School of Biochemistry 
and Molecular Biology

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CONTENTS: (This site is still under construction)

Text books


There is more information in some of these sources than you require for a first year course. Use this website or the handout sheets to identify topics that you really need to know.

Lecture 1: Bacterial movement, structure of flagella, flagellin, flagellar motor, chemotaxis.

Many common bacteria (including both gram positive and gram negative species) move with the aid of flagellae. These hollow corkscrew tubes act as simple propellers and are rotated at high speed (up to 100,000 rpm) by powerful molecular motors within the bacterial membrane. In most species the energy to drive these motors comes directly from the transmembrane proton gradient, although some bacteria use a sodium ion gradient instead. Flagellae are rigid structures, but have flexible universal joints (called "hooks") at their base where they insert into the bacterial membrane. Each flagellum contains multiple copies of a single protein called flagellin, but about 50 other genes are necessary for a fully functional motile system.

Gram negative bacteria (such as E. coli and S. typhimurium) have three distinct surface layers: phospholipid inner and outer membranes, between which lies the rigid peptidoglycan cell wall. The space between the inner and the outer membranes is called the periplasmic space. The torque generating assemblies consisting of the S, M and C rings protrude from the inner membrane into the cytoplasm, and are also anchored by ten studs to the peptidoglycan layer. Between the hook and the motor there is a rigid drive shaft which rotates within molecular bearings located in the bacterial membranes: the L ring in the outer membrane and the P ring in the peptidoglycan layer. The walls of gram positive bacteria lack the two outer layers, and in these species the L and P rings are also absent.

In addition to its impressive rotation speed, each flagellar assembly is hollow. It is synthesised from the inside outwards, and most of the new protein subunits are transported down the hollow core and then fitted into place at the growing tip. The protein component supply changes during flagellar synthesis as various parts of the structure are completed. It is a bit like building a tall chimney by hauling pre-fabricated components up the flue.

A typical E. coli or S. typhimurium cell has about 6 flagellae, which are about 10nm long. In these species the maximum rotation speed is about 18,000 rpm. Most of the time they all rotate in a counter-clockwise direction. This allows the individual rotating corkscrews to mesh comfortably together, forming a tight bundle which is an effective propeller. About once per second one or more of them changes direction and rotates briefly in a clockwise sense. This does not mesh with its neighbours and causes the bundle to fly apart, so that the bacterium tumbles randomly, and adopts a new direction.

This behaviour forms part of a strategy for finding food and avoiding noxious compounds. Bacteria are too small to detect any concentration gradients from one end of the cell to the other. Their only method for deciding which way to swim, is to move a short distance at random, and then check to see whether things have improved or not. If the situation is getting better then they keep going a little longer in the current direction, but if things are worse then they tumble and try a new tack.

[Eukaryotic cells are much larger and are able to detect tiny concentration gradients from one side of the cell to another. They consequently know which way to swim, but this requires a different internal signalling system which is covered in subsequent lectures.]

Eukaryotes can measure spatial concentration gradients, but bacteria measure concentration versus time, and then equate time with distance moved. They can remember the previous situation for about 3 seconds. We know this from rapid mixing experiments, where added attractants briefly suppress tumbling behaviour, even though there is no gradient to follow. There is no point in bacteria trying to swim in a "straight" line for more than a few seconds, because random Brownian motion will eventually knock them off course, whether they tumble or not.

Bacteria can follow more than one gradient at the same time, so they must have more than one type of detetector. They can also respond to concentrations of attractants spanning five orders of magnitude. This requires some method to adjust the sensitivity of the detector systems so that they do not become swamped at very high ligand concentrations.

Many components of the bacterial chemotaxis system were identified from mutants lacking various features of the wild type system. Some of these are listed on the next page:




effect of gene deletion

MCP I (tsr)

inner membrane

binds serine (attractant), or leucine (repellant) directly

no response to either serine or leucine, remainder OK

MCP II (tar)

inner membrane

binds aspartate, the maltose receptor protein (attractants) and Ni, Co (repellants)

no response to aspartate, maltose, Ni or Co, but all other systems operate OK

MCP III (trg)

inner membrane

binds the galactose and ribose receptor proteins

no response to galactose or ribose, all other systems OK

MCP IV (tap)

inner membrane

binds the dipeptide receptor protein

no response to dipeptides, but all other systems are OK

maltose receptor


binds maltose to MCP II

no response to maltose, but all other systems are OK

galactose receptor


binds galactose to MCP III

no response to galactose, but all other systems are OK

ribose receptor


binds ribose to MCP III

no response to ribose, but all other systems are OK

dipeptide receptor


binds dipeptides to MCP IV

no response to dipeptides, but all other systems are OK

Che A


phosphorylates itself if MCP empty, then Che Y & Che B

counterclockwise bias: swim smoothly, cannot tumble

Che B


MCP methyl esterase when phosphorylated

insensitive, no adaptation, no memory

Che R


MCP methylator (uses SAM)

hypersensitive, no adaptation

Che W


connects MCPs to Che A

counterclockwise bias

Che Y


motor turns clockwise if this protein is phosphorylated

counterclockwise bias

Che Z


Che Y phosphatase

incessant tumbling

Mot A

inner membrane

proton channel (4 crossings) part of stator creates torque

flagellae assembled but do not turn

Mot B

inner membrane

proton channel (1 crossing) part of stator creates torque

flagellae assembled but do not turn


M & S rings

major rotor component

incomplete flagellae


"M" ring

part of rotor: creates torque? causes direction switching?

incomplete flagellae


"C" ring

part of rotor?

incomplete flagellae


"C" ring

part of rotor?

incomplete flagellae

FlgB, FlgC and FlgG

minor rod components

parts of the hollow drive shaft (6 copies each per rod)

incomplete flagellae


major rod component

part of the hollow drive shaft (about 26 copies per rod)

incomplete flagellae


P ring

peptidoglycan bearing

incomplete flagellae


L ring

outer membrane bearing

incomplete flagellae



universal joint

incomplete flagellae


hook tip

scaffolding: controls hook assembly, then discarded

incomplete flagellae

FlgK [HAP1]

hook tip

caps hook, joins to flagellin

incomplete flagellae

FlgL [HAP3]

hook tip

caps hook, joins to flagellin

incomplete flagellae


flagella spiral

major structural component

incomplete flagellae


caps flagella

prevents loss of subunits

new subunits drop off

The central components in the bacterial chemotaxis system are the four varieties of methyl-accepting chemotaxis proteins or MCPs. The conformation of these transmembrane proteins changes from a clockwise-signalling [CWS] to a counter-clockwise-signalling [CCWS] state after attractant binding, either directly, or via one of the periplasmic receptor proteins. It should be noted that the effector ligand does not enter the bacterial cell cytoplasm in order to exert its effects.

The CWS state results in the activation of the Che A protein [via the Che W adaptor protein]. Che A is an autokinase which initially phosphorylates itself using ATP, but then proceeds to phosphorylate two other regulator proteins: Che Y and Che B.

Phosphorylated Che Y interacts with the motor control system at the base of the flagellum, and increases the probability of clockwise rotation and tumbling. A further protein, Che Z, promotes the de-phosphorylation of Che Y, thereby terminating this part of the response.

If the above mechanism were the whole of the chemotaxis system it would merely inhibit tumbling at high attractant concentrations. It has no memory and would not be sufficient for the bacteria to follow a gradient. The memory is supplied by the second regulator, Che B.

An enzyme Che R continuously methylates the MCPs, using S-adenosyl methionine as the methyl donor. It methylates multiple glutamate residues in both the CWS and CCWS states and progressively reduces the attractant binding affinity of the MCPs. Phosphorylated Che B is a methylesterase that removes these methyl groups from the MCPs.

The first effect of this arrangement is that the methylation level of the MCPs continuously tracks the effector concentration. At low attractant concentrations, most of the MCPs are in the CWS state, Che A is activated, and Che B removes almost all of the methyl groups. In this state the MCPs have high affinities for their ligands and detect very low concentrations of these effectors. If the attractant concentration increases, the MCPs tend to adopt the CCWS conformation, Che A and Che B thereby lose their activities, and methyl group removal becomes progressively less efficient. With increasing attractant concentrations, the MCPs are gradually converted into a fully methylated, low affinity state. Although the percentage of the MCPs in the CCWS state increases in proportion to the average ligand concentration, it does not alter nearly as much as might initially be expected if the negative feedback tracking system were not in operation.

The second effect is that the system has memory. It takes the tracking system a few seconds to catch up with any alteration in the ligand concentrations, and during that period the MCPs generate very large output signals via Che A. Instead of signalling absolute concentrations of the attractants, the detector systems have first derivative outputs, and signal changes in the attractant concentrations. This vital information enables bacteria to follow the concentration gradients, and is important for cell growth and survival.

In addition, the tracking system greatly extends the dynamic range of the MCPs as ligand detectors. The changing receptor affinity allows these proteins to respond to small fractional changes in attractant concentration, over a very wide range of absolute ligand concentrations.

The main features of the bacterial chemotaxis system are summarised in the following diagram:

How do eukaryotic cells move along a concentration gradient?

Lecture 2: Eukaryotic cilia, microtubules, dynein, organelle movement along microtubules.

Ancient origins: the principal elements of the cytoskeleton are present in both plant and animal cells, although there are no centrioles or cilia in higher plants.

There are three types of cytoskeleton: the thinest microfilaments about 7nm diameter based around actin and ATP, the thickest microtubules about 24nm diameter based around tubulin and GTP, and a diverse group of intermediate filaments about 10nm in diameter containing a wide range of protein components with a multiplicity of structural roles.

microfilaments microtubules

Actin (40kD monomer) and tubulin (50kD monomer) form very different structures, which nevertheless possess some common features:







dynamic instability:






bound nucleotides:











molecular motors: in addition to their "load bearing" structural functions, microfilaments and microtubules are associated with force generation and active movement, powered by at least three major classes of ATP-driven molecular motor - myosins, kinesins & dyneins. In contrast to this, the intermediate filaments appear to have only structural roles.




Function / location

myosin I



vesicle transport, Golgi

myosin II

large aggregates


voluntary muscles, cytokinesis

myosin V



vesicle transport, Golgi, major roles in melanocytes & neurones

myosin VI


cochlea hair cells [Snell's waltzer]

myosin VII


cochlea hair cells, retinal pigment cells and photoreceptors Usher syndrome

myosin IX


myeloid cells

myosin X



myosin XV


cochlea hair cells [shaker-2 mutation]


cytosolic kinesins



vesicle transport

KRP (+)



nuclear spindle, cell division

KRP (-)



nuclear spindle, cell division


cytosolic dynein

two heads


vesicles, kinetochore

outer arm dynein

three heads


cilia & flagellae

inner arm dynein

two heads


cilia & flagellae


Axonal transport:


Fast forward


Fast backwards






Very slow


Flagellae and cilia: These have fundamentally the same structure, but flagellae are longer.


Force generation in cilia:












Basal bodies:

















Cell division:


Lecture 3: Muscle and the use of ATP, myosin and actin. Calcium regulation of muscle contraction: sliding filament model. Smooth muscle myosin. Actin & myosin in non-muscle cells.

The diagram above shows the control of cardiac muscle contraction. The Na/K ATPase or sodium pump (1) works continuously, using the energy from ATP to maintain a high K concentration inside the cells and a high Na concentration in the extracellular fluid (ECF). The cell membrane (sarcolemma) is usually more permeable to potassium ions than to sodium ions, and this gives rise to a membrane potential of about 80mV (inside negative) in relaxed muscle. Calcium ions are also removed from the cytosol into the ECF by an ATP-driven calcium pump (2) in all tissues. Cardiac muscle possesses an additional sodium/calcium exchange protein (3). This export system is driven by the pre-existing sodium ion gradient. The calcium concentration inside resting cells is low (0.01mM) , but rises sharply to 1 mM during contractions. The sarcolemma is very thin (about 6 nm) so the 80mV membrane potential equates to a voltage gradient of about 13,000,000 volts per metre! All membrane components are subject to intense electric fields, and protein conformations are greatly influenced by the membrane potential. "Voltage gated" ion channels will only conduct over a narrow range of membrane potentials, whereas "ligand gated" ion channels (such as the acetylcholine receptor in voluntary muscle) require specific chemical activators.

Muscle contraction is triggered by a wave of membrane depolarisation which spreads from neighbouring cardiac cells. [In skeletal muscle the cells work independently and are triggered by their motor neurone via the motor end plate.] The change in electric field activates voltage gated sodium channels (4) in the sarcolemma, each of which allows a few hundred positively charged sodium ions to enter the negatively charged cytosol, further reducing the membrane potential until the whole sarcolemma is depolarised. These sodium gates close spontaneously after a few milliseconds, but slower voltage-gated calcium ion channels, thought to be identical with the dihydropyridine receptors (5) take over and maintain a positive inward current for several hundred milliseconds (in human ventricle) during the plateau phase of the cardiac action potential. [The plateau phase is absent in skeletal muscle.] The membrane potential is eventually restored to its resting value by a delayed loss of positive potassium ions (16) from the cells.

About 10% of the calcium needed to activate cardiac contraction enters during each beat from the ECF. The remainder is released from the sarcoplasmic reticulum through a channel known as the ryanodine receptor (6). Separate genes coding for this enormous protein (over 5000 amino acids) are active in heart and skeletal muscles. Operation of the ryanodine receptor depends in a mysterious way on the flow of calcium ions through the dihydropyridine receptors in cardiac muscle, but not in skeletal muscle.

Skeletal muscle can contract in the absence of extracellular calcium, and skeletal S.R, shows depolarisation-induced calcium release In contrast to this, cardiac S.R needs external "trigger calcium" to enter the cells via the dihydropyridine receptors during the plateau phase of each action potential to initiate calcium-induced calcium release (CICR). Cardiac and skeletal ryanodine receptors probably differ in their precise intracellular location. The two genes are on separate chromosomes and show substantial differences. They are also expressed in brain, egg cells and many other tissues, where they regulate calcium release from the smooth endoplasmic reticulum. Dihydropyridine receptors are also present in some smooth muscles. They are blocked by the important drugs verapamil and nifedipine, which reduce the force of cardiac contraction, while maintaining an adequate cardiac output by relaxing vascular smooth muscle and reducing the peripheral vascular resistance.

In striated muscles, cytosolic calcium ions bind to the regulatory protein troponin-C located in the thin filaments (7), leading to a change in filament shape. This allows flexible head groups from the protein myosin in the thick filaments (8) to interact with the protein actin in the thin filaments. A change in myosin conformation causes the thick and thin filaments to slide against each other and hydrolyse ATP, which provides the energy for contraction. Movement and ATP hydrolysis continue until the calcium ions are removed from the cytosol at the end of each contraction. Most of the calcium ions are returned to the sarcoplasmic reticulum by a calcium pump (9) but about 10% leave cardiac muscle cell via proteins (2) and (3) described above. Skeletal muscle almost completely lacks the plasmalemma calcium import and export routes. Calcium ions are stored within the sarcoplasmic reticulum loosely bound to calsequestrin (10), a high-capacity, low-affinity calcium buffer protein.

Troponin is absent in smooth muscle, so the high-speed thin filament regulatory system found in striated muscle is not operative in smooth muscle. However, a low-speed alternative system is present, based on the actin-binding protein caldesmon. When caldesmon is bound to actin it blocks the actin - myosin interaction and contraction is inhibited. Caldesmon can be removed from actin either by phosphorylation using protein kinase C or by binding to calmodulin in the presence of calcium ions. In addition, the thick filaments play a much larger role in regulating smooth muscle contraction: direct calcium binding to myosin light chains is important, and this is enhanced when the myosin light chains are phosphorylated by myosin light chain kinase in the presence of calcium and calmodulin. Protein kinase C inhibits smooth muscle contraction by phosphorylating an alternative myosin light chain site, and so can produce either contraction or relaxation depending on the precise pattern of smooth muscle gene expression. The role of protein kinase A in relaxing smooth muscle was described on page 2. Myosin light chain kinase is also found in striated muscles, but appears to play a subsidiary role in these cells.

Some actin binding proteins


Organisation & functions


Thymosin 5Kd

binds G actin, prevents polymerisation, helps to buffer the G actin pool.


Profilin 15Kd

binds G actin, promotes ADP / ATP exchange, replenishes G actin pool containing bound ATP


CapZ 30Kd

permanently caps the + ends of F actin at the Z line in striated muscle cells

Z lines, and membrane anchors

Tropomodulin 43Kd

permanently caps the free - ends of F actin towards the middle of each muscle sarcomere

muscle and many other cells (eg erythrocytes)

Tropomyosin 2x35Kd

binds together and coordinates groups of seven actin subunits in muscle thin filaments

all muscles and other cell types.

Troponins 18-36Kd

Know the difference between Tn-I; Tn-T & Tn-C

striated muscles only

Caldesmon 150 Kd

thin filament contractile regulation by calcium ions in smooth muscles and during cytokinesis

smooth muscles, and all dividing cells

Gelsolin 87Kd

severs F-actin in the presence of calcium ions, and remains stuck to the new + end. Inhibited by PIP2

widely distributed

Villin 92Kd


intestinal and kidney microvilli

Severin 40Kd


CapG 40Kd

caps but does not sever actin filaments


30Kd protein

filopodia, lamellipodia, stress fibres


EF-1a 50Kd



Fascin 55Kd

filopodia, lamellipodia, stress fibres,microvilli, acrosomal process


Scruin 102Kd

acrosomal process (in echinoderms!)

Sea urchin sperm

Dematin 48Kd



Fimbrin 68Kd

microvilli, stereocilia, adhesion plaques


a-actinin 102Kd

filopodia, lamellipodia, stress fibres, adhesion plaques, muscle Z disks

all motile cells

Spectrin 550Kd a+b

cortical networks


Dystrophin 427Kd

Anchors actin filaments to the sarcolemma trans-membrane proteins. Prevents muscle damage.

cortical networks in striated muscle cells

Utrophin 400Kd

Similar to dystrophin, but concentrated near the neuromuscular junction.

also present in non-muscle cells

ABP120 92Kd



Filamin 280Kd

filopodia, pseudopodia, stress fibres