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Ringvorlesung Bioorganische Chemie

Biomolekulare NMR-Spektroskopie

Armin GeyerFachbereich ChemiePhilipps-Universität

04. Feb. 20052

InhaltInhalt

1. Stunde (Studenten der Medizin, Folien 1-21)Wieso NMR ? Interpretation von NMR-Spektren Analyse von empfindlichen MolekülenFingerabdruck: Analyse von Molekülgemischen, von empfindlichen Molekülen in Gemischen (Cholesterin im Blut) Das NMR-Signal: FID (free induction decay), AudiofrequenzenStrukturaufklärung, 2D NMR Signale, große Moleküle

2. Stunde (Studenten der Chemie, Folien 22-64)Multinukleare NMR-Spektroskopie an Proteinen, Struktur + Dynamik = Funktion, Moleküle im Kristall und in Lösung, Cyclosporin A, Calonyctin A, Schleifenstrukturen in Proteinen, Peptidmimetika

Ringvorlesung Bioorganische Chemie

Biomolekulare NMR-Spektroskopie

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Wieso NMR ?

A proton has achemical shift, an intensity, and a multiplicity.

NMR observables depend on molecular size and shape.

NMR methods observe the ligand and/or the receptor. Isotope labeling in the case of large molecules.

11H NMR:H NMR:Even strong

magnetic fields cannot resolve

all proton resonances of

a large molecule

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Interpretation von NMRInterpretation von NMR--SpektrenSpektrenZwei grundverschiedene Vorgehensweisen zu Analyse von Spektren

2. Strukturaufklärung: Jedem Signal im Spektrum wird ein Atom des Moleküls zugeordnet. So können bisher unbekannte Moleküle identifiziert werden.

Wie eine Datenbank von Fingerabdrücken können bereits bekannte Moleküle identifiziert werden.

1. Fingerabdruck: Das Spektrum identifiziert eindeutig eine Molekülspezies.

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Analyse von empfindlichen MolekülenAnalyse von empfindlichen MolekülenFingerabdruck

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Ladungsmuster und Immunstimulation

Lipoteichonsäure (LTA)

Analyse von empfindlichen MolekülenAnalyse von empfindlichen MolekülenFingerabdruck und Strukturanalyse S. Morath, A. Geyer, T. Hartung.

Structure-function relationship of cytokine induction by lipoteichoic acid from Staphylococcus aureus.J. Exp. Med. 2001, 193, 393-397.

70% D-Ala

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Analyse von empfindlichen Molekülen in GemischenAnalyse von empfindlichen Molekülen in GemischenCholesterin

HDL 8-11 nmLDL 20-25 nm

VLDL 28-70 nm

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Analyse von empfindlichen Molekülen in GemischenAnalyse von empfindlichen Molekülen in GemischenCholesterin

Typical NMR Spectrum of Plasma Shown are the plasma methyl lipid signal (shaded) and a schematic representationof lipoprotein structure, depicted as a neutral lipid core ofcholesterol ester (CE) and triglyceride (TG) surrounded by ashell consisting of phospholipids (PL) and free (unesterified)cholesterol (FC).

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Analyse von empfindlichen Molekülen in GemischenAnalyse von empfindlichen Molekülen in GemischenCholesterin

HDL 8-11 nmLDL 20-25 nm

VLDL 28-70 nm

Spreizung des Methylbereiches im 1H NMR zwischen 0.5 und 1 ppm

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NMR signal: FID (free induction decay)

time axis [s]

The free induction decay detected by pulse NMR is in the kilohertz range.

The precision of +/- 1Hz on a 400 MHz spectrometer (GHz scale) can be compared tomeasuring the distance earth-moon with the precision of +/- 1 m.400 000 000 Hz NMR frequency380 000 000 m moon-earth distance

pulse

basic spectrometer frequency 400 000 000 Hz (reference)

10 µs on/off

400 004 000 Hz

signal at 10 ppm

stored signal: 4000 Hz (audio frequency)

subtraction

detector

transmitter

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The NMR-spectrum is obtained by frequency analysis of the FID. The appearance of the FID depends on the transmitter frequency.

NMR signal: FID (free induction decay)

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NMR ⇔ music

rf channels ⇔ instrumentsCα vs. CO ⇔ octavesselective pulses ⇔ notes

∆, τ ⇔ short breaks

Multi-pulse experiments

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2D Methoden

Two-dimesnional spectra resolve andidentify overlapping signals

C-H groups appear as `spots´ in the inverseinverse CHCH--correlationcorrelation

11HH dimensiondimension

1313 CC

dim

ensi

ondi

men

sion

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1D spectrum

projection

slice

2D spectra correlate two different frequencies - two spinsstacked plot contour plot

2D NMR Signale

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NMR spectroscopyNMR is different from other spectroscopic methods:The number of signals correlates with the number of atoms !

Advantage: Address every atomic position in a moleculeDisadvantage: NMR-spectra get crowded. A 10 amino acid peptide contains about 100 NMR chromophores (1H, 13C, 15N).

Cross signals in 2D spectra correlate pairs of atoms (pairs of frequencies)Example: 13C + 1H identify CHX groups in HMQC-type spectra.

Filtering of spectral informationExample: HACACONHN spectrum = magnetisation transfer from the α-proton(HA) to the α-carbon (CA) to the amide carbonyl (CO) etc...

How does this work? A combination of different frequency channels anddifferent coupling constants.

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NMR spectroscopy

Structural analysis

compositioncompositionnucleus, δ, integral

connectivityconnectivity andandstereochemistrystereochemistryJ-coupling

Conformational analysis

rotamer distributionrotamer distributionsingle and partial doublebonds

nonnon--covalent interactions covalent interactions hydrogen bondscomplexationsolvation etc...

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NOE-Spektren

Im NOE-Spektrum wird jedes Feld durch zwei Frequenzen charakterisiert: Zwei Wasserstoffatome.

Auf einem Schachbrett wird jedes Feld durch eine Zahl und einen Buchstaben charakterisiert.

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Vorgehensweise:Die Bestimmung der Abstände (Pfeile) zwischen Wasserstoffatomen (schwarze Punkte) in einem Protein liefert die Information über die dreidimensionale Struktur. Das Netzwerk von Abständen ist vergleichbar zum Bauplan für ein Haus.

NMRNMR--SpektroskopieSpektroskopieNobelpreis 2002 für K. Wüthrich

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The bovine prion protein (bPrP) is nearly identical tothe human prion protein (hPrP). K. Wüthrich et al.

PNAS 97, 2000, 8334.

The spatial structure of a protein determines its

properties

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NMR spectroscopy

Structural analysis

compositioncompositionnucleus, δ, integral

connectivityconnectivity andandstereochemistrystereochemistryJ-coupling

Conformational analysis

rotamer distributionrotamer distributionsingle and partial doublebonds

nonnon--covalent interactions covalent interactions hydrogen bondscomplexationsolvation etc...

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NOE-Netzwerke

NOE = Nuclear Overhauser EffectDie große Anzahl der Kontakte sind genug Information, um die Struktur des Proteins zu bestimmenRechts: Die schwarzen Striche sind Abstände zwischen Wasserstoffatomen, die kürzer als < 4 Å sind.

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Differences between the two prion proteins are mainly found inthe flexible loop regions.

e.g. amino acids 166-172

loop regions connect sequences which form secondary structures like α-helices and β-sheets.The β-turn (reverse turn) is the smallest and most abundant loop.

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⇒ folding

Glycosylation und phosphorylation at exposed turn structures.

⇒ post-translationalmodifications

Reverse turns are nucleation sites for theformation of α-helices in early stages of protein folding (µs). Turns or loops formjoints between secondary structural elements in native protein conformations.

⇒ proteolysis Proteolytic enzymes attack at exposedsites. Processing of peptide hormones.

A β−turn is the minimal secondary structural element. Protein evolution started from a peptide loop.

⇒ evolution

J. Jurka J. Mol. Evol. 1987, 15.

Reverse turns in proteins

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protein-protein-interactionsare the molecular fundament of many (patho)physiologicalprocesses.

Recognition processes as attractivetargets for therapheutic intervention.

Turn structures which present betweentwo and four amino acid sidechains arekommt bei der Interaktion eine besondere Bedeutung zu.

β-turn mimetics are a synthetic challenge

Molecular recognition

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Drug design: from the natural ligand towrds a synthetic drug

1. Identify the active sequencewithin the amino acid chain

3. And further steps:Exchange amino acids for non-peptide building blocks

2. Synthesis of short peptidesequences (cyclic)

Acitive sequence in the primary structure of the peptide ligand

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Integrins adhere to the extracellular matrix (protein 1 and protein 2). Different conformations of the RGD-epitope (R= Arg, G= Gly, D= Asp) result in differend binding selectivities.

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Linear RGD-peptides bind both integrins: No selectivity selectivity without a defined conformation.

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A rigid RGD-peptideselectively binds only

one integrin.

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4. A scaffold orientates functional groups

1. Nativepeptide

2. Rigid peptide

3. A templateorientates a tripeptide

H3N

NH NH3

NH

NH

ONH

O

O

NH

CO2O

NH

CO2

NO

NH2

CO2

NH

NH

H3NO

NN

OH

H H

H. KesslerTU München

NHNH3

NHO2C

S

N

O

N

O

H

N

O

H

N H

O

NH

O

NHNH3

NH

N

O

H

HN

NO

H

N H

O

NCO

HO

O2C

Stepwise modification of peptides

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Thermodynamics

affinity is a thermo-dynamic equilibrium

quantity.

A molecule which does not bind to the receptor cannot become a drug.

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No receptor is too big for NMR

... if you want to observe only the binding process

1 mM ligand (1)100 µM protein (224 kDa IMPDH)

Ki ~ 60 µM

LineLine--broadeningbroadening is semi-quantitative: T2 change and chemical shift change leads to differentbroadening for individual protons ofthe same ligand.

M. Moore, Biopolymers 1999, 221-243.

without proteinwithout protein::

with proteinwith protein::

approxapprox. 10000. 10000 protein protonsprotein protons

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Why can´t we see the protein ?Relaxation of NMR signals: matching frequencies The slow Brownian tumbling of proteins (ττcc ~ 10-9 s) matches theNMR frequency (10-9 Hz) while the fast tumbling of small molecules(ττcc ~ 10-12 s) does not.

long-lived excited statesof small molecules ⇒sharp resonancessharp resonances

short-lived excited statesof large molecules ⇒broad resonancesbroad resonances

ττcc

prot

ein

prot

ein

ligan

dlig

and

ττcc

ΤΤ22

ΤΤ22

ττcc : correlation timeaverage time to rotate about one rad

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Structure-based drug design

ligand

conformation anddynamics

`small-moleculeNMR´chemical shifts, coupling constants,hydrogen bonds,NOEs

receptor

structure of ligand-receptor complex

Protein NMRisotope labelingediting

complexation

affinities and ratesof binding

NMR titrationline-broadeningtransferred NOESTD, diffusion-based methods

angles, torsions,distances, rates

accumulate structural

information about your target

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Molecular interactions

The NMR parameters for a small molecule are very sensitive tointeractionsinteractions with other molecules.

Changes in signal positionsignal position (chemical shift), signal shapesignal shape (line-width), and NOENOE (cross-relaxation rates) are used to characterize and quantify the binding process.

Chemical shiftChemical shift:: measured with a precision of 0.1 Hz on a 103 Hz scaleLineLine widthwidth:: ligand signals broaden upon receptor bindingNOE:NOE: sign inversion of the ligand NOEs upon receptor binding

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Complex formationThree species are involved in a typicalprotein-ligand interaction, the proteinE, the ligand L, and the complex EL.

Kd = dissociation constant, koff = rate of dissociation of ligand from EL. A tight binding ligand (e.g. Kd ~ 10–9 M ) has an off rate koff ~ 10–1 s–1

⇒⇒ slow exchangeslow exchange on the NMR time scale.A weak ligand (e.g. Kd ~ 10–5 M ) has an off rate koff ~ 103 s–1

⇒⇒ fastfast exchangeexchange on the NMR time scale.

diffusion-limited on rate kon ~ 108 M-–1s–1.

kon

koffKd = =[ EL ]

[E ] [ L ]E + L EL

kon

koff

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Time scales

slow exchangeslow exchange : ∆δ » t1/2–1

fastfast exchangeexchange : ∆δ « t1/2 –1

t1/2 = half-life of state

example: signal separation of 1000 Hz ⇒ averaging of chemical shifts on the ms time scale∆δ ~ 1000 Hz ⇒ t1/2 ~ 10–3 s

coupling constants:∆ 3JH,H ~ 10 Hz ⇒ t1/2 ~ 10–1 s

NMR methods cover a wide range of ligand-receptor affinities.Slow exchangeSlow exchange:: separate chemical shifts for ligand and ligand in complex

FastFast exchangeexchange:: general formula for averaged parameter P = chemical shift,diffusion coefficient, etc.

Pobs = xfree ·Pfree+ (1 – xfree) ·Pbound

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DiffusionRotation and translation of a molecule ⇒ relaxation of the NMR signal

Rotational diffusionRotational diffusion changes the angle between a spin state (vector) and theB0 field axis and permits the separation of large and small molecules by NMR.Methods: STD, trNOE etc.

Translational diffusionTranslational diffusion changes the position of the molecule and it experiences B0 field inhomogenities. Controlled inhomogeneities (pulsed field gradients) permit a separation of large and small molecules by NMR.Methods: LED and DOSY ⇒ diffusion coefficientdiffusion coefficient

DDtt [m[m2 2 ss––11]]

select for speed ...not for spin

RotationRotationTranslation

Translation

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DOSY

HDOHDO

GlcGlc--OMeOMe

sucrosesucrose

ββ--CyclodextrinCyclodextrin

diffusion-ordered spectroscopy

11HH dimensiondimension

diff

usi

on c

oeff

icie

nt

Ddi

ffu

sion

coe

ffic

ien

t D

tt[m[m

22 ss––

11 ]]

Overlapping signalsin the 1H NMR are not resolved.A disadvantage for the analysis ofmixtures.

DOSY is fast, sensitive, and the indirect dimensionhas a good resolution.

C42

C12

C7

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2D methods

Two-dimesnional spectra resolve andidentify overlapping signals

C-H groups appear as `spots´ in the inverseinverse CHCH--correlationcorrelation

11HH dimensiondimension

1313 CC

dim

ensi

ondi

men

sion

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Multinukleare NMR-Spektroskopie an Proteinen

Mit zunehmender Molekülgröße werden − Signalüberlagerungen immer häufiger und − die Relaxationszeiten immer kürzer.

Beide Nachteile lassen sich durch heteronukleare multidimensionale Techniken bis zu gewissen Grenzen zurückdrängen. Dazu braucht man vollständig mit 13C und 15N angereicherte Substanzen. Diese gewinnt man durch Verfütterung von 13C6-Glucose und 15NH4Cl an Mikroorganismen (z.B. E.coli),

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HNCO Spectrum of Human UbiquitinA standard compound for testing 3D NMR is human ubiquitin, a 76 residue protein (8.5 kDa).The experimental 600 MHz spectra below of 0.2 mM 15N and 13C labelled ubiquitin (90% H2O/10% D2O, pH 5.8, 20oC) are a courtesy of George Gray, Varian, Inc. The HNCO triple resonance experiment is illustrated in the figure on the left. The HNCO data was acquired in 1 h 13 min with ProteinPack's ghn_co pulse sequence (32 x 16 x 512 phase sensitive points, two transients per increment, zerofilled to 128 x 64 x 512 points).

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Molekülspektroskopie

Woher wissen wir, wie ein Molekül aussieht?Wie kann man Abmessungen im Bereich von 10-10 m bestimmen?Wie kann man die Funktion eines Moleküls charakterisieren?

Es gibt zwei Strategien:Im Festkörper... ...oder in Lösung

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Spektroskopische Strukturaufklärung

Kristallstrukturanalyse:Kristallstrukturanalyse:Organische Moleküle bilden Kristalle und können dann exakt vermessen werden.Nachteil: Dichteste Packung, keine Aussage über die Beweglichkeit.

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Spektroskopische Strukturaufklärung

Kernresonanzspektroskopie,Kernresonanzspektroskopie,NMRNMR--Spektroskopie :Spektroskopie :Die Moleküle werden im gelösten Zustand charakterisiert.

Schwierigkeit: Das dynamische Verhalten führt zu einer Mittelung (verschmieren) der Messdaten.Eine Zeitachse kommt ins Spiel.

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Struktur + Dynamik = Funktion

Bildhafte Darstellung von BewegungAbmessungen, Frequenz, Amplitude?

M. Duchampnude No 2

G. Balla, dog on a leash

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Correlated motion

function:walking

feet:functional movement

ears, tail:random

Giacomo Balla : Dynamism of a Dog on a Leash 1912

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Molekulare Maschinen

Zwischen technischen Maschinen und Proteinen

bestehen prinzipielle Unterschiede:

Proteine besitzen keine glatten Oberflächen, sie haben eine dynamische

Struktur, sie sind verformbar und können

sich anpassen.

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Die Konformation kleiner Biomoleküle wird massgeblich durch die Umgebung bestimmt.

Was ist eine relevante Umgebungfür die Strukturanalyse?

Für ein Medikament ist die am Rezeptor gebundene Konformation die relevante Umgebung.

CsA ist ein cyclisches Peptid mit 11 Aminosäuren. 7 davon sind tertiäre Amide.

Die am Rezeptor gebundene Konformationunterscheidet sich von allen Lösungskonformationen.

NMRKristall

Am Rezeptor

56Blau: Wasserkanal im Kristall von Tricolorin A

Tricolorin A ist amphiphil, es hat polare und unpolare Gruppen.

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...The hydrophobic surface exposed externally and elongated along the axis of the water channel is ideally oriented for parallelinteraction with the lipids of a biological membrane upon insertion of tricolorin A into the membrane. This hypothesis differs from predictions made by molecular dynamics simulationsand NMR studies of micellar solutions of calonyctin A, anotherplant-growth-regulating resin glycoside,[18] which inserts perpendicularly into micelle lipid membranes. ...

[18] Z. H. Jiang, A. Geyer, R. R. Schmidt, Angew. Chem. 1995, 107, 2730

Die Kristallstrukturen von „großen“ Proteinen sind aussagekräftig, nicht jedoch die von „kleinen“ Biomolekülen: Die Oberfläche wächst quadratisch (m2), das Volumen kubisch (m3).Die Übertragung eines beliebigen Ausschnitts vom Kristall in die Lösung ist eine wissenschaftlich fragwürdige Vorgehensweise.

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The plant hormone Calonyctin A

calonyction aculeatumcalonyction aculeatum(moonlight flower), China

Calonyctin A is insoluble in water.

Z. H. Jiang, A. Geyer, R. R. Schmidt, Angew. Chem. 1995, 107, 2730

Calonyctin A is a plant growth hormone

There are only very few cyclic glycolipids in nature.What is the relative orientation between the oligosaccharide moiety and the alkyl chain?

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Calonyctin A text

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Quantification of NOEstext

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Precision

Interproton distances [pm]within the pyranose rings of the β-quinovoses (4C1-chair) and the α-rhamnose(1C4-chair) in Calonyctin A.

The distances in columns 4C1 and 1C4 are average values from 50 ps molecular dynamics simulations ofthe isolated methylglycosides without NOE distance restraints. Interproton distances of rings a - dof Calonyctin A were extracted froma NOESY spectrum. Due to signal overlap only three NOEs could be extracted for Qui a. The average deviation (∆) of the experimentaldistances and the calculated average distances are given below.

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Calonyctin AThe NOE network (red) spans the entire tetrasaccharide moiety ofcalonyctin A (blue).

The experimental NOEs were included as weak forces into the molecular dynamics simulation of Calonyctin A.

Weak Hook potentials about average values.

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Calonyctin AThe alkyl chain further stabilizes the main conformation of the tetrasaccharide moiety by excluding secondary conformers.

10 snapshots from a 100 ps molecular dynamics simulation. Conformational

dynamics of the fatty acid chain.