University of Rajshahi

Assignment of line for Proteins and polypeptides in

¹

H-NMR spectrum

1. Characteristics sets of lines at particular positions that can be recognized in complex spectra by simple inspection:

2. Comparison between the spectra of a protein with known sequence and its fragments

protein H₃NCHC

O

R

+ NHCHCO

O

R C –

O

Carboxypeptidase

NHCHC O

R'

NHCHC O

R"

NHCHC O

R

C C

Trypsin

4. Use of deuterated amino acid residue

bi-Arsenic complex

3. Chemical Reaction with a particular residue and binding of a ligand to the residue of a protein

D₂N CD C

CD₂

OD O

D

D D

D D

Deuterated Phenylalanine

5. Comparison of the spectrum of a protein with that of a mutant in which there is a single amino acid substitution

AGCCGUCAUGGCAUCGGCGUACGAUUGCCA

3' 5'

AGCCGUCAUGGCAUCGGCGAACGAUUGCCA

3' 5'

Peptide chain ^H_N CH C Peptide chain

CH₂ O

CH₂ C OH

O HN

Peptide chain ^H_N CH C Peptide chain

CH O

CH₃

HN

CH₃

Glutamic acid

Valine

6. Comparison between ¹H-NMR spectra of a protein with their X-ray diffraction analysis data

Ways to study proteins by NMR

1. To determine the fraction of amino acids in the α-helical configuration 2. To monitor helix-coil transitions and especially to observe local changes

during the transition

3. To determine the conformation of selected regions of the protein (e.g.

around a particular amino acid)

4. To observe the binding of small molecules and metal ions to selected regions of the protein (by observing either the ligand or the protein spectra)

5. To study paramagnetic active sites in electron- transfer proteins.

Fraction of residues of poly-(γ-benzyl-L-glutamate) in the α-helical configuration

poly-(γ-benzyl-L-glutamate)

H₂N C C

CH₂

OH O

CH

C

OH O H

n

C C

O HO

F F F

Trifluroacetic acid

x 100

A_H A_H + A_R A_H (%) =

Unfolding of a protein / Denaturation of a protein

Properties of the active site of Ribonuclease-A

Ribonuclease-A

Ribonuclease-A is an enzyme secreted by the pancreas into the small intestine, where it catalyzes the hydrolysis of certain bonds in the ribonucleic acids present in ingested food.

Ribonuclease-A

Total residues: 124; α-helical (%): 26; β-conformation: 35

3'-CMP / 2'-CMP / 5'-CMP (All are competitive inhibitors of ribonuclease-A)

O

OH O

H H

H CH₂

H OH

N N NH₂

O

P O

O O

- -

3'-Cytidine monophosphate (3'-CMP)

O

OH OH

H H

H CH₂

H

N N NH₂

O

5'-Cytidine monophosphate (5'-CMP)

P O

O

O

-

O -

OH OH

H H

H CH₂

H OH

N N NH₂

O

P O O

O

- -

2'-Cytidine monophosphate (2'-CMP)

H-C2 of three histidine residues (His-12, 48, 119) of RN-A (Ribonuclase-A)

3'-CMP +

Chemical shift change

H₂N CH C CH₂

OH O

C N

C NH CH

H

2 1

3 4 5

Histidine

H-C2 (Proton of C-2) of imidazole ring (instead of Histidine residue) Different concentration of Phosphate Group (Instead of 3'-CMP)

+

Same change in chemical shift like His-119

H-C2 of three histidine residues (His-12, 48) of RN-A (Ribonuclase-A)

3'-CMP / 2'-CMP/ 5'-CMP (competitive inhibitors of Ribonuclase-A) whose binding constant different

+

Chemical shift change is same for three inhibitors (3'-CMP / 2'-CMP/ 5'-CMP) Using different pH of medium

H₂N CH C CH₂

OH O

C N

C NH CH

H

2 1

3 4 5

Histidine

Proton of phenyl ring of phenylalanine residue (Phe-120) of Ribonuclase-A

3’-CMP +

Big change in chemical shift

Proton of phenyl ring of phenylalanine residue (Phe-120) of Ribonuclase-A

Phosphate Group +

No change in chemical shift

Proton of phenyl ring of

phenylalanine residue (Phe-120) of Ribonuclase-A

Cytosine / (3'-CMP / 2'-CMP/ 5'-CMP) +

Same big change in chemical shift

H2N CH C CH2

OH O

HC

HC CH

CH CH

Phenylalanine

Effect of a cofactor on enzyme-substrate binding

Nicotinamide proton E + NAD

Broadened

Nicotinamide proton other E + NAD

Not Broadened

Methyl proton of Et-OH and acetaldehyde

E + NAD

Broadened

Methyl proton of Et-OH and acetaldehyde

E Not Broadened

Methyl proton of Et-OH and acetaldehyde

NAD Not Broadened

C C

O H₃C

O

O^-

pyruvate

Pyruvate decarboxylase TPP, Mg²⁺

CO₂

HC

CH₃ O

Acetaldehyde

Alcohol dehydrogenase NADH + H⁺ NAD⁺

H₂ C

CH₃ HO

Ethanol

Binding of a polymer to a protein (DNA-histone complex)

Histone

Basic region

Acidic region

Nonpolar region

DNA + Histone DNA-histone complex

DNA strands wrap around proteins called histones

There are four types of histones, named: H2A, H2B, H3, and H4.

Spin Labeling

N H₃C

H₃C CH₃

CH₃ O

X

H₃C N

H₃C CH₃

CH₃ O

Piperidine ring

X

Pyrrolidine ring

Nitroxide group

The amount of broadening line decreases

α

Distance between the unpaired electrons¹ and the nucleus of interest increases

≤ 40 A ^o

H O

OH

OH

H H

NH H

OH H₂C

H

C O CH₃ OH

N-acetyl- -D-glucosamine (GlcNAc)

H O

OH

OH

H H

NH H

OR

H₂C

H

C O CH₃ OH

R= ^{O CH}

CH₃ CH₃

Nacetyl muramic acid (Mur2Ac)

Amino acids

Labeled Histidine-15 residue

MRI (Magnetic Resonance Imaging)

Fourier Transform NMR

Absorb photon (excitation)

Emit photon (relaxation)

Free induction decay

Fourier transform