Perform Basic Protein And Peptide Structure Research

Published Date: 02 Nov 2017

REPORT-1

For Practicum 1 and 1a

PENG ZHAO

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Abstract: This study used Swiss-PDB to study the application of this software to

perform basic protein and peptide structure research. The software functions and

interface were examined and explored. For the protein structure study. First, several

protein secondary structural elements were studied. It included type-I and type

beta-turn, alpha-helix and beta sheet (anti-parallel and parallel). The characteristics

contain hydrogen bonds pattern, main chain torsion angles and side chains

distribution profile in alpha-helix and beta-strand were studied and reported. Second,

the Fab of 4-4-20 antibody was studied. The ligand-fluorescein binding pattern was

examined. Then, the antibody structure was displayed as ribbon which facilitate the

structure description of beta-barrel for each domain. For the disulfide bond which

help stabilize protein structure, it was located and identified. This study also showed

the various potential binding forces which involved in the tightly binding between

antibody 4-4-20 and fluorescein. It turned out that there are VDW force, hydrogen

bond and electrostatic interaction involved in the binding between fluorescein and

antibody. Last, the secondary structural elements of RNase was examined and those

residues which has torsion angles out the common range was identified in

Ramachandran Plot. Also, those disulfide bonds which help stabilize RNase

structure was located and identified. For RNase’s possible mechanism involved in

RNA hydrolysis, the active site complex was identified out of the pool which has all

amino acids located within 10Ã… of the phosphate atom in CPA.

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1 Introduction ..................................................................................................... 1

1.1 Peptide Bond ......................................................................................... 1

1.2 Primary Structure of Peptide and Protein .............................................. 1

1.3 Secondary Structure of Peptide and Protein ......................................... 1

1.3.1 Alpha Helix ................................................................................... 1

1.3.2 Beta Strand and Beta Sheet ........................................................ 1

1.4 Tertiary structure of protein[1] ................................................................ 2

1.5 Quaternary structure of proteins ............................................................ 2

1.6 Protein Functions and Role ................................................................... 2

1.7 Functions of Protein Examined ............................................................. 3

1.7.1 Functions of Antigen-binding Fragment (Fab’) ............................. 3

1.7.2 Functions of Ribonuclease (RNase) ............................................ 3

2 Methods .......................................................................................................... 3

2.1 X-ray crystallography--Basis and Process[2] ......................................... 3

2.2 X-ray Crystallography Procedure for 4-4-20 Fab-fluorescein complex

(adapted from [3]) .......................................................................................... 4

2.3 X-ray Crystallography Procedure for RNase A (adapted from [4]) ......... 4

2.4 Molecular Modeling Using Molecular Graphics Software ...................... 5

2.4.1 General Introduction .................................................................... 5

2.4.2 Protein Data Bank—Introduction and Information ........................ 5

2.4.3 Swiss PDB Viewer—Introduction ................................................. 6

3 Results............................................................................................................ 6

3.1 Practicum-1-Exercise-1 ......................................................................... 7

3.2 Practicum-1-Exercise-2 ....................................................................... 10

3.3 Practicum-1-Exercise-3 ....................................................................... 14

3.4 Practicum-1a-Exercise-1 ..................................................................... 18

3.5 Practicum-1a-Exercise-2 ..................................................................... 30

4 Discussion .................................................................................................... 35

4.1 Protein Secondary Structural Elements Examined—Properties .......... 35

4.1.1 Alpha helix ................................................................................. 35

4.1.2 Beta sheet .................................................................................. 35

4.2 Protein Secondary Structural Elements in Protein Folding .................. 35

4.3 4-4-20 Anti-Fluorescein Antigen-Binding Fragment ............................. 36

4.3.1 Secondary, Tertiary, and Quaternary Structural Elements

Comprise the 4-4-20 Fab ...................................................................... 36

4.3.2 Architecture of the Antigen-Combining Site and Potential

Explanation for High Affinity Antigen Binding ........................................ 37

4.4 Bovine Pancreas Ribonuclease A (RNase) Structure .......................... 37

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4.4.1 Secondary and Tertiary Structural Elements that Comprise RNase

37

4.4.2 Architecture of the Active Site and Reaction Mechanism ........... 38

5 Reference ..................................................................................................... 38

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1 Introduction

1.1 Peptide Bond

A peptide bond is created by condensation reaction between two amino acids

when the condensation between carboxyl group of one amino acids and the amino

group of the other amino acids and generate a water.

Electrons delocalization from the double bond render the peptide bond to be

planar with torsion angle ω=180 ︒. Dihedral angles indicate the internal degrees of

protein freedom and control the protein's conformation. Therefore, the dihedral

angles phi φ and psi ψ should fall into certain range common for certain secondary

structure elements.

1.2 Primary Structure of Peptide and Protein

Proteins’ primary structure is the amino acid linear sequence from the

N-terminal (N) to the C-terminal. The primary structure determines those secondary,

tertiary and quaternary structures of protein.

1.3 Secondary Structure of Peptide and Protein

Secondary structure is local sub-structures which highly regular and include

the alpha helix, beta strand and random coils etc. their regular geometry indicates

that the dihedral angles ψ and φ are constrained to certain range. The secondary

structural elements are always stabilized by hydrogen bonds between peptide.

1.3.1 Alpha Helix

Alpha helix has amino acids arranged to a right-handed helix with 3.6 residues

each turn and the pitch= 5.4 Å. Therefore each residue has rotation= 100° and 1.5 Å

(0.15 nm) raise along the heliex axis.

The hydrogen bonds are formed between the amino group (N-H) of i residue

and the carboxylic group (C=O) of i+4 residue. This repeated hydrogen bond help

stabilize alpha-helix significantly.

1.3.2 Beta Strand and Beta Sheet

The chirality of component amino acids help twist Beta strands. The side chains

point roughly perpendicularly outward from the beta-sheet plane, it sometime could

create hydrophobicity at one side of the beta-sheets and hydrophilicity at another

side.

There are the extensive hydrogen bond network between amine groups in one

strand’s backbone and the carboxyl groups in the neighbor strands’ backbone. Again,

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like alpha-helix case, this hydrogen bond network helps stabilize the beta strand a

lot. The preferred dihedral angles of amino acids consist of beta-strand are near (φ,

ψ) = (–135°, 135°)

For the anti-parallel strands. They are typically connected to each other through

a short loop (2-5 residues) and form a Beta hairpin.

Besides the typical beta sheet, there are structural motifs including Greek key

motif, β-α-β motif and Beta-meander motif which are common secondary structure

made by beta strands.

1.4 Tertiary structure of protein[1]

The term tertiary structure is the unique three-dimensional conformations

assumed by globular proteins when adapt native structures. The protein's primary

structure largely determine it.

This process turns an unorganized, nascent molecule to a highly organized

structure through building up interactions between the side chains in primary

structure. Also, by excluding most water molecules from the protein’s interior it

could generate new interactions between both polar and nonpolar groups possible

and could have a significant difference in the relative geometry of those amino acid

residues in primary structure. This interaction lead to hydrophobic interactions

which favor protein folding.

Moreover, electrostatic interactions between ionic groups of opposite charge.

For example, salt bridge.

Also, there are van der waals forces in including dipole-dipole, ion-dipole forces.

Again, there are hydrogen bonds which are significant in the interior of the folded

protein and may help hold subunits into protein tertiary structure.

Besides those non-covalent one, the covalent bonds could alter a polypeptide’s

structure either during or after its synthesis. For example, the bisulfide bridges is

widely spread in tertiary structure of extracellular proteins. For example, antibody

in which is indispensable for adapting the right conformation.

1.5 Quaternary structure of proteins

The final structure which the multi-subunits of a protein assemble to is called

quaternary structure. This is seen commonly in proteins with more than one

polypeptide chains.

1.6 Protein Functions and Role

The ability for protein to bind other molecules specifically and tightly serves a

basis for protein’s diverse functions.

First, protein could be enzymes to catalyze chemical reactions. Enzymes could

involve in reactions in metabolism, DNA replication, DNA repair, transcription etc.

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Besides enzyme function, proteins play critical roles in cell signaling

transduction. It could be extracellular signals that have signal transmitting functions

between cells even between distant organs. For example, protein hormones. Some

proteins are membrane receptors that induce ensuring cellular response by

initiating cell signaling pathway by binding certain signaling molecules.

The cell also need cellular scaffolding or skeleton formed by proteins to control

the movement, shape and behaviors in the cell. Those functions are crucial in some

disease process like cancer metastasis.

1.7 Functions of Protein Examined

1.7.1 Functions of Antigen-binding Fragment (Fab’)

The Antigen-binding fragment consists of one constant and one variable

domain of each of the heavy and the light chain. The function of those

Antigen-binding fragment is to bind antigen since they contain the antigen binding

sites of the antibody. The specificity of antigen binding is determined by the

particular combination of V and V .

H L

The Antigen-binding fragment was once generated by papain digestion which

breaks the immunoglobulin molecule from the hinge region and generate 2 Fab’ and

1 Fc.

1.7.2 Functions of Ribonuclease (RNase)

Ribonuclease (RNase) is the nuclease that help degrade RNA into smaller pieces

or even nucleotide. The RNase is catalyst in those reaction.

Moreover, RNases is crucial in the RNA molecules maturation including mRNAs

and non-coding RNAs to provide diversely functions in cellular processes, e.g. RNAi.

Also, as suggested by its function, RNase could help defense host against RNA

viruses by catalyzing the degradation of RNA.

2 Methods

2.1 X-ray crystallography--Basis and Process[2]

X-ray crystallography can generate the information of detailed 3D structures of

proteins of interest at fairly an atomic level resolution.

The analysis elements are a protein crystal, a source of x-rays, and a detector.

To investigate molecular structures through X-ray crystallography need growth

of solid crystals of the molecules of interest. After acquiring proper crystals, a beam

of X-rays with high power was aimed at a tiny crystal which have numerous identical

protein molecules. At the same time, it is necessary to record the images in desired

orientation precisely in order to capture 3D pattern that crystal scatters. The

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electronic detector capture X-rays is similar as a digital camera in capturing images.

The X-ray crystallography generate the distances between atoms in angstroms

which reflexes the distance between atoms in a molecule.

2.2 X-ray Crystallography Procedure for 4-4-20 Fab-fluorescein

complex (adapted from [3])

The 4-4-20 Fab-fluorescein complex crystals were grown in either 47% (v/v)

MPD or 16% (w/v) PEG-3350. The samples crystallized with PEG early in the

project were abandoned since it were found to have monoclinic space groups and

were mostly twinned. This did not meet the requirement for proper crystals growth.

The purification is also a crucial step. Later samples purified under more

stringent requirements of light exclusion and temperature control (13 ± 2°C)

produced crystals with tri-clinic space groups and crystal morphology nearly

identical with MPD forms.

Then, it comes to collection of diffraction data which were collected with a

Siemens-Nicolet P21 diffractometer equipped with a scintillation detector and

helium chamber. Space groups were initially determined using 80 or 10° precession

photos, although the lattice parameters used for structure determination were

obtained from diffractometer data. In cases where indexing was difficult, 30

precession photos were used to align the crystal and adjust the arcs of the

goniometer. Diffraction data were collected using an omega step scan procedure and

intensities were corrected for background, absorption, and radiation damage as

previously described. Four crystals were used to collect the data set for the MPD

form. An attempt was made to extend the data to d spacings of 1.5-A, but the

reflections were mostly unobserved beyond 1.75-A. The PEG triclinic form was

particularly resistant to radiation damage, and only one crystal was required for

data collection. One crystal was sufficient for the PEG mono-clinic form, but the data

extended only to 2.5-A resolution.

2.3 X-ray Crystallography Procedure for RNase A (adapted from

[4])

RNase-A (Sigma, type XIIA) was purchased from Sigma. Trigonal crystals of the

native enzyme were obtained by the hanging drop vapor diffusion method. A

solution containing 35 mg/mL protein in 20 mM sodium phosphate, 20 mM sodium

acetate, pH 6, was equilibrated against 35% ammonium sulfate, 1.5 M sodium

chlorine. Crystals of 1×1× 1 mm3 grew after 1 week and were of space group P3221.

Crystals of the 3'-CMP complex of RNase A were obtained by soaking native

crystals in mother liquor to which 3'-CMP was added at a final concentration of 10

mM for 24 h. The monoclinic d (CpA) complex of RNase A was crystallized in sitting

drop setups. A solution containing 35 mg/mL protein and 10 mM d (CpA) in 20 mM

phosphate, 20 mM acetate, pH 5.2, was equilibrated against 50% MPD. Data on the

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trigonal crystals were collected on a MAR Image Plate mounted on a rotating anode

generator operated at 40 kV, 70 mA, with a Ni filter, and a 0.3-mm collimator. The

crystal-to-screen distance was 100 mm. Data were collected at 2"Iframe with an

exposure of 30 mid-frame. The native crystal was mounted with the c-axis along the

oscillation axis, and 15 frames (30") were collected. The crystal of the 3’-CMP

complex was mounted in a random orientation, and 47 frames (94") were collected.

The data were processed with MOSFLM and other programs from the CCP4 package

(Collaborative Computer Project 4, SERC Daresbury Laboratory, UK). The data on the

monoclinic d (CpA) complex were collected on an Enraf Nonius FAST Area detector

mounted on a rotating anode operated at 45 kV and 50 or 70 mA. Data were

collected at 22 or 60 s/frame, at 0.12"/frame. The shorter data collection times and

lower amperages were used in order to collect the low- resolution data. The data

reduction was performed with the program MADNESS.

2.4 Molecular Modeling Using Molecular Graphics Software

2.4.1 General Introduction

Molecular modeling consists of theoretical approaches and computational

methods for the simulation of the molecules’ behavior. Also, it is capable to study

molecular systems include small chemical, complicated macro-molecules and even

biomolecules assemblies. They are widely used in computational chemistry, drug

design, computational biology and materials science.

There are lots of molecular graphics software used in this molecular modeling.

For example, if you go to Protein Data Bank website, there are numerous common

molecular graphics software listed. For example, Chimera, Protein Scope, SPADE etc.

2.4.2 Protein Data Bank—Introduction and Information

The Protein Data Bank (PDB) stores the 3-D structural data of large biological

molecules, such as proteins and nucleic acids. It plays a critical role in structural

biology thus major scientific journals and some funding agencies require their

structure be submitted by to PDB ([5]).

There are free access in the PDB member organizations (PDBe, PDBj, and RCSB)

which provide X-ray crystallography structure or NMR spectroscopy structure for

scientist who need them.

In detail, the PDB file include the many aspects information of a biomolecule:

primary structure, heterogen, secondary structure, connectivity annotation,

miscellaneous features, crystallographic and coordinate transformation information,

coordinate information, connectivity and bookkeeping ([5]).

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2.4.3 Swiss PDB Viewer—Introduction

Swiss PDB Viewer has a number of self-explanatory windows which could

appear in the workspace by the Wind pull down menu. It includes Control Panel and

the layer Infor window and the most common Tool Bar.

Control panel is used to control the display of each residue and ligand. One or

many groups may be selected simply by clicking and use CTRL to select more than

one groups. Moreover, it could control the display options of the main chain, the

residues’ side chain, residues’ name labels, residues’ van der Waals surface, the

ribbons structure and options for color. It also have different secondary structural

elements labeled which will facilitate any study that focus on some of them.

The Prefs menu allows users to adjust those parameters in the software

conveniently.

The Toolbar provides basic tools useful in typical manipulation of protein and

peptide structure. For example, the distance measuring tool, the focus tool and the

button which allows users to display only groups fall into certain distance from a

designated group. This could help focus on the part which may involve potential

interaction with certain residue or ligand.

The Tools pull down menu consists of functions which could help study the

interaction and structure of the protein displayed. For example, the compute

hydrogen bond function is quite common used when we study the hydrogen pattern

which help protein fold and stabilize those secondary, tertiary and quaternary

structure.

By the Display and Color pull down menu, we could easily visualize the protein

structure with some features shown. For example, in the displaying of colorful

ribbon structure, we need use several command in Display and Color pull down

menu then finally generate a ribbon model and distinguish those secondary

elements precisely.

At last, another most powerful menu is the Select pull down menu. In order to

manipulate and study the structure of a protein, we need select some parts for the

software to focus on. The Select pull down menu offers flexible selection choice that

realize our goal. For example, if we want to study disulfide bond which stabilize the

secondary structure of a protein, we could select those Cys by the "Select Certain

Group" menu. Then, we could turn the side chains of those cysteine on and generate

a structure with Disulfide Bridge shown clearly.

For this Swiss PDB Viewer, there are more functions which still wait for us to

explore deeply.

3 Results

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3.1 Practicum-1-Exercise-1

For each file to be loaded by SPDBV, the txt file should looks like this:

>TA

TA

Figure1. 1.X-ray Structure loaded from text file TA

Load the raw sequence from the amino acids in the SwissModel pull down menu.

The program displays the molecule without hydrogen atoms which is standard for

x-ray structure (Figure1. 1).

The "Rock and Roll" and the hot key ">" works well, but it may be better to use

mouse to rotate the peptide to get a 3D idea.

Turn the residues on and off and label the skeletal model by Control Panel, the

individual residues were labeled by Lev 41 button in the toolbar. (Figure1. 2)

Figure1. 2 TA-Residues Labeled

Set the VDW and surface dot densities to 10 or higher by using Display from the

Prefs pull down menu. Then the Van der Waals (VDW) surface are turned on in

Control Panel (Figure1. 3).

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Figure1. 3 VDW Surface of TA

Then, the solvent accessible surface were turned on in Control Panel (Figure1.

4).

Figure1. 4 Solvent Accessible Surface of TA

Van der Waals (VDW) surface, it is contact surface of each atom controlled by

the Van der Waals radius. While, the solvent accessible surface is surface formed at

the center position of a water molecule rolling over the protein with a 1.4-Ã… radius

(approximately the radius of a water molecule). So, they are different in definition.

Then, turn off the dot surfaces and create a solid, 3-dimensional representation

of TA molecule by selecting Render in Solid 3D from the Display pull down menu. In

addition, use Slab to help focus on one part of the TA. Before using Slab, modify

default value of slab using the Display option of the Prefs pull down window to 5 Ã….

From the Figure1. 5, the atoms closest to us are brighter than ones farther away.

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Figure1. 5 Slab and 3D Rendering of TA

The ball-and-stick representation for TA was created by selecting 3D Rendering

in the Prefs pull down menu and decreasing the bond radius to 0.1 Ã…, and clicking

the box entitled "Keep atoms proportions by multiplying atom radius by…" (Figure1.

6)

Figure1. 6 Ball-and-Stick Representation of TA

Then, the Render in 3D was turned off. The phi and psi angles for beta sheet was

set by selecting "Strand" from the Set Omega/Psi/Phi option in the Tools pull down

menu. Then, the distances of all the bonds (Figure1. 7) and bond angles (Figure1. 8)

in the dipeptide were measured.

Figure1. 7 Distances of All the Bonds

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Figure1. 8 Bond Angles of TA

Angles correspond to phi, psi and omega for each amino acid residue were

identified and measured. The torsion angles are labeled in the central bond which

define it, there are there angles, 1 psi, 1 omega and 1 phi in the main chain, see

Figure1. 9 :

Figure1. 9 All Torsion Angles in TA

Modify the phi and psi angles. If choose the torsion angles as indicated by

Figure1. 10 (psi=phi=180°), the oxygen in THR1 will have close contact with the

nitrogen atom in THR1. As shown in Figure1. 10.

Figure1. 10 Atoms Come Into Close Contact when psi=180° and phi=180°

3.2 Practicum-1-Exercise-2

For the tetrapeptide KNAQ, first, modify the torsion angles for type-I beta turn

as in the table on page 4 of Practicum-1. The main chain torsion angles were

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measured (Figure1. 12) and the fixed torsion angles location and the hydrogen bond

between main chains that stabilizes KNAQ loop shown (Figure1. 11).

All the omega torsion angles in the main chain of peptide KNAQ belongs to

torsion angles, since omega are limited to trans or cis configuration by peptide bond

resonance.

Moreover, those torsion angles which are intentionally modified in order to

generate a type-I beta turn are also fixed torsion angles. In detail, the torsions of

ASN2—phi=-70°, psi=-30°. The torsion angles of ALA3—phi=-90°, psi=10°. Without

those fixed torsion angles, there is no way to form a type-I beta turn and stabilized

by hydrogen bond.

Figure1. 11 below indicated the hydrogen bond stabilizes the loop. This

hydrogen bond is between i to i+3 residue.

Figure1. 11 Hydrogen Bond in Main Chain that Make the KNAQ loop Stable and Torsion Angles of

Type-I beta Turn--Sketch

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Figure1. 12 Hydrogen Bond in Main Chain that Make the KNAQ loop Stable and Main Chain Hydrogen

Bond that Stabilizes the Loop KNAQ and Torsion Angles of Type-I beta Turn

Then, model the KNAQ tetrapeptide into a type-II beta turn by those special

parameters for it. We could see there are hydrogen bond which help stabilize the

loop, it is between i to i+2 residue (Figure1. 13).

Figure1. 13 H bond in type-II beta Turn

The distance of groups in type II beta turn was measured. If compare it with the

above figure for type-I beta turn, we could conclude that the oxygen atom in LYS-1

came into close contact with the nitrogen in ALA-3 (Figure1. 14).

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Figure1. 14 Groups that come into Close Contact at Type-II beta turn

Then, the TPGN peptide main chain torsion angles were measured (Figure1. 15)

and sketched (Figure1. 16) with the location of the fixed torsion angles and the

hydrogen bond between main chains that make the KNAQ loop stable.

For the fixed torsion angles, all the omega in the main chain are fixed torsion

angles since omega are limited to trans or cis configuration due to peptide bond

resonance.

For other fixed torsion angles, the torsion angle of Pro2—phi=-60°, psi=130°.

Also, the torsion angle of GLY3—phi=80°, psi=0°.

The Proline’s cyclic structure makes its φ dihedral angle at about −60° .

Therefore, it leads to and exceptional rigidity in peptide conformation. This rigidity

will constraint the direction of the residue after proline. Moreover, glycine is a very

flexible amino acid since it has no side chain. It will not interfere with the directions

of a turn.

Therefore, a rigid amino acid with a flexible amino acid residue will create an

atom distribution profile where break close contact between the oxygen in proline

and the nitrogen in threonine.

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Figure1. 15 Torsion Angles and Hydrogen Bond in Main Chains that Stabilizes the Loop

Figure1. 16 Torsion Angles and Hydrogen Bond in Main Chain that Stabilizes the Loop--Sketch

3.3 Practicum-1-Exercise-3

Modify the torsion angles oligopeptide LSKVLKSLVKTL into an alpha helix

based on the Page4 in Practicum-1. The helix (Figure1. 17) and its distribution of

side chains (Figure1. 18) was examined.

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Figure1. 17 LSKVLKSLVKTL alpha-helix

Figure1. 18 LSKVLKSLVKTL Distribution of Side Chains

Alpha-helix could be found in motifs for binding DNA, for example, the zinc

finger motifs. This could explained that the Alpha-helix has a diameter about 12Ã…

(1.2 nm) which matches the B-form DNA major groove width [6].

Moreover, alpha-helix common that in biological membranes crosses proteins,

due to hydrophobicity distribution profile of the helical structure. The hydrogen

bonding pattern (Figure1. 19) was identified which is between i and i+4 residue.

.

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Figure1. 19 LSKVLKSLVKTL Alpha-helix Hydrogen Bonding Pattern

The distance between two atoms involved in hydrogen bonds are measured,

and they are between 3.17Ã… and 3.20 Ã…. (Figure1. 20)

Figure1. 20 LSKVLKSLVKTL Alpha-helix Hydrogen Bonding Distance

Those peptide bond will create a individual dipole by their C=O group. All those

individual dipole accumulate will lead to a aggregate effete and give the helix a net

overall dipole[7].

Build the oligopeptide (SVNLTFQIK) into a strand of anti-parallel beta sheet by

using angles provided. Open the file twice and merge those two identical layers to

create a layer with two molecules of SVNLTFQIK together. Then align the two strands

into an anti-parallel beta sheet. Then, examine the pattern of hydrogen bonds

(Figure1. 21, Figure1. 22).

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Figure1. 21 SVNLTFQIK Anti-Parallel and H-Bond Pattern

Figure1. 22 SVNLTFQIK Anti-Parallel and H-Bond Pattern-Sketch

For the anti-parallel beta-sheet, we could see from Figure1. 23, the upper side

of the beta-sheet is hydrophobic and the lower side is hydrophilic based on the side

chain distribution.

Figure1. 23 Anti-Parallel Beta-sheet Side Chain Configuaration Determines the Hydropilicity on Each

Side

Beta-sheets are could help make structures in proteins. For example, β barrels,

β sandwiches, β prisms, β propellers, and β-helices are all structures formed based

on beta-sheets.

Delete one strand and modify the torsion angles of the others to be consistent

with parallel beta sheet by the angles provided in handout. Duplicate the strand as

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above. Align the two strands into a parallel sheet. The pattern of hydrogen bonds is

staggered parallel (Figure1. 24, Figure1. 25).

Figure1. 24 Pattern of Hydrogen Bonds of Parallel Beta Sheet

Figure1. 25 Pattern of Hydrogen Bonds of Parallel Beta Sheet-Sketch

3.4 Practicum-1a-Exercise-1

The Fab fragment of 4-4-20 anti-fluorescein antibody is a 1.7 Ã… X-ray diffraction

structure that determined by Dr. Herron [3]. The light chain is labeled L1 - L219 and

the heavy chain is labeled H1 - H216; fluorescein is labeled FDS1 (fluorescein

disodium salt).

After turning the Slab function on, the atoms which are far more than 20 Ã… away

from fluorescein will become faint compared with those within 20 Ã… distance. This

could help find the fluorescein. Please see the Figure1a. 1 to see the fluorescein.

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Figure1a. 1Fab of 4-4-20 antibody Slab On and Fluorescein Labeled

Turn on a VDW surface for only fluorescein. Next, turn on Render in Solid 3D

and the fluorescein with doted VDW surface changes to a solid VDW model, while

the rest of the Fab is rendered as a ball-and-stick model (Figure1a. 2).

Figure1a. 2 VDW surface for only Fluorescein is on and Render in Solid 3D on

The Fab fragment contains four domains: 1) light chain variable domain L1 -

L110; 2) light chain constant domain L111 - L219; 3) heavy chain variable domain

H1 - H121; and 4) heavy chain constant domain H122 - H216.

Use the Col column in the Control Panel to color the four domains differently

and the fluorescein molecule a fifth color (Figure1a. 3).

The active site and hydrogen bonds involved in the interaction was labeled and

shown in Figure1a. 5.

The disulfide bonds which contribute to connect the heavy and light chains

were labeled at white color (Figure1a. 4 and Figure1a. 3).

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Figure1a. 3 Fab of 4-4-20 Antibody with Four Domains Colored

Figure1a. 4 Fab of 4-4-20Antibody with Four Domains Colored and Disulfide Bonds Labeled as White

Figure1a. 5 Fab of 4-4-20 Antibody with Active Sites Showed and Hydrogen Bonds involved labeled as

Green Dotted Line

Select Show Backbone as Carbon Trace in the Display pull down and then

turning off the side chains to display only the Cα trace. See belowFigure1a. 6.

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Figure1a. 6 Fab of 4-4-20Antibody with Cα trace Shown Only

The β-barrels are best visualized using ribbons. This could be done by following

steps. First, turn off both main and side chains by Control Panel for both "L" and "H"

chains. Then click the plus sign above the ribn column to display low-resolution

white ribbons showing the Fab’s secondary structure. To display solid ribbons, select

Ribbons in the Prefs pull down menu and click the Render as Solid Ribbons box and

the Also in Real Time box; then click both Arrows at C-termini boxes, and set Quality

to its highest value. Next, select Backbone and Side chains from the Color pull down

menu and change it to Act on Ribbons. Finally, select Secondary Structure Succession

from the Color pull down menu. (Part of this paragraph was adapted from handout

Practicum 1a).

Please see Figure1a. 7 for the ribbon model figure.

Figure1a. 7 Fab of 4-4-20Antibody with Ribbon Model Shown

Sketch the β-barrel for each domain, the start and end of each strand were

obtained from the secondary structure listings in the second column of the Control

Panel). Please refer toFigure1a. 8, Figure1a. 10, Figure1a. 12, Figure1a. 14, Figure1a.

12, Figure1a. 13, Figure1a. 14 and Figure1a. 15 to see each beta-barrel in those four

domains.

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Figure1a. 8 Fab of 4-4-20 Antibody with Beta-Barrel in L1-L110 Shown

Figure1a. 9 Fab of 4-4-20 Antibody with Beta-Barrel in L1-L110 Shown-Sketched

Figure1a. 10 Fab of 4-4-20Antibody with Beta-Barrel in L111-L219 Shown

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Figure1a. 11 Fab of 4-4-20Antibody with Beta-Barrel in L111-L219 Shown-Sketched

Figure1a. 12 Fab of 4-4-20Antibody with Beta-Barrel in H1-H121 Shown

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Figure1a. 13 Fab of 4-4-20Antibody with Beta-Barrel in H1-H121 Shown-Sketched

Figure1a. 14 Fab of 4-4-20 Antibody with Beta-Barrel in H122-H216 Shown

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Figure1a. 15 Fab of 4-4-20 Antibody with Beta-Barrel in H122-H216 Shown-Sketched

First beta-strand in VL1-110 is from VAL3 to VAL13, it has a kink between THR7

and LEU9 position. Last beta-strand in VL1-110 is from THR102 to GLU110, it has a

kink between GLY104 and THY107 position.

First beta-strand in VH1-121 is from LYS3 to VAL12, it has a kink between THR7

and GLY10 position. Last beta-strand in VH1-121 is from MET105 to SER117, it has a

kink between GLY109 and THR112 position.

There are mainly 4 types of residues which could cause kink in beta-strand:

THR, LEU, GLY, THY

The reason why there is a kink is due to the inconsistency of amino acids which

flank the kink. For example, the torsion angle of LEU9-L falls into alpha-helix range.

Also, the GLY104-L, GLY105-L also has torsion angle falls into the alpha-helix region.

Moreover, the GLY10-H and GLY8-H also has torsion angle which not belong to the

beta-strand region. The GLY109-H, GLN110-H, GLY111-H also have torsion angle

which not belong to the beta-strand region. Those violation cause the kink in a

continuous beta strand.

Please refer toFigure1a. 16, Figure1a. 17 below to see kicks in VL1-110 and VH1-121,

respectively.

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Figure1a. 16 Fab of 4-4-20 Antibody with Kicks Shown in Beta-Barrel from VL1-VL110

Figure1a. 17 Fab of 4-4-20 Antibody with Kicks Shown in Beta-Barrel from VH1-VH121

The disulfide bridge that stabilizes each beta barrel and the sequence number

of each cysteine were identified and located (Figure1a. 18). Each of these disulfide

bonds has a tryptophan within a few angstroms of it. They were located and

identified of their sequence numbers (Figure1a. 19) (they are Try-36, 40, 159, 153).

The reason why this nearby tryptophan could stabilize disulfide bond is

probably due to their electrostatic interaction. On paper actually mentioned that the

disulfide bond could quench the fluorescent signals from tryptophan [8]. This

indicates electrostatic or other kind of interactions which affect both the stability of

disulfide bond and tryptophan.

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Figure1a. 18 Fab of 4-4-20 Antibody with Disulfide Bridge that stabilizes each Beta Barrel and the

Sequence Number of each Cysteine Labeled

Figure1a. 19 Fab of 4-4-20 Antibody with Disulfide Bridge that stabilizes each beta barrel and the

Sequence number of each Cysteine and Neighbor Tryptophan Labeled

Using the Control Panel display the backbone atoms of the VL-VH dimer and

then display all the atoms in the six hypervarible loops

(complementarity-determining regions; CDRs) (Figure1a. 23)

Those residue close to fluorescein are HIS-L27, GLY-L29, TYR-L32, ARG-L34;

LYS-L50; SER-L91, TRP-L96; TRP-H33; ARG-H52, TYR-H53; SER-H95, TYR-H96,

TYR-H97, GLY-H98.

Among those residues, there are several residues which have greater role in

binding with fluorescein. Please see following paragraphs for details.

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1 Van der vaals interaction

â—‹

By turned on the VDW surface, we could see that the Tyr-H56 interact with

fluorescein by van der vaals interaction. Tyr-H56’s phenolic group interact with

fluorescein’s xanthenone ring by van der vaals interaction (Figure1a. 20).

Figure1a. 20 Tyr-H56’s Phenolic Group Interact with Fluorescein’S Xanthenone Ring by Van Der Vaals

Interaction

2 Electrostatic Interaction

â—‹

The guanidine group of ARG-H52 has electrostatic interaction with O1 enolic

group in fluorescein. Moreover, there is also a salt link between Arg-L39 and

fluorescein's C3 enolic group (Figure1a. 21).

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Figure1a. 21 Electrostatic Interaction between Guanidine Group of ARG-H52 and O1 Enolic Group in

Fluorescein; Salt-Link between Arg-L39 and Fluorescein's C3 Enolic Group

3 Hydrogen bond

â—‹

There is hydrogen bond between His L31 and the fluorescein enolic group. The

second hydrogen bond was between phenylcarboxylate group in fluorescein and the

hydroxyl group in side chain of Tyr-L37 (Figure1a. 22).

Figure1a. 22 Hydrogen Bond Between His L31 and; the Hydrogen Bond Between Fluorescein and the

Hydroxyl Group in Side Chain of Tyr-L37

From results above, we could conclude that there are three types of interaction:

VDW interaction, electrostatic interaction and hydrogen bond. That’s why this active

site has a high binding constant [Ka (intrinsic) = 2 ×1010M-1] for fluorescein.

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Figure1a. 23 Fab of 4-4-20 Antibody with The Backbone Atoms of the VL-VH Dimer and All the Atoms

in the Six Hypervarible loops Displayed

3.5 Practicum-1a-Exercise-2

Lookup the X-ray coordinates for the bovine pancreas ribonuclease A (RNase)

structure in Protein Data Bank (1RPG) solved to 1.4 Ã… resolution.

The primary information stored in the PDB archive consists of coordinate files

for biological molecules. It includes the 3D location of atoms in each protein. This

contains the structure solution, the sequence, a long list of the atoms and their

coordinates. Also, it mentions the experimental observations that are used to

determine these atomic coordinates.

In detail, the PDB file include the primary structure section, heterogen section,

secondary structure section, connectivity annotation section, miscellaneous features

section, crystallographic, coordinate transformation section, coordinate section,

connectivity section and bookkeeping section.

The PDB file contains coordinates for RNase and two ligands:

2'-deoxycytidine-2'-deoxyadenosine-3', 5’-monophosphate (CPA) and

methylpentanediol (MPD). CPA is a dinucleotide inhibitor of RNase that was

co-crystallized with the protein to investigate active site structure and catalytic

mechanism. MPD is a crystallizing agent. We could just leave it turned off.

Display the VDW surface for CPA (Figure1a. 24)

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Figure1a. 24 RNase with VDW surface for CPA Displayed and MPD Display Turned Off

Each secondary structural element were identified and located in the

polypeptide chain by using the 2nd and 3rd columns of the Control Panel. Also display

a solid, colored ribbon model of the RNase as described Problem 1d above (Figure1a.

25). In Figure1a. 25, all the secondary structure elements include helix, strand and

coil are labeled with their starting and ending residues.

Figure1a. 25 RNase with Solid, Colored Ribbon Model Displayed

In details, helixes are from ALA4 to GLN11, ASN24 to SER 32, LEU51 to VAL57.

Coils are from LYS1—THR3, HIS12—SER23, ARG33—LYS41, GLU49--SER50,

CYS58—GLN60, ALA64—THR70, TYR76—THR78, GLY88—CYS95,

ASN113—PRO114.

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Strands are from PRO42—HIS48, LYS61—VAL63, ASN71—SER75,

MET79—THR87, ALA96—GLY112, TYR115-VAL124.

Next, construct a Phi-Psi (Ramachandran Plot) of RNase by using the Control

Panel to select the entire RNase chain (LYS1 to OXT124) and then select

Ramachandran Plot from the Wind pull down menu. Please see Figure1a. 26 below

for Ramachandran Plot generated (Figure1a. 26).

Figure1a. 26 RNase with Ramachandran Plot Displayed

The Ramachandran Plot only shows torsion angles for selected amino-acids.

The cross means amino acids and the square means glycine.

There are three amino acids residues which have phi and psi angles outside the

allowed ranges. From the Ramachandran Plot, they are identified as GLN60, GLY68

and GLY112.

Location of disulfide bonds in the RNase polypeptide chain. The RNase does

have several disulfide bonds, which are located and labeled in Figure1a. 27 below.

From the Figure1a. 27, we could know its position Cys26-84, Cys58-110, Cys40-95

and Cys65-72.

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Figure1a. 27 RNase with Location of Disulfide Bonds in the RNase Polypeptide Chain Labeled in

White

The Cys26-84 and Cys58-110 are fundamental in the right folding conformation.

Each of help connect alpha helix distributed in different layers.

The Cys40-95 and Cys65-72 are less crucial with only one of them been reduced

without disrupting the RNase structure. Also, they are relatively exposed to solvent

which may become easier to be reduced under certain condition. The reduction

could affect the protein folding since the disulfide bond is a crucial bond in holding

the secondary structural elements together.

Remove the VDW surface from CPA and use the Toolbar and display only those

residues that are within 10 Ã… of the phosphate atom. Please see Figure1a. 28 below

for only those residues that are within 10 Ã… of the phosphate atom. Those residues

are listed below.

Residues within 10 Ã… are ALA4, LYS7, PHE8, GLU9, ARG10, GLN11, HIS12,

LEU35, ARG39, LYS41, VAL43, ASN44, THR45, ASN67, ILE107, VAL108, ALA109,

PRO117, VAL118, HIS119, PHE120, ASP121, and ALA122.

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Figure1a. 28 RNase with Only those Residues that are within 10 Ã… of the Phosphate atom of CPA

Displayed

Based on its reaction mechanism, the residues which are probably involved

with hydrolysis of the phosphodiester bond are HIS12, HIS119 and LYS41.

In detail, the deep cleft between two lobes is where the positive charges of

RNase A mainly locate. The RNA substrate locked in this cleft when been hydrolyzed

by two catalytic histidine residues, His12 and His119. They form a 2', 3’-cyclic

phosphate intermediate in which the His 12 interacts with the 2’ oxygen and His 119

in it’s A conformation. Then, this intermediate is stabilized by nearby Lys41.

Please see Figure1a. 29 below for active site complex involved in catalysis.

Figure1a. 29 RNase with Only Residues His12, His119 and LYS41 which are involved in hydrolysis of

the phosphodiester bond Displayed—Active Site Complex

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4 Discussion

4.1 Protein Secondary Structural Elements Examined—Properties

4.1.1 Alpha helix

The hydrogen bonds are formed between the amino group (N-H) of i residue

and the carboxylic group (C=O) of i+4 residue. This repeated hydrogen bond help

stabilize alpha-helix significantly (Figure1. 19).

The torsion angles in alpha helix are typically phi=-57°, psi=-47°. This could be

used to modify a random peptide chain to alpha helix.

Those peptide bond will create an individual dipole by their C=O group. All

those individual dipole accumulate will lead to an aggregate effete and give the helix

a net overall dipole [7].

The side chains of an alpha helix stick out from the helix axis, where they are

available to interact with the surroundings which could help constitute highly

organized structures like coiled-coil [9].

4.1.2 Beta sheet

There are the extensive hydrogen bond network between amine groups in one

strand’s backbone and the carboxyl groups in the neighbor strands’ backbone. Again,

like alpha-helix case, this hydrogen bond network helps stabilize the beta strand a

lot. The preferred dihedral angles of amino acids consist of beta-strand are near (φ,

ψ) = (–135°, 135°) (Figure1. 21, Figure1. 24).

For the hydrogen bonds pattern which stabilize beta-sheet, the antiparallel beta

sheet has hydrogen bonds parallel to each other (Figure1. 21) while the parallel beta

sheet has hydrogen bond staggered parallel (Figure1. 24) to each other.

The torsion angles for a parallel beta-sheet is phi=-119°, psi=113°. While, the

torsion angles for an anti-parallel beta-sheet is phi=-139°, psi=135°. Those angles

could be used to model peptide chain to a beta sheet in the Swiss-PDB viewer.

The side chains point roughly perpendicularly outward from the beta-sheet

plane, it sometime could create hydrophobicity at one side of the beta-sheets and

hydrophilicity at another side.

4.2 Protein Secondary Structural Elements in Protein Folding

The term tertiary structure is the unique three-dimensional conformations

assumed by globular proteins when adapt native structures. The protein's primary

structure largely determine it.

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This process turns an unorganized, nascent molecule to a highly organized

structure through building up interactions between the side chains in primary

structure. Also, by excluding most water molecules from the protein’s interior it

could generate new interactions between both polar and nonpolar groups possible

and could have a significant difference in the relative geometry of those amino acid

residues in primary structure. This interaction lead to hydrophobic interactions

which favor protein folding [1].

Moreover, electrostatic interactions between ionic groups of opposite charge

could also help hold protein subunits together. For example, salt bridge.

Also, there are van der waals forces in including dipole-dipole, ion-dipole forces.

Again, there are hydrogen bonds which are significant in the interior of the folded

protein and may help hold subunits into protein tertiary structure [10].

Besides those non-covalent one, the covalent bonds could alter a polypeptide’s

structure either during or after its synthesis. For example, the bisulfide bridges is

widely spread in tertiary structure of extracellular proteins. For example, antibody

in which is indispensable for adapting the right conformation.

4.3 4-4-20 Anti-Fluorescein Antigen-Binding Fragment

4.3.1 Secondary, Tertiary, and Quaternary Structural Elements Comprise

the 4-4-20 Fab

After examined by Swiss-PDB viewer, the 4-4-20 Fab contains several

beta-strand as its secondary structure, those beta-strands are connected to each

other through random coil structure which also belongs to secondary structure of

proteins.

Also, the first and last beta strands in each variable domain have a kink in the

middle of them which is an interruption in the middle of the beta strand. This could

be discerned easily either in control panel or from the ribbon structure. The kinks

are typically caused by the in consistence between two amino acids, which disrupt

the smooth beta strand.

Those beta-strands arranged in an anti-parallel way and form a beta-barrel in

each domain. This is the tertiary structure of 4-4-20 Fab. The beta barrel are

stabilized by disulfide bond between them which also stabilized by nearby

tryptophan.

The 4-4-20 anti-fluorescein antigen-binding fragment has 2 polypeptide chains,

one is heavy chain and another is light chain. Both of them comprise one variable

domain and constant domain. Those domains are tertiary structure of 4-4-20 Fab

which consists of beta-barrels. The folding of those two peptide chains to adapt a

36

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certain overall structure is the quaternary structure of this 4-4-20 anti-fluorescein

antigen-binding fragment. The light and heavy domain are connected by a disulfide

bond.

4.3.2 Architecture of the Antigen-Combining Site and Potential

Explanation for High Affinity Antigen Binding

The architecture of the fluorescein binding cite of 4-4-20 anti-fluorescein

antigen-binding fragment involved several residues which are strongly associated

with the fluorescein through force including Van der vaals interaction, electrostatic

interaction and hydrogen bonds.

All of those diverse and strong interactions help increase the affinity activity of

the CDRs to fluorescein. Among those forces, the electrostatic force is extremely

crucial in generating such a high affinity [3].

Van der vaals interaction

By turned on the VDW surface, we could see that the Tyr-H56 interact with

fluorescein by van der vaals interaction. Tyr-H56’s phenolic group interact with

fluorescein’s xanthenone ring by van der vaals interaction (Figure1a. 20).

Electrostatic Interaction

The guanidine group of ARG-H52 has electrostatic interaction with O1 enolic

group in fluorescein. Moreover, there is also a salt link between Arg-L39 and

fluorescein's C3 enolic group (Figure1a. 21).

Hydrogen bond

There is hydrogen bond between His L31 and the fluorescein enolic group. The

second hydrogen bond was between phenylcarboxylate group in fluorescein and the

hydroxyl group in side chain of Tyr-L37 (Figure1a. 22).

From results above, we could conclude that there are three types of interaction:

VDW interaction, electrostatic interaction and hydrogen bond. That’s why this active

site has a high binding constant [Ka (intrinsic) = 2 ×1010M-1] for fluorescein [3].

4.4 Bovine Pancreas Ribonuclease A (RNase) Structure

4.4.1 Secondary and Tertiary Structural Elements that Comprise RNase

The RNase comprises helix, coil and beta-strand in its secondary structure.

In detail, Helixes are from ALA4 to GLN11, ASN24 to SER 32, and LEU51 to

VAL57.

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Coils are from LYS1—THR3, HIS12—SER23, ARG33—LYS41, GLU49--SER50,

CYS58—GLN60, ALA64—THR70, TYR76—THR78, GLY88—CYS95,

ASN113—PRO114.

Strands are from PRO42—HIS48, LYS61—VAL63, ASN71—SER75,

MET79—THR87, ALA96—GLY112, TYR115-VAL124.

For the tertiary structural elements of RNase, it is the structure which consists

of all those secondary structural elements including helix, coil and beta-strand. The

coils are mainly used in connecting all the helix and beta-strand structures together.

Moreover, there are several disulfide bond which help stabilize the tertiary structure

and hold those secondary structures together.

4.4.2 Architecture of the Active Site and Reaction Mechanism

Based on modeling experiments with software and literature, the architecture

of the active site involve hydrolysis of the phosphodiester bond probably comprises

HIS12, HIS119 and LYS41.

In detail, the deep cleft between two lobes is where the positive charges of

RNase A mainly locate. The RNA substrate locked in this cleft when been hydrolyzed

by two catalytic histidine residues, His12 and His119. They form a 2', 3’-cyclic

phosphate intermediate in which the His 12 interacts with the 2’ oxygen and His 119

in its A conformation. Then, this intermediate is stabilized by nearby Lys41 [4].

Please see Figure1a. 29 below for active site complex involved in catalysis.