Crystallogaphy -- lecture 25
Protein guided tours: the meaning of “Life”
All life is based on reduced carbon
anoxic
4*109 y
oxidizing
3*109
2*109
1*109
present
origin of life
Chloroplasts
origin of
oxygenic
phototrophs
oxygenated
environment
Thanks to Doug Whittet, RPI Physics
O2 in the atmosphere provides a strong natural
selection for CO2 fixation
A narrowly avoided
catastrophe for life on earth:
•Cyanobacteria evolved a
means to use light for
energy, with O2 as the byproduct. PHOTOSYSTEM I
•O2 oxidizes everything in
sight.
•No reduced carbon left.
•Everything would have
died if not for
Rubisco/Nitrogenase!!
chloroplasts are descendents of the
early cyanobacteria.
Photosystem I: 1JB0
Classes of membrane proteins
•Single transmembrane helix
•several transmembrane helices
•beta-barrel or channel
•Anchored by one (not-transmembrane) helix or a covalently
attached fatty acid
Photosystem I: Guided tour
Download and display 1JB0.pdb (one jay bee zero)
restrict !protein and !hoh
color cpk
Display -> ball and stick
select magnesium
label %r
set fontsize 12
set fontstroke 2
color labels yellow
Find the pseudo 2-fold axis
How many Mg are there?
What are the residue numbers of the
“special pair” of chlorophylls?
Photosystem I : Guided tour
(select the special pair using select XXX or YYY)
spacefill
select hetero and !hoh
How are the B-factors
labels off
distributed?
color temperature
Which side is more
ordered? Chain A or
chain B?
Was NCS 2-fold symmetry
enforced during refinement?
Guess what:
2-fold symmetry was not
enforced during evolution!
Photosystem I : Guided tour
Find the name of the lipid that does not have
a phosphate group.
Unix shortcut: use grep
grep ^”HETNAM” 1JB0.pdb
Characterize the environment
of the lipid. Could it have a role in
the light harvest process?
select [LMG]
restrict selected
center selected
select within (11., [LMG]) and protein
Display -> ball_and_stick
color cpk
select within (11., [LMG]) and ligand
Photosystem I : Guided tour
select within (11., [LMG]) and ligand
spacefill 1.5
color green
select within (11., [LMG]) and *.MG
spacefill 1.5
color white
select within (11., [LMG]) and [PQN]
color red
select within (11., [LMG]) and solvent
spacefill 1.0
What is PQN?
color cyan
How close is it to the nearest
magnesium
Photosystem I : Guided tour
restrict ligand
wireframe
color cpk
Display-> ball and stick
select [CL1] or [CL2]
wireframe
color green
select [PQN]
color magenta
spacefill 1.0
select [BCR]
color orange
spacefill 1.5
select *.MG
spacefill 1.0
color white
Light harvesting complex
Trace the path of the electrons
from the special pair to the two
quinones.
Are are of the pigments
connected to the special pair?
Photosystem I : Guided tour
restrict [PQN]
Environment of the quinones
spacefill
color cpk
Which quinone is more
select within (11.,[PQN]) and protein
wireframe 0.5
loosely-bound
color cpk
select within (11.,[PQN]) and ligand and not [PQN]
color green
wireframe 0.5
select within (11.,[PQN]) and solvent
spacefill 0.6
How does the electron get
color cyan
from one quinone to the
other? What protein sidechain
forms a bridge?
Photosystem I : Guided tour
Draw a TOPS diagram of chain D
residues 24-95 only
Look in scop.berkeley.edu for the
fold under alpha+beta
What fold is this?
Rubisco
8RUC
Rubisco
Rubisco
RUBISCO = Ribulose Bisphophate Carboxylase/Oxygenase
Animals
---> sugars, etc.
Rubisco fixes CO2
5+1=3+3
One of the substrates is small and feature-less. Gets confused with O2.
Competing oxygenase activity
normal
reaction
Unwanted
competing
reaction
RUBISCO-FAQ
•Rubisco is notoriously inefficient. Why?
Oxygenase activity is an unwanted side-effect, but unavoidable since O2 is
of similar shape and is a better electron sink than CO2. Also, CO2 has a
lower partial pressure in the atmosphere.
•Carbon fixation evolved exactly once. Right?
Right. Otherwise we would see multiple proteins having this function.
Rubisco must have been an enormous advantage over its contemporary
competition.
•How does rubisco overcome the inefficiency problem?
Rubisco forms large complexes in order to have a larger concentration in
the cell.
•Why does rubisco require chaperonins to fold?
The ultra-high concentrations of rubisco in the chloroplasts mean that misfolded
rubisco quickly aggregates. Chaparones prevent aggregation.
Guided tour: 8RUC xtal symmetry
Download 8RUC.pdb from www.rcsb.org
rasmol 8RUC.pdb
color --> chain
display--> cartoons
set unitcell on
8RUC space group is C2221
What crystal class is this?
Guided tour: edit the pdb file
edit 8RUC.pdb (vi or jot)
Note resolution of the data. Number of reflections.
Number of atoms. Number of solvent atoms.
Find “HETNAM” records.
These are the “hetero” groups.
Find the CAP and KCX “ATOM” records.
Check the B-factors. How well ordered are they?
Guided tour: the A chain
Rasmol commands:
restrict :A
center :A
display-->cartoons
stereo -7
Adjust the window to eye separation. Relax eyes to see
stereo.
Trace the chain from N to C.
How many domains are there?
What “fold” is each domain? Find it in SCOP and/or CATH.
Guided tour: the active site
Rasmol commands:
restrict within (10., 201:A)
center within (10., 201:A)
wireframe 50
color-->CPK
select 201:A | CAP:A
wireframe 80
select hetero & within (10., 201:A) & MG
spacefill 1.0
select hetero & within (10., 201:A) & HOH
spacefill 0.5
options-->slab mode
Guided tour: the active site (cont’d)
Which residues are coordinating the magnesium?
Where is the CO2 that was added to the ribulose?
Rasmol commands:
select :A & not within (10., 201:A)
options-->slabmode (off)
display-->backbone
color-->group (this colors from N to C)
Where is the active site relative to the domain?
select not :A & protein
display-->backbone
color --> chain
Where is the active site relative to the oligomeric contacts?
Guided tour: protein-protein
contacts
select not :A & within (10., :A)
display-->sticks
color red
What chains are in contact with the A chain?
What residues are involved?
What command would select only waters that are
within 5A of both chain A and chain L?
Nitrogenase
anoxic
4*109 y
1N2C
oxidizing
3*109
2*109
1*109
present
Like CH2, NH3 was plentiful before oxygenic phototrophs.
Since then, most N in the atmosphere is in the form of N2.
Nitrogenase converts N2 to NH3
Nitrogenase is a hetero-tetramer. 2 Fe-proteins, 2
MoFe-proteins. With 3 Iron-containing clusters.
Feprotein
MoFeprotein
The Fe-protein cluster passes e- to the P-cluster, which passes them to
the FeMo-cluster. e- accumulate at the FeMo-cluster, where the
reduction of N2 takes place.
There are two Fe-containing clusters in the MoFe-protein
FeMo
cluster
Reduced
P-cluster
Oxidized
P-cluster
dithionite
A protein conformational change might
favor oxidized over reduced, driving the
reaction forward.
from Rees & Howard (2000) Current Opinion
in Chemical Biology, 4(5):p559-566.
Mechanism: simplified
2ATP
ATP
2ADP
slow
dissociation
of complex is
rate-limiting
ATP
ADP
Fe-protein (red)
ADP
Fe-protein (ox)
+
MoFe-protein (red)
fast
+2Pi
MoFe-protein (ox)
N2 + 6H+
H2
2H+
NH3
This reaction won’t
happen until there are
an accumulated 6e–
Mechanism: over-simplified
Conformational
changes couple
hydrolysis of ATP with
oxidation potential of
Fe in Fe-protein
2NH3
N2
FeMo
Fe2+
oxidized
distorted
FeMo
reduced
MoFe
protein
Fe2+
undistorted
ATP
Fe-protein
ADP
The high cost of nitrogen fixation
The stoichiometry of nitrogenase is still not completely known.
N2 + (6+2n)H++ (6+2n)e– + p(6+2n)ATP
2NH3 + nH2 + p(6+2n)ADP + p(6+2n)Pi
-->
n=number of H2 molecules formed (1 or 2, unknown)
p=number of ATP required per electron (probably 2)
2H+
H2
oops.
nitrogenase
Many small molecules bind to the FeMo-cluster
O2
(molecular oxygen)
inactivates
CO
(carbon monoxide)
inhibits (competitive)
HCCH
(acetylene)
substrate
HCN
(cyanide)
substrate
N3-
(azide)
substrate
N2 binds with KM = 0.02 atm
Interesting mutants of nitrogenase
His 195A --> Glu
Blocks N-fixation but allows
reduction of acetylene.
Gly 69A --> Ser
Blocks reduction of acetylene,
but allows N-fixation.
Guided tour of Nitrogenase (1N2C)
Download 1N2C.pdb from www.rcsb.org
In RasMol answer the following questions using the
scripts provided plus any additions of your own:
load 1N2C.pdb
Display-->backbone
color-->chain
select 50 and alpha
label %c
set fontsize 20
set fontstroke 4
color labels yellow
There are 8 chains in the
asu. Which chains are
related to which by noncrystallographic
symmetry?
line up the molecule along the non-crystallographic 2fold.
Guided tour of Nitrogenase (1N2C)
labels off
select 68-69:A
wireframe 50
restrict selected
center selected
color cpk
Does Gly69A have a
positive phi-angle?
Line up atoms 69:A n and 69:A ca to measure the phi
angle. R-handed is positive.
Mutating G69 blocks reduction of acetylene, but
allows N2-fixation. Would mutating Gly69A to a
Serine possibly change its conformation?
Guided tour of Nitrogenase
Draw a TOPS diagram of chain E.
restrict :E
center selected
Display-->cartoon
color-->structure
Line the structure up with the beta sheet perpendicular to the screen.
Ignore short helices (they are not really helices). Draw strands as up or
down arrows and the helices as circles. Then draw connecting lines, to the
middle if the connection is toward you, to the edge if the connection is
away from you. Find the N-term. Number the strands from N to C. Find
the fold class from SCOP (scop.berkeley.edu). (goto “top of the heirarchy”
then class 3, “alpha and beta proteins”)
Finding the fold name in SCOP
Chain E is “3-layer”. When you have numbered the
strands, look in SCOP for the “Fold” with the observed
strand order (for example: 43125, etc). (cute trick: use the
browser’s “search in page” function).
The strand order can be read from right-to-left or left-toright. If a terminal strand is at the edge of the beta sheet
it might be missing. Also, extra strands might be added at
the C-term or N-term is it occurs at the sheet edge.
Write the fold name here ______________________
Guided tour of nitrogenase
Characterize the environment of the ADP and metal
clusters.
select within (6., hetero) and (:A | :B | :E | :F)
restrict selected
Display -->ball and stick
color white
select selected and hetero
spacefill
color cpk
select within (6., hetero) and (:A | :B | :E | :F)
select selected and acidic
color red
select within (6., hetero) and (:A | :B | :E | :F)
select selected and basic
color blue
Guided tour of nitrogenase
Characterize the environment of the ADP and metal
clusters.
select within (6., hetero) and (:A | :B | :E | :F)
select selected and polar and not basic and not acidic
color green
How would you characterize the binding sites? (check one for each het group)
mostly mostly mostly mixed nonbasic acidic polar charges polar
ADP
FS4
CLF
CFM
CA