Dynamics in Chemiosmotic Systems L3-4 Flashcards
My are we using MYOGLOBIN as the case study?
Has provided us with molecular insights into the molecular motions that take place on the ultrafast timescales of catalysis
How many aa in Myoglobin?
153 aa encasing a haem prosthetic group
What is the function of myoglobin?
binds oxygen in muscle tissue
Does myoglobin bind O2 or CO with higher affinity?
CO
How is the Fe-CO bond broken?
With visible light
Bound CO can be dissociated from the haem of myoglobin by a pulse of visible light, and study of the kinetics and structural basis of this dissociation and re-association have given insights into fast timescale protein dynamics (flash-photolysis).
MYOGLOBIN
Experiments with myoglobin have provided insight into the molecular motions that take place on the ultrafast timescales of catalysis.
Myoglobin comprises a single polypeptide chain of 153 amino acids encasing a haem prosthetic group that binds oxygen in muscle tissue.
Surrounding the haem are 5 cavities, the ‘heam cavity’ and 4 cavities denoted Xe1 to Xe4 (identified through Xenon binding).
Myoglobin can also bind CO (with greater affinity than O2) and the Fe-CO bond is broken by absorbance of visible light.
A histidine ligand is present on the other side of the iron.
A notable feature is that there are no open apertures in the protein surface, so O2 or CO must access or leave the haem cavity in the protein interior via a channel that is gated by protein dynamics.
(No channels exist that connect O2/CO binding cavities to the outside). During the lifetime of the enzyme as its moving about, apertures must transiently appear in order for the O2 to get in and out.
In the 1970’s the kinetics of rebinding of flash-dissociated CO were studied by…
…. kinetic absorbance spectroscopy, taking advantage of distinctive differences in the absorbance spectra of CO-bound and CO-free haem.
Multiphasic kinetics at cryogenic temps…
Multiphasic kinetics of CO rebinding between 10µs and 10^3s at cryogenic temperatures suggested the presence of multiple protein/cofactor conformers that are frozen-in at low temperatures (range of sub-states/conformations).
The process was subsequently studied by absorbance spectroscopy, infrared spectroscopy, molecular dynamics simulations, and other techniques.
In the 1980’s X-ray crystallography revealed …
…differences in the structures of myoglobin (Mb), and myoglobin with CO bound (MbCO)
When is the harm group planar or domed in Mb?
In the Mb complex the haem is domed with the iron lying ~0.5Å out of plane of the macrocycle (haem has undergone a conformational change).
In the CO poisoned Mb, the haem is planar since the CO is binding on the other side of the haem and the iron was lying close to the plane of the macrocycle, and so the globin structure (protein surrounding the haem) differs somewhat from the Mb complex.
The transitions between the MbCO and Mb states have been discussed in the context of “protein quakes” of radiating conformational changes relieving the strain of binding CO (binding of CO to system creates strain).
Binding of CO to the haem→flattening→conf strain inside protein→series of conf changes release strain
What happens to structure of Mb after photolysis?
Photolysis was accompanied by changes in the positions of the CO, distal histidine (#64) and the haem, with smaller changes for the proximal histidine (close to binding site) and residues of the haem pocket.
Subtle changes in positions of the haem as well.
In the Mb*CO structure the CO was moved away from away from the haem Fe, and into the centre of the haem pocket.
Also the haem was partially domed, with the Fe moved out of the plane of the ring, unlike the MbCO structure but similar to the Mb structure.
Time-resolved Laue crystallography
Allows you to obtain BOTH structure and kinetics simultaneously
Used to further characterise changes in Mb MbCO Mb*CO structure.
A polychromatic X-ray beam is used (shows more structural information), enabling more detailed information to be obtained in a shorter time than standard crystallography using monochromatic X-rays (single wavelength of X-rays to interrogate your protein crystal).
Diffraction pattern is measured.
At some newer synchrotrons a single X-ray pulse of ∼100 ps duration can produce interpretable Laue data.
Exposure of the crystal structure to the X-ray pulse is coordinated with an excitation light pulse to initiate photo-dissociation of MbCO.
Can vary the time delay between the light pulse and the X-ray pulse. (Pump-Probe Crystallography).
Are there 2 different fates for CO within a Mb molecule?
YES - 2 different fates for CO exist (controlled by the position of phenylalanine which models the heterogeneous kinetics) when you flash it off – can move either left or right.
Rxn Kinetics
To measure the rate of a reaction you need to be able to initiate it on a timescale that is short compared to that of the reaction.
If a reaction is initiated by light, and its progress can be monitored using light, then it is possible to access timescales down to attoseconds (10-18 seconds) with a suitably sophisticated and expensive laser system.
For other reactions you may be limited by the rate at which you can initiate the reaction by mixing the components together or the rate at which you can detect the product.
For very slow reactions you may be able to (e.g.) inject a substrate into a cuvette containing an enzyme and monitor conversion to the product.
For faster reactions specialist apparatus for rapid mixing of components may be needed-a widely used technology is called stopped-flow spectrophotometry. (Mechanical means of mixing things together very rapidly to initiate reaction and follow the formation of the products.)
Hydrogen-Deuterium exchange allows you to detect….
…global or local unfolding on timescales of milliseconds or longer being analysed by either mass spec or NMR
