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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:
• 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.