Crystallogaphy -- lecture 25

Protein guided tours: the meaning of “Life”

All life is based on reduced carbon

Thanks to Doug Whittet, RPI Physics

present1*1092*1093*1094*109 y

origin of life

origin of oxygenic phototrophs

anoxic

oxygenated environment

oxidizing

Chloroplasts

O2 in the atmosphere provides a strong natural selection for CO2 fixation

•Cyanobacteria evolved a means to use light for energy, with O2 as the by-product. 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.

A narrowly avoided catastrophe for life on earth:

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 !hohcolor cpkDisplay -> ball and stick

select magnesiumlabel %rset fontsize 12set fontstroke 2color 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)spacefillselect hetero and !hohlabels offcolor temperature

How are the B-factorsdistributed?

Was NCS 2-fold symmetry enforced during refinement?

Guess what: 2-fold symmetry was not enforced during evolution!

Which side is more ordered? Chain A or chain B?

Photosystem I : Guided tour

Find the name of the lipid that does not havea phosphate group.

Characterize the environmentof the lipid. Could it have a role in the light harvest process?

Unix shortcut: use grepgrep ^”HETNAM” 1JB0.pdb

select [LMG]restrict selectedcenter selectedselect within (11., [LMG]) and proteinDisplay -> ball_and_stickcolor cpkselect within (11., [LMG]) and ligand

Photosystem I : Guided tourselect within (11., [LMG]) and ligandspacefill 1.5color green select within (11., [LMG]) and *.MGspacefill 1.5color white select within (11., [LMG]) and [PQN]color red select within (11., [LMG]) and solventspacefill 1.0color cyan What is PQN?

How close is it to the nearestmagnesium

Photosystem I : Guided tourrestrict ligandwireframecolor cpkDisplay-> ball and stickselect [CL1] or [CL2]wireframe color greenselect [PQN]color magentaspacefill 1.0select [BCR]color orangespacefill 1.5select *.MGspacefill 1.0color white

Trace the path of the electronsfrom the special pair to the twoquinones.

Light harvesting complex

Are are of the pigmentsconnected to the special pair?

Photosystem I : Guided tourrestrict [PQN]spacefill color cpkselect within (11.,[PQN]) and proteinwireframe 0.5color cpkselect within (11.,[PQN]) and ligand and not [PQN]color greenwireframe 0.5select within (11.,[PQN]) and solvent spacefill 0.6color cyan

Which quinone is more loosely-bound

Environment of the quinones

How does the electron getfrom one quinone to theother? What protein sidechainforms a bridge?

Photosystem I : Guided tour

Look in scop.berkeley.edu for thefold under alpha+betaWhat fold is this?

Draw a TOPS diagram of chain D

residues 24-95 only

Rubisco 8RUC

RubiscoRubisco

Animals

---> sugars, etc.

RUBISCO = Ribulose Bisphophate Carboxylase/Oxygenase

Rubisco fixes CO2

One of the substrates is small and feature-less. Gets confused with O2.

5 + 1 = 3 + 3

Competing oxygenase activity

Unwanted competing reaction

normal 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 fileedit 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 chainRasmol 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 siteRasmol 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 arewithin 5A of both chain A and chain L?

Nitrogenase 1N2C

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

present1*1092*1093*1094*109 y

anoxic oxidizing

Nitrogenase is a hetero-tetramer. 2 Fe-proteins, 2 MoFe-proteins. With 3 Iron-containing clusters.

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.

Fe-protein

MoFe-protein

from Rees & Howard (2000) Current Opinion in Chemical Biology, 4(5):p559-566.

FeMo cluster

Reduced P-cluster

Oxidized P-cluster

dithionite

There are two Fe-containing clusters in the MoFe-protein

A protein conformational change might favor oxidized over reduced, driving the reaction forward.

Mechanism: simplified

Fe-protein (red)

MoFe-protein (ox)

ATP ATP

+

Fe-protein (ox)

MoFe-protein (red)

ADP ADP

N2 + 6H+

NH3

+2Pi

2ADP2ATP dissociation of complex is rate-limiting

This reaction won’t happen until there are an accumulated 6e–

H2

2H+

fast

slow

less than 6 e-?

Mechanism: over-simplified

Fe-proteinMoFe

protein

ATP

ADPFe2+

undistorted

Fe2+

distorted

FeMooxidized

FeMoreduced

6e-

N2

2NH3

Conformational changes couple hydrolysis of ATP with oxidation potential of Fe in Fe-protein

The high cost of nitrogen fixation

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)

The stoichiometry of nitrogenase is still not completely known.

nitrogenase

H22H+

oops.

O2 (molecular oxygen) inactivates

CO (carbon monoxide) inhibits (competitive)

HCCH (acetylene) substrate

HCN (cyanide) substrate

N3- (azide) substrate

Many small molecules bind to the FeMo-cluster

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-->backbonecolor-->chainselect 50 and alphalabel %cset fontsize 20set fontstroke 4color labels yellow

line up the molecule along the non-crystallographic 2-fold.

There are 8 chains in the asu. Which chains are related to which by non-crystallographic symmetry?

Guided tour of Nitrogenase (1N2C)

labels off

select 68-69:Awireframe 50restrict selectedcenter selectedcolor cpk

Line up atoms 69:A n and 69:A ca to measure the phi angle. R-handed is positive.

Does Gly69A have a positive phi-angle?

Mutating G69 blocks reduction of acetylene, but allows N2-fixation. Would mutating Gly69A to a Serine possibly change its conformation?

Guided tour of NitrogenaseDraw a TOPS diagram of chain E.

restrict :Ecenter selectedDisplay-->cartooncolor-->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-to-right. 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 nitrogenaseCharacterize the environment of the ADP and metal clusters.

select within (6., hetero) and (:A | :B | :E | :F)restrict selectedDisplay -->ball and stickcolor whiteselect selected and heterospacefillcolor cpkselect within (6., hetero) and (:A | :B | :E | :F)select selected and acidiccolor red select within (6., hetero) and (:A | :B | :E | :F)select selected and basiccolor blue

continued...

Guided tour of nitrogenaseCharacterize 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 acidiccolor green

How would you characterize the binding sites? (check one for each het group)

mostly mostly mostly mixed non-basic acidic polar charges polar

ADPFS4CLFCFMCA