... understanding life in molecular detail

Prof John Trinick

Muscle, motility, titin, microscopy


We are sad to announce that Professor John Trinick passed away in January 2019.

We are interested in how living systems generate movement, including muscle sarcomere structure, intra-cellular transport by non-muscle myosins, and passive muscle force due to titin. We use a wide range of biochemical and biophysical methods, but specialise in electron microscopy, including cryo- and time-resolved EM.

The electron micrograph above shows titin molecules combed out by the force of a receding meniscus (left to right) during drying. The ends of titin tend to stick to the substrate leaving the remainder free to be pulled by the meniscus force of a few hundred pN. The upper molecule is stretched by ~150%, which demonstrates elasticity, one of titin's roles in muscle. Two of the molecules in the trimer below attached by both ends. All three join through their head (C-terminal) ends. Titin is the largest polypeptide yet found in nature (up to 3.7 MDa, >30,000aa) and the third most abundant protein of muscle, after myosin and actin. It gives sarcomeres passive elasticity and controls their assembly. Mutations in titin are the commonest cause of dilated cardiomyopathy (DCM), which affects the heart in ~1 in 250 people and is a major cause of death. Picture by L Tskhovrebova.

Current major projects include:
  • Roles of the giant protein titin in muscle assembly and elasticity
  • Electron microscopy of mosin superfamily shape and flexibility
  • Studies of isolated Z-discs
  • EM of large protein complexes, including cryo- and time-resolved

We mainly study the structure and functioning of the contractile apparatus of muscle, as determined by the properties of its constituent proteins. This includes active and passive force generation by acto-myosin and titin and the role of titin in controlling muscle assembly. It has also led to study of contractile proteins outside muscle, such as myosin V which operates as a single molecule cargo carrier in neurones. We are also studying the structures of isolated Z-discs. We use a wide variety of biochemical and biophysical methods but specialise in electron microscopy, including cryo- and time-resolved EM.

Research Areas

Titin

Titin is the largest polypeptide yet found in nature (3.7 MDa) and the third most abundant protein of muscle. It consists mainly of ~300 domains similar to I-set immunoglobulins and type III fibronectin. Single molecules span half the sarcomere, the repeating unit of the muscle contractile apparatus.

In the thick filament region of the sarcomere titin binds to myosin. Here we have proposed it acts as a 'protein-ruler' regulating exact assembly of the 294 myosin molecules in the polymer. We are exploring this hypothesis by studying the interactions of expressed titin domains with myosin and the other thick filament proteins.

The remainder of titin forms elastic connections between the ends of the thick filament and the Z-disc. These connections are the main source of the passive elasticity of relaxed muscle. They also centre thick filaments between Z-lines; without them there would be myosin force imbalances in opposing halves of thick filaments during active contraction.

We were the first to propose that the elastic mechanism of titin involves unfolding of its polypeptide. We subsequently demonstrated mechanical unfolding in single titin molecules, both in the immunoglobulin and fibronectin domains and in a short region of unique sequence high in P, E, V and K residues, the PEVK region. Methods used were optical tweezers (collaboration with King's College, London), atomic force microscopy and electron microscopy after 'molecular combing' by a receding meniscus (see picture above). Single molecule studies allow examination of protein mechanics and folding in physiological environments (i.e. non-denaturing). Our 1997 paper in Nature using optical tweezers is cited >500x.

Myosin

The molecular origin of muscle force is a gross shape change in the heads of myosin while attached to actin. Using negative staining, we obtained the most detailed electron micrographs of myosin II from muscle. which is the most studied member of the myosin superfamily. Images showed the motor and regulatory domains in the heads and points of flexibility in the coiled-coil alpha-helical tail. Image averaging by single particle processing was used to greatly improve detail and to make 3D head models.

Nearly 50 non-muscle myosin classes have now been identified in the super-family and we have applied negative stain electron microscopy several of  these, including myosins I, V, VI and XVIII. Myosin V is widely distributed and abundant in neurones. We obtained the first pictures of myosin V making linear 'processive' walking strides along actin filaments. These were the first pictures of the myosin head at the beginning of its power stroke, which is difficult to observe in muscle myosin II. We recently also showed that myosin V heads can execute their powerstroke without cargo movement by severely distorting (PNAS, 2010). Myosin VI moves backwards along actin and is important in hearing and cancer invasiveness.

Cryo and time-resolved EM

Most proteins do not function singly but as parts of larger complexes, which are hard to crystallise for X-ray diffraction studies. We use cryo-electron microscopy of unstained frozen-hydrated specimens to study complexes >~0.5 MDa. The ultra-rapid freezing (~106degC/sec) required to retain the vitreous state of water allows transient states in mechanisms to be trapped for EM (collaboration with H White, E. Virginia Med. School). One way to do this is to layer one reactant onto the grid and spray on another just before freezing. This methodology is widely applicable, since a wide range of reactants can be delivered as aerosols, from small molecules to macromolecules and even macromolecule assemblies; time resolution is ~5 msec. Information recovery from micrographs is maximised by single particle image processing, which improves resolution and gives 3D models.

Detailed research programme                  Close ▲

Selected Publications

  1. Oke OA; Burgess SA; Forgacs E; Knight PJ; Sakamoto T; Sellers JR; White H; Trinick J Influence of lever structure on myosin 5a walking P NATL ACAD SCI USA 107 2509-2514, 2010

  2. Walker ML; Burgess S; Sellers JR; Wang F; Hammer JA; Trinick J; Knight PJ Two-headed binding of a processive myosin to F-actin Nature 405 804-807, 2000

  3. Tskhovrebova LA; Trinick J; Sleep JA; Simmons RM Elasticity and unfolding of single molecules of the giant muscle protein titin. Nature 387 308-312, 1997

  4. Labeit S; Barlow Dp; Gautel M; Gibson T; Holt J; Hsieh Cl; Francke U; Leonard K; Wardale J; Whiting A; Trinick J A Regular Pattern Of 2 Types Of 100-Residue Motif In The Sequence Of Titin Nature 345 273-276, 1990