in the construction and analysis of large-scale protein–protein interaction networks (4).

Effects of pressure and temperature on the binding of RecA protein to single-stranded DNA

The binding and polymerization of RecA protein to DNA is required for recombination, which is an essential function of life. We study the pressure and temperature dependence of RecA binding to single-stranded DNA in the presence of adenosine 5'-[γ-thio]triphosphate (ATP[γ-S]), in a temperature regulated high pressure cell using fluorescence anisotropy. Measurements were possible at temperatures between 5-60 °C and pressures up to 300 MPa. Experiments were performed on Escherichia coli RecA and RecA from a thermophilic bacteria, Thermus thermophilus. For E. coli RecA at a given temperature, binding is a monotonically decreasing and reversible function of pressure. At atmospheric pressure, E. coli RecA binding decreases monotonically up to 42 °C, where a sharp transition to the unbound state indicates irreversible heat inactivation. T. thermophilus showed no such transition within the temperature range of our apparatus. Furthermore, we find that binding occurs for a wider range of pressure and temperature for T. thermophilus compared to E. coli RecA, suggesting a correlation between thermophilicity and barophilicity. We use a two-state model of RecA binding/unbinding to extract the associated thermodynamic parameters. For E. coli, we find that the binding/unbinding phase boundary is hyperbolic. Our results of the binding of RecA from E. coli and T. thermophilus show adaptation to pressure and temperature at the single protein level.

Simultaneous production of single cell protein and killer toxin by Wickerhamomyces anomalus HN1-2 isolated from mangrove ecosystem

The yeast Wickerhamomyces anomalus (the previous name was Pichia anomala) HN1-2 isolated from the mangrove ecosystem was found to be able to produce high level of both killer toxin and single cell protein. When the killer yeast cells were grown by batch cultivation in 5-l fermentor, crude protein in the cells, cell mass, reducing sugar, and diameter of the inhibition zone reached 56.0 g per 100 g of cell dry weight, 7.3 g per liter, 9.5 g per liter, and 19.0 mm, respectively within 12 h and this yeast synthesized a large amount of the essential amino acids, such as lysine (7.8%), methionine (1.8%), and leucine (9.0%). The crude killer toxin produced by the killer yeast isolate HN1-2 could kill the cells of Lodderomyces elongisporus, Candida albicans, Metschnikowia bicuspidata, Pichia guilliermondii, Saccharomyces cerevisiae, Yarrowia lipolytica, and Kluyveromyces aestuarii, which were widely distributed in natural marine environments. The results also showed that the undesirable yeast could be avoided during cell growth of the killer yeast.

Uncovering ubiquitin and ubiquitin-like signaling networks.

Proteomes are significantly more complex than genomes and transcriptomes due to protein processing and extensive post-translational modification (PTM) of proteins. Hundreds of different modifications exist. Release 66 of the RESID database(1) (http://www.ebi.ac.uk/RESID/) contains 559 different modifications, including small chemical modifications such as phosphorylation, acetylation, and methylation and modification by small proteins, including ubiquitin and ubiquitin-like (UBL) proteins that are covalently coupled to proteins to regulate their activity. A wide variety of cellular processes are regulated by these reversible modifications, including transcription, replication, cell-cycle progression, and responses to DNA damage. Protein modifications have been studied for many years at the level of single target proteins, but currently available technologies enable proteome-wide studies of these modifications by mass spectrometry (MS).2,3 Powerful proteomics tools are available to study phosphorylation and acetylation at a systems-wide level in a site-specific manner. It is more challenging to study ubiquitin targets and targets for ubiquitin-like proteins at a proteome-wide level in a site-specific manner due to the relatively large size of these modifications, but hundreds of potential target proteins have been uncovered over the past eight years, mainly in a non-site-specific manner. This review is focused on uncovering signaling networks for ubiquitin and ubiquitin-like proteins by mass spectrometry and highlights the site-specific studies published in 2010 and 2011. Site-specific methodologies will likely have a major impact on the ubiquitin field in the near future. The methodology, results, challenges, pitfalls, crosstalk with other PTMs, and future directions are discussed in this review. 1.1. Ubiquitin and Ubiquitin-like Proteins Ubiquitin was first discovered in the mid-1970s, and the 2004 Nobel Prize in Chemistry was awarded for this finding. Ubiquitin is a 76 amino acid protein that is highly conserved from yeast to plants and mammals. Many ubiquitin-like proteins have been uncovered, including Nedd8, small ubiquitin-like modifier 1 (SUMO-1), SUMO-2, SUMO-3, FUBI, HUB1, ISG15, FAT10, URM1, UFM1, Atg12, and Atg8. Ubiquitin-like proteins are also found in prokaryotes and archaea; PUPs are prokaryotic ubiquitin-like proteins, and SAMPs are ubiquitin-like small archaeal modifier proteins. Despite limited sequence homology of some family members with ubiquitin, all ubiquitin family members display structural homology via the characteristic β-grasp ubiquitin fold.4−9 These small proteins are covalently coupled to target proteins via isopeptide bonds between C-terminal diglycine motifs and e-amino groups in lysines of target proteins using an enzymatic cascade that consists of an E1 enzyme,(10) an activator of ubiquitin and UBLs, an E2 enzyme,11,12 and a ligase, known as an E3 enzyme(13) (Figure ​(Figure1).1). Humans express 8 E1 enzymes(10) (including 1 dedicated to ubiquitin, 1 shared between ubiquitin and the UBL FAT10, and 6 dedicated to other UBLs) and 35 active E2 enzymes (including 28 dedicated to ubiquitin, 3 shared between ubiquitin and the UBL ISG15, 3 dedicated to other UBLs, and 1 putative E2).(12) Ubiquitin E3 enzymes are subdivided into HECT-type E3 enzymes (homology to E6AP carboxyl terminus)(14) and RING-type E3 enzymes (really interesting new gene).(15) HECT-type E3 enzymes form thioesters with ubiquitin, whereas RING-type E3 enzymes lack catalytic cysteines. Over 600 human genes encode components of RING-based E3 ligases.(15) Figure 1 Ubiquitylation cascade. Ubiquitin precursors are processed by proteases to generate mature ubiquitin containing a C-terminal diglycine motif for conjugation to target proteins. Three different classes of enzymes are involved: E1, E2, and E3 enzymes. Ubiquitin ...

Thermal Performance Curves, Phenotypic Plasticity, and the Time Scales of Temperature Exposure

Thermal performance curves (TPCs) describe the effects of temperature on biological rate processes. Here, we use examples from our work on common killifish (Fundulus heteroclitus) to illustrate some important conceptual issues relating to TPCs in the context of using these curves to predict the responses of organisms to climate change. Phenotypic plasticity has the capacity to alter the shape and position of the TPCs for acute exposures, but these changes can be obscured when rate processes are measured only following chronic exposures. For example, the acute TPC for mitochondrial respiration in killifish is exponential in shape, but this shape changes with acclimation. If respiration rate is measured only at the acclimation temperature, the TPC is linear, concealing the underlying mechanistic complexity at an acute time scale. These issues are particularly problematic when attempting to use TPCs to predict the responses of organisms to temperature change in natural environments. Many TPCs are generated using laboratory exposures to constant temperatures, but temperature fluctuates in the natural environment, and the mechanisms influencing performance at acute and chronic time scales, and the responses of the performance traits at these time scales may be quite different. Unfortunately, our current understanding of the mechanisms underlying the responses of organisms to temperature change is incomplete, particularly with respect to integrating from processes occurring at the level of single proteins up to whole-organism functions across different time scales, which is a challenge for the development of strongly grounded mechanistic models of responses to global climate change.

Resolving RAD51 Filament Nucleation, Extension and Disassembly on ssDNA and dsDNA One Protein at a Time

Homologous recombination is essential for the preservation of genome stability. The core protein in this process, RAD51, drives homology search and DNA strand exchange, processes that requires the nucleation, assembly and disassembly of a RAD51 filament on single-stranded (ss) and double-stranded (ds)DNA, coupled to ATP binding and hydrolysis. Here we show that we can characterize all these RAD51 DNA transactions on long individual DNA molecules, in real-time, at the single-protein level using a combination of single-molecule fluorescence microscopy and optical tweezers. These experiments show that the sizes of RAD51 nuclei on ssDNA vary and display a broad Poissonian distribution with an average size of 4 monomers. Filament extension tracked in time with single-protein resolution reveals that nuclei extend by one RAD51 monomer at a time with a rate independent of tension on the ssDNA. This is in contrast to force-dependent monomeric extension on dsDNA. Counting and timing individual RAD51 monomers disassembling from nucleoprotein filament on ssDNA also yields contrasting results compared with dsDNA, reflecting the difference in the underlying mechanical properties ssDNA and dsDNA based nucleoprotein filaments. Together, these results yield unprecedented quantitative insight in the mechanical rearrangement during formation and collapse of RAD51 nucleoprotein filaments.

Paclitaxel effects on the proteome of HL-60 promyelocytic leukemic cells: comparison to peloruside A

Paclitaxel (Taxol®), a drug used to treat solid tumors of the breast, ovary and lung, stabilizes microtubules and arrests cells in G(2)/M of the cell cycle. Using two-dimensional differential in-gel electrophoresis (DIGE), we examined the proteomic response of a human HL-60 promyeloid leukemic cell line to paclitaxel. Our intention was to compare the effects of paclitaxel to those of a new-generation microtubule-stabilizing agent, peloruside A, investigated in an earlier study. In response to 100nM paclitaxel treatment for 24h, 21 identified proteins changed in abundance, with 13 increases and 8 decreases. In addition, 21 other unidentified proteins were also changed by treatment with paclitaxel. Using Western blotting, the transcription factor c-Myc was shown to be reduced in abundance by both drugs. Our results showed both differences and similarities at the single protein level between paclitaxel and peloruside A, although the same general classes of proteins: cytoskeletal, nucleic acid binding, stress, and apoptotic proteins, changed following exposure. The proteomic response to paclitaxel was more extensive than the response to an equipotent dose of peloruside A.

Optical detection of the interaction of N-type Ca2+ channels with G proteins at the single protein level

Optical detection of the interaction of N-type Ca2+ channels with G proteins at the single protein level

The manganese/iron-carboxylate proteins: what is what, where are they, and what can the sequences tell us?

The manganese/iron-carboxylate proteins make up a recently discovered group of proteins that contain a heterodinuclear Mn/Fe redox cofactor. The chemical potential of the heterodinuclear metal site is just starting to be characterized, but available data suggest that it may have capabilities for similarly versatile chemistry as the extensively studied diiron-carboxylate cofactor. The presently identified members of the manganese/iron-carboxylate proteins are all sequence homologues of the radical-generating R2 subunit of class I ribonucleotide reductase, canonically a diiron protein. They are also commonly misannotated as such in databases. In spite of the sequence similarity, the manganese/iron-carboxylate proteins form at least two functionally distinct groups, radical-generating ribonucleotide reductase subunits and ligand-binding Mn/Fe proteins. Here, the presently available sequences for the manganese/iron-carboxylate proteins are gathered, grouped, and analyzed. The analysis provides sequence determinants that allow group identification of new sequences on the single-protein level. Key differences between the groups are mapped on the known representative structures, providing clues to the structural prerequisites for metal specificity, cofactor formation, and difference in function. The organisms that encode manganese/iron-carboxylate proteins are briefly discussed; their environmental preference suggests that the Mn/Fe heterodinuclear cofactor is preferred by extremophiles and pathogens with a particularly high relative presence in Archaea.

Mechanical Signaling on the Single Protein Level Studied Using Steered Molecular Dynamics

Efficient communication between the cell and its external environment is of the utmost importance to the function of multicellular organisms. While signaling events can be generally characterized as information exchange by means of controlled energy conversion, research efforts have hitherto mainly been concerned with mechanisms involving chemical and electrical energy transfer. Here, we review recent computational efforts addressing the function of mechanical force in signal transduction. Specifically, we focus on the role of steered molecular dynamics (SMD) simulations in providing details at the atomic level on a group of protein domains, which play a fundamental role in signal exchange by responding properly to mechanical strain. We start by giving a brief introduction to the SMD technique and general properties of mechanically stable protein folds, followed by specific examples illustrating three general regimes of signal transfer utilizing mechanical energy: purely mechanical, mechanical to chemical, and chemical to mechanical. Whenever possible the physiological importance of the example at hand is stressed to highlight the diversity of the processes in which mechanical signaling plays a key role. We also provide an overview of future challenges and perspectives for this rapidly developing field.