Biophotonics - nanoimaging.de€¦ · Biophotonics Lecture #2, 2013 Building Blocks of Biological Material Prof. Dr. Stefan H. Heinemann Zentrum für molekulare Biomedizin, CMB Lehrstuhl

Biophotonics Lecture #2, 2013

Building Blocks of Biological Material

Prof. Dr. Stefan H. Heinemann

Zentrum für molekulare Biomedizin, CMB Lehrstuhl für Biophysik

FSU Jena Hans-Knöll-Straße 2

D-07745 Jena

This lecture presents modern methods in spectroscopy and

imaging dedicated to biological samples. The biological part

introduces to molecular and cellular properties of living organisms,

explains some major components of physiological function and

diseases and sets the stage for biophotonics applications by

highlighting some key methods necessary to prepare biological

material for photonics experiments and by showing several

examples of how biophotonics can help to shed light on biologically

and clinically relevant processes.

Script available at:

http://www.biophysik.uni-jena.de/ Lehrveranstaltungen

user: Student

passw: Biophysik13

Aim

From genome to organism

Genome

FISH

chromosomes

Proteome

Cells Organs

Organism

Overview “Omics”

Organism, Taxonomy

Man, animal, plant, …

Genetic fingerprint: Genome

Organ, tissue, cell:

Expressed genes (mRNA)

Site-specific snapshot of active

genome: Transcriptome

Translated proteins All active proteins: Proteome

(including posttranslational modifications)

Low-molecular weight

mediators (energy, signals)

Products and effectors of the metabolic

pathways: Metabolome

Communication and

signaling

Exchange of information within and

between cells / organisms

Something goes wrong Diseases / Medicine

Building blocks of biological material

• Introduction

• Polymers

• DNA / RNA

• Peptides, Proteins Protein folding, 3D structure Folds, domains and protein complexes

Posttranslational modification Special-purpose proteins: prosthetic groups

Pigments and fluorescent proteins

• Polysaccharides

• Lipids and Membranes

A prototypical cell: some dimensions

Axon,up to 1 m

5 μm

Mitochondrion, 1 μm

Membrane thickness, 5 nm

Vesicles, 50 nm

30 μm Cell body, soma

Nucleus

Length and time scales

Length and time scales

Molecules in a cell

Targets for light-matter interactions

% of total weight

About 70% water and 26% macromolecules: rest = 4%

Simple bacterium, e.g. E. coli: Human cell:

>1,000 > 100,000 different types of molecules

Macromolecules, polymers

Guanine Thymine Adenine Cytosine Adenine

Nucleic acid Nucleotides

Tyrosine Alanine Leucine Aspartate Proline

Protein Amino acids

Glucose Mannose Galactose

Fructose Glucose

Mannose

Polysaccharide Sugars

Central dogma of molecular biology

DNA

Replication

mRNA

Transcription

Protein

Translation

Nucleus Cytosol

Ribosomes

Information

Firmware

Function

Hardware

Nucleic acids: storage of genetic information

DNA: deoxyribonucleic acid (nucleus), 2 strands

DNA has 4 letters: GTAC

4n possibilities, n = number of bases or base pairs

RNA: ribonucleic acid ( m = messenger, t = transfer, r = ribosomal), 1 strand

U(racil) instead of T(hymine)

Human genome: 3 billion base pairs = 3 Gbp

3 bases (triplet, codon) determine an amino acid in a protein: genetic code

Gene: sequence Protein: function

Human genome: about 22,000 genes, i.e. only 2% of the entire sequence

Genes (consisting of exons) are assembled by gene splicing,

introns are removed: alternative splicing = increased variability

Guanine Thymine Adenine Cytosine

Nucleotides

N

N

H2N

N

N

O

Guanine, G

O N

HN

OCH3

Uracil, U

Thymine, T

O

OH

H HH H

CH2

1

23

4

5

OPO2–33 end

NH2

O N

NCH3

Cytosine, C

methylated

Deoxyribose

Ribose

pyrimidine

O

OH

H HH H

HOCH2

1

23

4

5

O

P

O

–O O

5 end

N

N

NH2

N

NAdenine, A

Phosphodiester

bond

purine

Nucleic acids: structure

Nucleotide = phosphate + sugar + base (variable)

Major groove

Minor groove

3.4 nm

2 nm

A

T

T

C

C

G

G

ADeoxyribose

PhosphateBases with

hydrogen bondsA B

Base pairing: double-stranded DNA

Base pairing ensures producing unambiguous complementary copies.

Condensation of DNA

Human genome:

1) DNA double strand 1 m

Extendend DNA per chromosome 50 mm

2+3) Chromatin: DNA – histone complexes 1 mm

5) Chromosome: folded chromatin 5 m

22 human chromosomes + sex chromosomes (X,Y)

FISH nucleus

DNA stability and mutations

DNA has to be extremely stable as it holds the blueprint information for the

entire orgamism – for the entire life.

Very many DNA modifications happen during a day:

e.g. single-strand breaks 50,000; double-strand breaks much harder to repair

Specific repair systems reduce the errors substantially.

“Surviving“ DNA changes give rise to mutations

Germ cells evolution Somatic cells often cancer

Deviations from “normal“ genomic sequence: SNP (single-nucleotide polymorphism)

Biophotonics: UV light (directly or via reactive species) can damage DNA!

Peptides & proteins

Proteins are the working horses of the orgamism. Small proteins are called peptides.

20n possibilities, n = number of residues. Example: n = 10 1013 possibilities!

Many proteins have more than 1000 residues - basically infinite variability possible.

Normally protein sequences have 20 letters: amino acids

Tyrosine Alanine Leucine Aspartate Proline

Protein Amino acids

DNA sequences are translated into protein sequences in two steps:

1) Production of a complementary copy: mRNA 2) Translation of this mRNA sequence, triplet by triplet, into a polypeptide

by means of the Genetic Code

Purpose: Enzymes (catalytic function)

Structural material Energy harvesting, consuming

Communication, …

UUUUUCUUAUUG

Phe

Leu

UCUUCCUCAUCG

Ser

UAUUACUAAUAG

Tyr

stopstop

UGUUGCUGAUGG

Cys

stopTrp

CUUCUCCUACUG

Leu

CCUCCCCCACCG

Pro

CAUCACCAACAG

His

Gln

CGUCGCCGACGG

Arg

AUUAUCAUAAUG

Ile

Met

ACUACCACAACG

Thr

AAUAACAAAAAG

Asn

Lys

AGUAGCAGAAGG

Ser

Arg

GUUGUCGUAGUG

Val

GCUGCCGCAGCG

Ala

GAUGACGAAGAG

Asp

Glu

GGUGGCGGAGGG

Gly

UCAGUCAGUCAGUCAG

U C A G

U

C

A

G

Pos 1 Pos 2 Pos 3

Genetic code

Translation starts at

“ATG“.

Uniqueness ensures in-frame reading

degenerate:

not every DNA mutation

codes for protein mutation. (silent mutation)

unique

Amino acids: structure

Typically proteins only consist of 5 elements: C, H, O, N, S

(As we will see later, some also harbor Se and many P.)

There are different codes:

Full name: Tyrosine 3-letter code: Tyr

single-letter code: Y

There are many ways of classifying amino acids:

Charge Size (mass)

Volume Hydrophilicity

Note: 1 Dalton (1 Da or 1 D) is the mass of 1/12 of a 12C atom = 1.66 10-27 kg.

Useful unit in biology: kD = 1,000 Dalton.

Amino acids: non-polar

C H

COO–

NH3

H+

GlycineGly, G57.0 D

C CH3

COO–

NH3

H+

AlanineAla, A71.1 D

C

COO–

NH3

H+

ValineVal, V99.1 D CH

CH3

CH3

C CH2

COO–

NH3

H+

LeucineLeu, L113.2 D CH

CH3

CH3

CH

COO–

NH3+

IsoleucineIle, I113.2 D C* CH2 CH3

CH3

H

C CH2

COO–

NH3

H+

MethionineMet, M131.2 D CH2 S CH3

C CH2

COO–

NH3

H+

PhenylalaninePhe, F147.2 D

C CH2

COO–

NH3

H+

TryptophanTrp, W186.2 D

NH

CH2

CH2

CH2

CCOO–

NH+

ProlinePro, P97.1 D

H2

Molar extinction: 220/M/cm at 257 nm

Molar extinction: 5,050/M/cm at 280 nm

Note: Maximal absorption of DNA at 260 nm. Ratio of absorbance 260 nm / 280 nm provides estimate for purity of DNA/RNA sample.

Amino acids: polar & uncharged

C CH2

COO–

NH3

H+

SerineSer, S87.1 D OH

C

COO–

NH3

H+

ThreonineThr, T101.1 D C* CH3

H

OH

C CH2

COO–

NH3

H+

AsparagineAsn, N114.1 D C

O

NH2

CH2C

COO–

NH3

H+

CysteineCys, C103.1 DpKR = 8.37

SH

C CH2

COO–

NH3

H+

TyrosineTyr, Y163.2 DpKR = 10.46

OH

C CH2

COO–

NH3

H+

GlutamineGln, Q128.1 D CH2 C

O

NH2

Molar extinction: 1,440/M/cm at 274 nm

Note: Absorption sequence: Trp > Tyr > Phe

Amino acids: charged

C CH2

COO–

NH3

H+

Aspartic acidAsp, D115.1 DpKR = 3.90

CO

O–

C CH2

COO–

NH3

H+

Glutamic acidGlu, E129.1 DpKR = 4.07

CO

O–

CH2

Negatively charged

C CH2

COO–

NH3

H+

HistidineHis, H137.1 DpKR = 6.04 N

H

NH+

C CH2

COO–

NH3

H+

LysineLys, K128.2 DpKR = 10.54

CH2 CH2 CH2 NH3+

C CH2

COO–

NH3

H+

ArginineArg, R156.2 DpKR = 12.48

CH2 CH2 NH CNH2

NH2+

Positively charged

Positively charged, pH dependent

Peptide bond

Dihedral angles determine the overall structure

C-CO-N-H: backbone

Side chains, residues

N-terminus C-terminus

Secondary structural elements

-helix -sheet coiled coil -hairpin

turn

Structural levels

Protein structure:

primary - sequence of amino acids

secondary - structural elements ( -helix etc.) tertiary - 3D structure of protein unit

quaternary - 3D structure of protein complexes

Trypsin

ball & stick cartoon space-filling, surface

Hemoglobin

Protein stabilization

Salt bridges Asp – Lys, Glu – Arg, …, His – (pH dependent)

Hydrogen bonds …H… van der Waals forces hydrophobic forces

Stabilizing metal ions Ca2+ (mostly dynamic), Zn2+ (mostly stable), …

Disulfide bridges Cys – Cys (redox regulated)

Protein folds

TIM barrel

Immunoglobin

http://xray.bmc.uu.se/~lars/biowww/Proteinfolds.html

a/b domains

Rossmann fold

Jelly roll

Of the nearly unlimited number of theoretical protein folds

only a subset (>1,000) actually occurs in nature.

Domains

Lacking information on 3D structures, proteins are often depicted as an

array of structural (often based on homology) or functional domains.

Functional domains of phospholipase 1 (PLC 1):

PH, Pleckstrin homology domain; EF, Ca2+-binding EF-hand domains; SH, Src homology domains; C2, Ca2+ dependent phospholipid binding domain.

Posttranslational modification

Covalent modification of amino acids during or after translation tremendously

increases protein variability and makes proteins “tunable“. This is a very important prerequisite for cell signaling.

Some prominent examples: cleavage (peptidases, not shown)

phosphorylation

acetylation

oxidation

nitration

Prosthetic groups

Many proteins acquire specific function by incorporating small molecules,

so-called prosthetic groups. Many are very important for biophotonics, as they often strongly interact with light.

Rhodopsin with light-harvesting

complex retinal (green).

Prosthetic groups

Many proteins acquire specific function by incorporating small molecules,

so-called prosthetic groups. Many are very important for biophotonics, as they often strongly interact with light.

Hemoglobin with O2- or CO-binding heme group (Fe2+-porphyrine)

Living colors

Some proteins (in particular from jelly fish) acquire fluorescence properties

upon posttranslational modification. Most prominent example: Green Fluorescent Protein (GFP)

http://gfp.conncoll.edu/cooluses0.html

Available for many different wavelengths

Ser-Tyr-Gly.

The resulting conjugated double

bonds are fluorescent with an ideal excitation at 400 nm and

an emission at 508 nm (green).

Carbohydrates

Carbohydrates or saccharides: polymers of monosaccharides

Glucose Mannose Galactose

Fructose Glucose

Mannose

Polysaccharide Sugars

Elements: C, H, O. Structurally very complex.

Purpose: Energy source

Structural material Part of protein/lipid: signaling & recognition

H3

OH

OH2

HOH

1

H

HO4

H H

5

CH2OH6

O

OH4

H

H3

OHH5

HOH2C6

OH2

1CH2OH

O

-D-glucopyranose

-D-fructofuranose

Cyclization

H1

OH

CH2OH2

1

OH

Anomers

CH

1

O

CH OH2

CHO H3

CH OH4

CH OH5

CH2OH6

CH2OH1

C=O2

CHO H3

CH OH4

CH OH5

CH2OH6

D-glucose

D-fructose

Epimers

CH

1

O

CHO H2

CHO H3

CH OH4

CH OH5

CH2OH6

D-mannose

Ald

oh

exo

se

Nomenclature of monosaccharides

Number of carbons determines name: triose, tetrose, pentose, hexose, heptose

CH2OH1

C=O2

CH OH3

CH OH4

CH OH5

CH2OH6

D-psicose

Keto

hexo

se

Nature of carbonyl: aldehyde = aldose, ketone = ketose

Di- and polysaccharides

Glucose Mannose Galactose

Fructose Glucose

Mannose

Polysaccharide Sugars

Su

cro

se

H3

OH

OH2

HHO

4

H H

5

CH2OH6

OH3

H

H4

OH2

OH

5

6CH2OH

OHOCH21

O1

HO

D-glucose D-fructose

D-glucose D-glucose

Ce

llu

los

e

H3

OH

OH2

HH

14

H H

5

CH2OH6

O

H3

OH

OH2

HH

14

H H

5

CH2OH6

O

O O

n

N-linked

H

OH

NHCCH3

H

O

HO

H H

CH2OH

=

H

O CCH2

C=O

NH

HCO

NH

Protein glycosylation

Asn

O-linked

H

OH

NHCCH3

H

O

H

HO H

CH2OH

=

HO

O CCH2

C=O

NH

H

Ser

Thr

Sialic acid

Galactose

Mannose

GlcNAc

Peptide determined by genome

great statistical variation

GlcNAc = N-acetylglucosamine

Lipids & membranes

Lipids do not polymerize.

They form large complex by means of self-aggregation.

Purpose: Energy source (fat)

Formation of membranes (electrical insulators, diffusion barriers)

Scaffolds for biochemical reactions Signaling molecules

Extremely diverse group of molecules.

Those forming membranes: fatty acids, triacylglycerols, glycerophospholipids,

sphingolipids, cholesterol

Typically: Hydrophobic tail + polar headgroup

O

P

O

O–O

CH2 CH CH2

O

OCCH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH3

O

OCCH2

CH2

CH2

CH2

CH2

CH2

CH2

CHCH

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH3

N(CH3)3

CH2

CH2Choline

Phosphate

GlycerolF

atty

aci

d c

hai

n 1

Fat

ty a

cid

ch

ain

2

Po

lar

head

gro

up

No

np

ola

r ta

ils

PLA1 PLA2

PLC

PLD

Ethanolamine

CH2

CH2

NH3

Serine

COO–

CH2

CH2H

NH3

Choline

CH3

CH2

CH2

NH3CCH3

Inositol

H

OH

OH

HHO

H H

OH

R

H

H

OH

R

R

R

Phospholipids

Phosphatidylcholine

O

P

O

N(CH3)3

O–O

CH2

CH2

CH

OH

CH CH2

CH

CHCH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH3

NH

OCCH2

CH2

CH2

CH2

CH2

CH2

CH2

CHCH

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH3

Sp

hin

go

sin

e

Fat

ty a

cid

ch

ain

2

Sphingomyeline

Polar headgroup

Hydrophobic tail

4-6 nm

Micelle

Vesicle

Bilayer

Selfaggregation, membranes

Asymmetry

Curvature

Biomembranes

Dynamic: fluid mosaic model

Protein content up to 50%

Further reading

• Heinemann, S.H., R. Schönherr, T. Hoshi. 2011. Biology.

In: J. Popp, V.V. Tuchin, A. Chiou, S.H. Heinemann (edts), Handbook of Biophotonics, Vol. 1: Basics and Techniques,

WILEY-VCH Verlag & Co. KGaA, Weinheim, p. 489–648

• Voltage-Gated Ion Channels as Drug Targets. WILEY-VCH.

Weinheim 2006. Edts: Triggle, Gopalakrishnan, Rampe, Zheng

• Ion Channels: Molecules in Action. The Rockefeller University Press. 1996. Aidley, J., Stanfield, P.R.

• Ion Channels of Excitable Membranes, 3rd Ed. Sinauer, Sunderland. 2001. Hille, B.

• Ion Channels and Disease. Academic Press, San Diego, 2000,

Ashcroft, F.M.