How most People Know LSU LSU Chem Images Macro Lab SAACS Meet & Greet.

How most People Know LSU

LSU Chem Images

MacroLab

SAACS Meet & Greet

LSU Chem Images

Team ExamTeam Meeting

Grad Studentsand REU’s in San Diego

STSCPutting The P In Ph.D.

LSU Graduate Students Factoids

A NOBEL SUCCESSChemistry graduate students and postdocs from around the world meet with Nobel Laureates to discuss research, discovery, and life in general

MADELEINE JACOBS, C&EN WASHINGTON

● 127 Ph.D. students; 7 M.S. students (62 female, 72 male)

● 70 US Citizens: 32 African Americans; 2 Hispanic Americans; 1 Asian American

● 64 International Students from 20 different countries: 1 (Benin), 1 (Bulgaria), 21 (China), 3 (Cyprus), 1 (France), 1 (Ghana), 4 (India), 1 (Iran), 9 (Kenya), 4 (Korea), 3 (Mexico), 1 (Nicaragua), 1 (Philippines), 3 (Romania), 2 (Saudi Arabia), 1 (Senegal), 1 (Sierra Leone),

1 (Tanzania), 4 (Turkey), 1 (Vietnam)

● 15 special State Fellowship Holders

● 6 NSF and 3 NIH Fellows● Former students employed at: Companies (e.g., ExxonMobil, Dow, Dupont, Abbott, Air Products, GE, Syngenta, Proctor & Gamble, Pfizer, Ferro, Martin Marietta, Chevron) National & Private Research Labs (e.g., NIST, Pacific Northwest National Lab, Fox-Chase Cancer Center) Colleges & Universities (e.g., Mississippi State, Fayetteville State, Houston Baptist, Univ. of Texas Pan-Am, Many International)

LUNCHTIME U.S. delegates (from left) Vernessa M. Edwards, Robin Macaluso, and Michael W. Blair took a break from the meeting.

Enough about LSULet’s Talk about

Undergraduate Student Research

• Ask and ye shall receive.• NSF REU Sites (many of them all across

the U.S., including several at LSU). • Just Google NSF REU or send me an e-mail:

[email protected]

• Private non-profit (e.g., Howard Hughes)• Industrial (many companies)

Graduate Studies• Are you Ph.D. material? • Heavily subsidized. Rumors of

starving graduate students are not true.

• Does it pay off? • In Chemistry?

– Why not Chemical Engineering?– Why not Polymer Science or Materials

Science?– Why not MD-PHD?– Why not Interdisciplinary?

IGERT

• Integrative Graduate Education Research Training.• All areas supported by NSF…including new areas

NSF didn’t think of yet. • 130 sites nationwide, including LSU.• All are interdisciplinary, well-equipped, global,

experiments in graduate education reform.• Learn to work independently and in teams. • Emphasis on creativity and/or entrepreneurship. • $30,000 minimum stipend plus tuition. • Macromolecular IGERT’s: LSU, USM, VT

www.igert.org or igert.LSU.edu

Macromolecules for The Demented

and methods for their studyHelp from Keunok Yu, Jirun Sun, Bethany Lyles, George Newkome and LSU’s Alz-Hammer’s Research Team

Krispy Kreme Donut Day, September 2003Supported by National Institutes of Health-AG, NSF-DMR and NSF-IGERT

• How Alzheimer’s happens• Attempts to prevent or reverse it• Characterization challenges• Alzheimer’s model systems with materials implications

Amyloid Diseases

• Several diseases are caused by the misfolding of proteins into self-associating structures (fibrils) w/ predominantly -sheet secondary structure

• Alzheimer’s Disease: Amyloid -protein (Ain neurons/brain

• Type II Diabetes: amylin in Islets of Langerhans• Mad Cow Disease (BSE): PrpSc in brain --

transmittable by protein aggregates• Huntington’s Disease, triplet repeat expansion:

(Gln)n

PET images courtesy of the Alzheimer's Disease Education and Referral Center/National Institute on Aging; Postmortem images

courtesy of Edward C. Klatt, Florida State University College of Medicine

Positron emission tomographyAge: 20 -- 80 Normal -- 80 AD

Postmortem Coronal Sections

NormalAlzheimer’s

http://www.bmb.leeds.ac.uk/staff/nmh/amy.html

APP = Amyloid Precursor Protein

APP = the larger, lighter pink one

•Transmembrane protein•Normal function not known•Educated guesses

May help stem cells develop identityOr help relocate cells to final locationMay “mature” cells into structural typeMay protect brain cells from injurySynaptic actionCopper homeostasis

•Anyway, you need it.•Normal “clipping” of APP by a “secretase” enzyme (in red, and also assumed to be a transmembrane protein) is shown.•There are several secretases, also associated proteins, and they seem to mutate easily: there is a genetic link. •It is not exactly clear why things go awry with advanced age.

Unlike the α-secretase enzyme, which cuts APP into shorter protein fragments in the cell's cytoplasm, the γ-secretase cleaves its target within the hydrophobic membrane of the cell. The transmembrane proteins called presenilins permit access to the γ site on APP. Mutations in the genes coding for presenilins are frequent causes for autosomal dominant Alzheimer's disease.

NH2 terminus

Clipping APP the right & wrong ways

Feature article by Vernon M. IngramAmerican Scientist on-Line

Vol. 91, #4 July-August 2003http://www.americanscientist.org/template/IssueTOC/issue/394

Correct Incorrect

Feature article by Vernon M. Ingram

American Scientist on-LineVol. 91, #4 July-August 2003

http://www.americanscientist.org/template/IssueTOC/issue/394

Figure 6. The Aβ1-42 protein fragment is exported from the cell immediately after being cut from the parent APP molecule. Once it reaches the extracellular space, the peptide refolds to form a sticky shape that clumps together as an insoluble aggregate.

Exporting the dangerous fragments

Amyloid hypothesis: fibrils or protofibrils cause cell death, possibly as the body’s own defenses tries to

clear such “foreign” matter.

Peter Lansbury Grouphttp://focus.hms.harvard.edu/1998/June4_1998/neuro.html

Competing hypothesis: channel formation disrupts Ca+2 metabolism

10 mm x 10 mm scanning probe microscope images (on mica) of 300 mM A10–35 incubated for 8 days at room temperature in 15 mM phosphate buffer containing 50 mM salt.

Introduction of Varying Salts to Increase β-amyloid Aggregation, A 10-35

NaCl NaNO3 NaF

Alz-Hammer’s Team goal:Mediate the Aggregation of A

R = regularH = hydrophobic

H-(Lys)-Val-Leu-Phe-Phe-(Lys)6-NH2

Peptide-based Mediation Requires a Specific Sequence

Hydrophobic KLVFF region is responsible for β-amyloid aggregation

Incorporation of such region for β-sheet breaking or capping

A peptide construct incorporating the KLVFF region developed by Professor Regina Murphy at the University of Wisconsin-Madison

Peptide-based Inhibitors of A Fibrillogenesis

H

HN N

H

O

O H

HN N

H

H O

O H

HN NH2

H O

H3NO

H

H3N

H-Lys-Leu-Val-Phe-Phe-(Lys)6-NH2

6

Murphy

NH3

H

NH3NH

O

OH

HNN

H

HO

OH

HNH2N

HO

H-DLeu-DPhe-DLeu-DArg-DArg-NH2

Norstedt

HN

HN

H2NNH2

NH2H2N

H

N NH

O

O H

N NH

H O

O H

N NH

H

CH3

O

H3NO

H

H3N

H-Lys-(Me)Leu-Val-(Me)Phe-Phe-(Me)Ala-Glu-NH2

Meredith

CH3 CH3 CH3

O

NH2

H

CO2

A16-20 core "Disrupter" A16-22 core

"Disrupter"

A core analog"Disrupter"?

LSU Peptide-based Mediators

AMY-1 x = 1, y = 6AMY-2 x = 6, y = 1AMY-3 x = 1, y = 1

Mediators Developed by Professor Robert Hammer & Professor Mark McLaughlin

MCP 1

MCP 2

K L V F F

K L V F F

H2NO

OHH2N

O

OH

Dbzg Dibg

Synthesis & Vetting of Peptide with AA-Blocker

HPLC of crude peptide

MALDI-MS of purified peptideCalc’d for (M + Na) = 1731.3

H-Lys-Dibg-Val-Dbzg-Phe-Dpg-(Lys)6-NH2

iBu

N

HN

iBu

O

O Bn

N

HN

HHV

Bn

O

O Pr

N NH

HHF

Pr

O

H2NO

HK H

O

NH2

HK

( )6

Determining Mediator Efficacy Using Transmission Electron Microscopy

Control50 mM A1-40

4.5 months

50 mM A1-40

50 mM AMY-14.5 months

TEM image after 4.5 months at Room Temperature50 mM phosphate buffer/ 150 mM NaCl pH 7.4

10 10 MM 10 10 MM 10 10 MM

50 mM A1-40

5 mM AMY-14.5 months

Control 1:1 A:Inhibitor

10:1 A:Inhibitor

Even a sub-stoichiometric amount of AMY-1 inhibitor is effective

But such limited success is very after-the-fact.

Can we use diffusion-based and other methods to determine the early stages of aggregation?

Can we follow it in real-time in vitro? Two Possible choices:• Dynamic light scattering • Fluorescence photobleaching

recovery

0 10000 20000 30000 40000 50000 60000 700000

10000

20000

30000

40000

50000

-Amyloid1-40 incubated in 100% DMSO followed by dilution in PBS pH 7.4

Addition of 5L of 500M Murphy Peptide dissolved in water

Sonicate in water bath for 10 mins with probe sonicator

R h

,app /

Å

Time/s

A series of dynamic light scattering runs can identify a peptide that has an effect on large fibrils.

That’s OK for simple screening, but there are problems with

DLS 1) the size is only an apparent value,

because of the single angle used for measurement;

2) the presence of small protofibrils, and the effect of inhibitors on them, is difficult to ascertain, especially in the presence of larger fibrils that dominate the scattering;

3) reversibility is not easily studied; and,4) experimentally tedious for early stages

of aggregation.

Modulation FPR Device Lanni & Ware, Rev. Sci. Instrum. 1982

*

*

*

*

AOM

M

M

D

RR

DM

OBJ

S

PMT

PA

SCOPE

TA/PVD

ARGON ION LASER

* = computer link

IF

X

c

5-10% bleach depth

Cue The Movie

H2N-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIAL-COOH

Labeling β-Amyloid fragment 1-43

Sticht, H.; Bayer, P.; Willbold, D.; Dames, S.; Hilbich, C.; Beyreuther, K.; Frank, R.; Rösch, P.Eur. J. Biochem. 1995, 233, 293-298.

When fluorescein is attached, we call it L-A

Fluorescein has about 7% the mass of

A

Diffusion Results – Great Reproducibility But Dye Shrinks it…and may stabilize against

aggregation.

100 μM Mixture β-amyloid1-40 in phosphate buffer – pH 2.7, 6.9 and 11

0 5 10 15 20 25 30 350.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0D

/10-6

cm

2 s-1

Days

pH 2.7 pH 6.9 pH 11

Theoretical/Experimental D0 for monomeric β-amyloid1-40

Sodium Fluorescein Dye

Theory/Experimental result for monomeric A1-40 from: Massi, F.; Peng, J.W.; Lee, J.P.; Straub, J.E. Stimulation Study of the Structure and Dynamics of the Alzheimer’s Amyloid Peptide Congener in Solution. Biophysical Journal 2001, 80, 31-44.

0 25 50 75 100 125 150 175 2000.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0100 M -Amyloid

1-40 in PBS, pH 7.4

D/1

0-9 c

m2 s-1

Ionic strength/ mM

Back to DLS: L-A does not prevent formation of large fibrils when mixed with unlabeled material and fibrils increase in size with added salt.

Mixed labeled & unlabled

Epifluorescence also shows L-A is actually incorporated into

macrofibers.

Bottom Line: we think L-A is OK to study.

1 10 100 1000

1E-3

0.01

0.1

1

Co

ntr

ast

/ A

rbitr

ary

t/s

pH 2.7 pH 6.9 pH 11

Two FPR Contrast Decay Modes are Often Observed: Fast = small; Slow = large.

Doing More Experiments Faster with Less Precious Amyloid:

Dialysis FPR

Cover slip

PTFE spacer Dialysis membraneO-ring

Sample

Exchange Fluid

Pump

0 20 40 60 80 100 120 140

0.0

0.5

1.0

1.5

2.0

2.5FPR of 50M labeled -amyloid

1-40 in 10 mM KOH

dialysis againt 10mM Citrate buffer pH 5.0

Amplitude 18.5% ; 18%

Amplitude 81.5% ; 82%

Diff

usi

on

Co

eff

icie

nt/ 1

0-6 c

m2 s-1

Time/min

Evolution of protofibrils from labeled monomer after dialysis against a weak citrate buffer at pH 5.0. After one hour, large aggregates appear and represent ~ 18% of the signal.

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

1E-9

1E-8

1E-7

1E-6

Diff

usio

n C

oef

ficie

nt/ c

m2s-1

Time/102 mins

Slow Fast

0.1N HCl pH1

50mM PB PH 7.2

5mM CaCl2

10mM CaCl2

15mM CaCl2

25mM CaCl2

100M Acetate buffer pH4

2 Exponential Analysis

One Pot Dialysis FPR of 50M 5CF-Amyloid1-40

in 10mM KOH

0 5 10 15 20 25 30 3505

101520253035404550556065707580859095

100

Per

cent

Am

plitu

de

Scan #

Fast Slow

Finding a convenient buffer for controlled self assembly. This run is at pH 4 Acetate Buffer. Adding calcium hastens aggregation.

Amplitudes

Diffusion from in situ FPR of 5-carboxyfluorescein-A1-40 (25% mixed with unlabeled 75% A1-40) starting at pH 11, then alternately dialyzed between 50 mM phosphate (pH 2.7) and 50 mM phosphate (pH 7.4).

0 200 400 600 800 1000 1200 1400 1600 1800

1E-8

1E-7

1E-6

FPR Study: Reversibility of -Amyloid Aggregation100M 5-CF--Amyloid

1-40+ -Amyloid

1-40 pH 11

dialysis against 50mM PB pH 7.4

dialysis against 50mM PB pH 2.7

D/1

0-6 c

m2 s-1

Time/min

Reversing Amyloid Aggregation…by pH

Bolaform amphiphiles have a dumb-bell shape.

hydrophilichydrophilic

hydrophobic

A = $400,000/gramNeed cheaper model systems.

They also have materials applications.

OH

OH

OH

NH

OH

OH

OHNH

OO

O

OH

OH

OH

NH OH

OH

OH

NH

OH

OH

OHNH

OO

O

OH

OH

OH

NH

Arborol example: [9]-10-[9]

9 watery hydroxyl groups

10 oily methylene groups

Stacked dumbbell model

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

1E-4

1E-3

0.01

0.1

1

qI(

q)

q2/ nm-2

a

b

x y

z

2N

3N

4N (back)

N

6 N+6

4 N+4

2 N+2

4N+1

4N+2

1 N+1

3 N+3

5 N+5

N-1

N+N-1

2N+6

3N+6 (back)

2N+4

3N+4 (back)

2N+2

3N+2 (back)

2N+1

3N+1 (back)

2N+3

3N+3 (back)

2N+5

3N+5 (back)

2N+(N-1)

3N+(N-1) (back)

Origin(0,0,0)

radius, r

S qM

qr

qrj j i

Mij

iji

M

( )sin( )

( )

1

21 1

P qrx

x x x( ) sin cos

33

2

I(q) = S(q)P(qr)

Based on molecular modeling, SAXS, FF-SEM, DSC, AFM, POM…and common sense.

Synthesis of Inhibitor [9]-6

Br

OOEt

NaCO

OEt

OOEt

OOEt

OOEt

OOEt

OH

OH

OH

NH2

OH

OH

OH

NH

OH

OH

OHNH

OO

O

OH

OH

OH

NH

+ DMF/Benzene

bromohexane

triethyl carbonate

DMSO

3

[9]-6

Self-assembly of [9]-12-[9]Starting point is “extruded”

fibers

0 1 2 3 4400

500

600

700

800

900

1000

1100

1200

1300

Rh/

(Å)

Number of Days

[9]-10-[9]only [9]-10-[9] plus [9]-6

Rh from linear fit of gamma vs q2 of DLS data at five angles: 40, 50, 60, 70 and 90.

Scientific Conclusions• Promising inhibitors have been designed and

constructed. Probably even more expensive than A itself.

• DLS can screen promising ones.• Dialysis FPR can observe A deconstruction in

real time. So far, only by pH, but dialysis experiments with precious inhibitor are coming.

• Model systems to practice with can teach us better methods…and have some materials science applications.

• Many things not shown: e.g., A slows diffusion of the lipids that make cell membranes. Is this important?

Broader Conclusions• Membrane proteins (or fragments) are

hard to study.• Don’t expect a cure soon and you won’t be

disappointed. • Take your statins once the doctor tells you

to start, then hope for the best. • Science in the service of practical

problems is increasingly multidisciplinary. • Scientists spend a lot more time

scratching their heads and wondering what’s going on than it must seem from textbooks.

Discussion Points

• We have spent $1.4 M for this research (so far).

• Perhaps 10 papers will appear eventually.

• About six Ph.D. students will be trained. • Could 100,000 teams like ours (that’s

2000 in every state!) cure Alzheimer’s, Mad Cow, Huntington’s and other related afflictions?

Thank you

What one thing did you learn?

What one thing do you wish you understood better?

Action Item: Find REU or similar for Summer 2005

Synthesis ( illustrated for [9]-10-[9] )

BrBr

OOEt

NaCO

OEt

OOEt

OEtO

OEtO

OEtO

OOEt

OOEt

OOEt

+DMF/Benzene

2

Hexaethylcarboxylate

1,10-dibromodecane

OH

OH

OH

NH2

OEtO

OEtO

OEtO

OOEt

OOEt

OOEt

OH

OH

OH

NH

OH

OH

OHNH

OO

O

OH

OH

OH

NH OH

OH

OH

NH

OH

OH

OHNH

OO

O

OH

OH

OH

NH

+DMSO

K2CO36

Hexaethylcarboxylate

[9]-10-[9]

APP’s normal function

• No one knows for sure• May help stem cells develop identity• Or relocate cells from birth site to normal

location• May “mature” cells into structural type

rather than neuronal• May protect brain cells from injury• Synaptic action• Copper homeostasis

Figure 4. Amyloid precursor protein (APP) can be cut harmlessly or cleaved into toxic fragments. When the full-length APP is cut at the α site (left), the resulting pieces do not harm the cell. However, when APP is cut at the β and γ sites (right), the excised fragment, known as Aβ1-42, aggregates to form toxic plaques.

Emma Skurnick

http://www.bmb.leeds.ac.uk/staff/nmh/amy.html

XXXXThe title I promised

Nadia & Sun PhotosAlzheimer’s Group Photo

Paul S. Russo

Nadia J. Edwin, U.S. Virgin Islands

Jirun Sun, China

How does FPR work?

Bleaching Diffusion

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.50.0

0.1

0.2

0.3

0.4

0.5

0.6

Y = A + B * XParameter Value Error----------------------------------A 0.01643 0.00441B 1.68536E-6 2.30047E-8----------------------------------R SD N P----------------------------------0.99981 0.0056 4 1.86263E-4----------------------------------

/Hz

K2/105 cm-2

0 50 100 150 2000

1

2

3

4

5

DC

Sig

nal

C

on

tras

t

t/s

Techniques

Fluorescence Photobleaching Recovery (FPR)Dynamic and Static Light Scattering (DLS/SLS)Diffusion Ordered Spectroscopy (DOSY)Small Angle X-ray Scattering (SAXS)Analytical Ultracentrifugation (AU)

To determine the kinetics of β-Amyloid aggregation in aqueous buffer systems (in vitro environment) for accurate studies of aggregation inhibition by synthetic peptides in such environments.

Major Goal of this Research

OutlineThe role of β-Amyloid in the onset of Alzheimer’s Disease

The β-Amyloid peptide sequence

Sample Handling Issues

- Preventing non-amyloid nucleation

- Sample preparation

Results

Summary

Future Work

What is Amyloid?

1853 – Rudolf Virchow named cerebral deposits as amyloid

Amyloid –proteinaceous aggregates associated with diseases (Alzheimer’s, Parkinson’s, spongiform encephalopathies).

β-Amyloid peptide : (39- 42 amino acids)

β-Amyloid Precursor Protein : (~ 695 amino acids)

β-APP – an inhibitory molecule that regulates the activity of proteases

Dobson, C.M. Protein misfolding, evolution and disease. Trends Biochem.Sci. 1999, 24, 329-332.

APP

Enzymes

APP- The Cause of Alzheimer’s Disease?

- Amyloid plaque

Over-active protease producing excess -amyloid? or Alzheimer’s patients simply cannot rid the brain of theaccumulating by-product?

Amyloid Hypothesis

Neurodegeneration in Alzheimer’s disease (AD) may be caused by deposition of amyloid β-peptide (Aβ) in plaques in brain tissue.

Current studies probe effects of physical conditions (differing pH, temperature, salt concentration) on Aβ aggregation.

Hardy, J.; Selkoe, D.J. Science, 2002, 297, 353-356.

Importance of Sample Preparation

• Impurities within the sample can initiate aggregation.

-autoclave pipet tips, microcentrifuge tubes

-pre-wet filters

• Introduction of “salt” induces aggregation.

-use pure materials to eliminate heavy metals

• Proper sample containment is important for creating stable stock solutions.

• Dissolve A in filtered 10 mM KOH at high pH

• Add phosphate buffered saline to filtrate

• Filter A stock solution to remove dust using 0.02 m syringe filter

• Place stock solutions within 1.5 ml microcentrifuge tubes

Preparation of Stock Solutions

Sample Preparation for Kinetic Studies

• Mix Aliquots of filtered A (both labeled and unlabeled) and buffer with appropriate ionic strength for desired sample ratio in microcentrifuge tubes

• Vortex sample

• Check pH with Micro-pH electrode from Microelectrodes, INC.

• Load samples in 0.2-mm-path-length rectangular microslide (VitroCom Inc.) by capillary, and flame-seal for FPR analysis.

Future Work

Effect of pH on fibril formation

FPR/DLS Dialysis study of FABAB1-40

Test of β-Amyloid peptide in the presence of inhibitors

Temperature study

Pressure study

Instrumental studies – DOSY, AUC, KDLS

DLS Diffusion Results

0 1 2 3 4 50

500

1000

1500

2000

Y = A + B * XParameter Value Error----------------------------------------A 70.42302 27.69701B 4.1051E-8 1.09481E-9----------------------------------------R SD N P----------------------------------------0.99823 40.65853 7 <0.0001----------------------------------------

/s-1

q2/1010cm20 1 2 3 4 5

0

1

2

3

4

5

6

7

8

Y = A + B * XParameter Value Error---------------------------------------A 4.86309 0.11086B -1.39935E-11 4.38203E-12---------------------------------------R SD N P----------------------------------------0.81915 0.16274 7 0.02417---------------------------------------

Dap

p/10

-8 c

m2 s-1

q2/1010 cm2

β-amyloid Aggregation

β-amyloid Aggregation Mediation

+ M + MM M

M

• Particulate• Metal Ion• Surface Effect

X Non-amyloid seed

Beta-strand

Fibril

Mediator

Peptides Designed by Professor Mark McLaughlin

LSU & USF Fibril Forming Cyclic Peptides

MCP 1

MCP 2

K L V F F

K L V F F

CD again shows labeled doesn’t prevent unlabeled from

aggregating

Day 1Day 3

100 M -amyloid1-40 in Phosphate Buffer (pH 7)

Note: Mixture – 50/50 FABAB

L-A is random coil, stays that wayNormal A alone makes beta sheetMixture suggests mixed conformations

Arborol self-assembly issues & materials science rationale/agenda

• Can we control self-assembly?

• Can we make an inhibitor? • Use for methods development related to

Alzheimer’s disease fibril formation? • Self-assembled structures that form lyotropic

LC’s?• Removable templates for porous media?• Removable stationary phase in separations?

Terminator

“Space” of Peptides and Proteins

C

C

O

N

H

C

C

H(C)

C

O

N

H

C

-Helix= -57°, = -47°3.6 residues/turn

1.5 Å rise/res.

310-Helix= -49°, = -26°

3 residues/turn2.0 Å rise/res.

-Sheet/extended= -135 – 180° = 113 – 180°

2 residues/repeat3.4 Å rise/res.