Clay Clark - @biochemprof
Since I’m traveling to San Diego this week, I thought that I’d leave a reading list. These publications may or may not make it into a blog, but here’s a list of what I’m reading this week and why.
Clay Clark - @biochemprof
A new paper published in PLoS Biology characterizes two bacterial death pathways
Programmed cell death (PCD) in eukaryotes is a well-studied process that is used by organisms to maintain homeostasis. The mechanisms of PCD are under intense study because altered regulation leads to excessive cell death (inflammatory diseases) or excessive cell growth (cancer). The classic pathway in eukaryotes is apoptosis. Likewise, some bacteria also contain PCD pathways, but the reactions are less well characterized. In bacterial PCD, it is also not clear how a death program in single-celled organisms provides an evolutionary advantage. It is important to note that PCD refers to any form of cell death mediated by an intracellular program, regardless of whether the program displays all of the hallmarks of eukaryotic apoptosis.
In bacteria, PCD is mediated through modules that consist of a pair of genes, called “addiction modules” or toxin-antitoxin (TA) systems. One gene of the module encodes for a toxin, and the second gene encodes for a labile antitoxin. Under normal growth conditions (that is, non-stressed), the continual production of the antitoxin inhibits the activity of the toxin.
Rapid bioorthogonal labeling of proteins
Clay Clark
By Clay Clark, @biochemprof
There are a number of current methods for labeling proteins for imaging either in vitro and/or in live cells and organisms, including fusions with fluorescent proteins, dyes, tags (such as SNAP, HALO, CLIP), ligases, spin-labels, or unnatural amino acids. Some of the methods are summarized in the figure from Chen & Ting, which shows several approaches to labeling proteins with small molecules, either through fusion proteins (A), chemical or enzymatic labeling of a protein tag (B,C) or site-specific labeling using genetically encoded amber mutations (D).
Protein labeling strategies, from Chen & Ting, DOI 10.1016/j.copbio.2004.12.003
In recent years, several chemical probes have been developed that allow the incorporation of reactive tags into proteins. The tags can then be modified within the complex mixture of the cellular milieu, providing a powerful technique to examine protein structure and function as well as interaction networks in native conditions (see review of the tags by Best).
While some of the tags allow rapid labeling and have been used in numerous cell biological studies, many of the methods result in the addition of extra amino acids to the protein, which may affect the protein structure or function. In addition, some of the chemical probes have slow reaction times, which limits their use.
Clay Clark
By Clay Clark – @biochemprof
Small GTPases are important for regulation of a host of cellular functions, from gene transcription and other signaling cascades to cell motility, proliferation, and vesicle transport.
In their simplest form, the proteins work as binary switches, where the “on-state” depends on the type of nucleotide bound. For example, with guanosine triphosphate (GTP) bound, the protein forms a conformation that allows it to interact with other proteins (called effectors) that, in turn, convey a molecular signal. In contrast, when the terminal phosphate is hydrolyzed by the GTPase, such that guanosine diphosphate (GDP) is bound, the protein forms a conformation considered the “off-state.” In this form, the protein does not interact with other signaling proteins.
Here’s a short movie that shows a morph between the two nucleotide-bound states of the small GTPase Arf: Arf Conformational Change
The process starts a so-called “molecular clock” in which the amplitude of the signal depends on the length of time GTP is bound to the GTPase because the GTP determines whether the protein is in the “on-state.”
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When nano first met biology
Using biological molecules in nanotechnology
by Xun Lu
Lysozyme is an enzyme that helps to protect us from getting bacterial infections because it can degrade and utilize the sugars in the bacterial cell wall. A good source of lysozyme is human tears.
A single lysozyme molecule is so small that one can’t really see it with the naked eye or even under the most powerful microscope. However, scientists at UC Irvine now can use an electronic chip to record the dynamic motions of a lysozyme molecule hydrolyzing its favorite substrate in REAL TIME. It’s like using an iPod to monitor your pace during a workout except that the iPod only gives you an average rate. In contrast, this electronic chip shows the pace of a lysozyme molecule every 10 micro-seconds throughout its entire workout. Just as we hold the iPod to monitor our pace, the lysozyme molecule has to stick to the electronic chip so that its pace can be measured.
Using TIRF to examine the assembly of a viral DNA packaging motor
Graduate Student Rashid Riboul
By Rashid Riboul
Phones, music players, and cars: nearly everything these days is getting smaller. Even big screen televisions are smaller and thinner than their ancient counterparts. Now scientists are working on nano motors: machines nanometers thick that are capable doing work.
Although it is unknown how these motors are generally assembled, these powerful motors, based on viral machinery, have massive potential in the nanotechnology field. Scientists are analyzing the structure of the machinery, but no real consensus can be made on the general structure. A radical group of researchers lead by Peixuan Guo at the University of Cincinnati, however, thinks that they have ascertained the structure of the motor.
Graduate Student Christie Cade
HAT=histone acetyl transferase
One modification which generally makes the DNA more accessible is acetylation of an amino acid called lysine at various positions on the histone. Many techniques exist for studying the cellular effects of acetylation at each position. However, it is difficult to understand at a molecular level how this works. Several techniques have recently emerged to allow researchers to study homogeneous populations of modified histones: enzymatic modification of histones, native chemical ligation, and unnatural amino acid mutagenesis.