SUBJECT

Title

Motor Proteins

Type of instruction

lecture

Level

master

Faculty

Faculty of Science

Part of degree program

Biology MSc

Credits

Recommended in

Semesters 1-4

Typically offered in

Autumn/Spring semester

Course description

1. Motor proteins - definition. Chemomechanical energy transduction. Cell motility, cytoskeleton. Classical linear motors: myosin, kinesin, dynein. Rotors: ATP synthase, bacterial flagella. Other motos: polymerisation motors, processive enzymes (RNA and DNA polymerase, helicase), macromolecule translocators, dynamin.

2. History of motor proteins. Theories of muscle contraction: Machina carnis. Significance of Prof. Szent-Györgyi's school. Discovery of cytoplasmic myosins, kinesins and dyneins.

3. Investigation of motor proteins. Interdisciplinary methodical arsenal. Muscle fibres: mechanics, small-angle X-ray diffraction, fluorescence etc. In situ localization. 3D image reconstruction techniques. Isolated proteins: protein chemistry, spectroscopic methods, molecular genetics (recombinant proteins). Transgenic organisms. In vitro motility assays. Single molecule methods: optical trapping and other nanotechniques.

4. Structural basis of conventional myosin function. Structure and filaments of the sarcomere. Swinging cross-bridge and swinging lever arm models. Atomic structures and models of myosin and actomyosin. Conformational transitions in myosin during the ATPase cycle. The role of actin in force generation - communication between the actin and ATP binding sites of the myosin head.

5. Kinetic mechanism of conventional myosin. Chemomechanical cycle of muscle myosin 2. The Lymn-Taylor model. Kinetic cycle of the myosin ATPase - the Bagshaw-Trentham model. Relation of the powerstroke to ATP hydrolysis. Fluorescent sensors in the myosin head.

6. Diversity of myosin mechanisms. Functions, working environments and family tree of myosins. Myosin pools in different genomes. Role of the duty ratio, actin-attachment lifetime and processivity in the working mechanism of different myosins. Functional adaptations of the enzymatic and mechanical parameters of myosins. Mechanism and load-sensitivity of non-muscle myosin 2. Processive stepping mechanism of myosin 5. Reverse directionality of myosin 6.

7. The myosin superfamily. Conventional and unconventional myosins. Single-headed myosins (myosin 1). Myosins from 3 to 18. Modular architecture: motor domain, IQ motif, coiled-coil structure, signal transduction and other motifs. Role of myosins in cell motility: cytokinesis, cytoplasmic streaming, contraction of microvilli, exo- and endocytosis etc.

8. Mechanism of action of kinesins. Diversity and cellular functions of kinesins. Structure, enzymatic and processive stepping mechanism of conventional kinesin. Potential mechanisms of single-head processivity.

9. Mechanism of action of dyneins. Classes and functions of dyneins. Enzymatic mechanism and mechanical properties of dyneins.

10. In vitro and in vivo motility assays in batch and on single molecules. Total internal reflection fluorescence (TIRF) microscopy. FIONA (fluorescence imaging with one nanometer accuracy).

11. Molecular mechanics. Laser trapping arrangements. Measurement of the step size of motor proteins. Measurement of the load-dependence of motor kinetics. Measurement of force effects in solution conditions.

12. Types of actomyosin regulation. Actin-linked regulation (vertebrate skeletal muscle): troponin-tropomyosin. The steric blocking model. Myosin-linked regulation: light and heavy chain phosphorylation (vertebrate smooth and non-muscle myosin 2, invertebrate myosins); direct Ca-binding (molluscan and Physarum myosins).

13. The actin polymerisation motor. Structure of G- and F-actin. Microfilament dynamics: actin treadmilling. The diverse family of actin-binding proteins (Arp2/3 complex, cofilin, profilin, capping proteins etc.) Cortical actin networks. Organelles of cell motility: lamellopodia, filopodia, memrane ruffles. Intracellular rocket motility: Shigella, Listeria, Legionella. Mechanisms of the actin polymerisation motor.

14. Motor proteins and disease. Diseases linked to conventional myosins: FHC, asthma etc. Unconventional myosins: Griscelli- and Usher-syndrome, etc. Diseases linked to axon transport: the linkage between CMT, SPG, Alzheimer, Huntington, ALC and other neurodegenerative disorders and motor protein function (cause-effect?). Disorders related to cilia: policystic renal failure, situs inversus etc. Motors as therapeutic targets. Motors in the targeting of drug molecules.

Readings

C. R. Bagshaw: Muscle contraction. 2nd edition. Chapman and Hall, 1993.
J. Howard: Mechanics of motor proteins and the cytoskeleton. Sinauer Associates, 2001.