TIBS 17 - NOVEMBER 1992
/ii! i
0
TECHNI UES =
ELECTRON SPIN RESONANCE (ESR) has been one of the core techniques in biochemistry and biophysics for nearly three decades. It directly detects the presence of free radicals and reveals information on structure and motional dynamics at the site of one or more unpaired electrons. The field of ESR is presently undergoing considerable rejuvenation in terms of novel new techniques as well as inventive new applications. In particular, nitroxide spin labels are proving to be remarkably useful in solving m o d e r n problems. This resurgence of the use of spin labels is fueled by modern chemical and molecular biological approaches that allow the researcher to specifically place a nitroxide at points of interest in biomolecules. This review will focus on just a few of these modern uses of nitroxide spin labels. (For more detail on experimental techniques and labeling methods see Refs 1,2.) Nitroxides are stable, organic freeradical reporter groups that can be chemically attached to strategic points on biomolecules to provide detailed information on the local molecular environment. They can be used to probe local dynamics, exposure to solvent, exposure to oxygen, molecular collisions, local ordering a n d local structure. There is a large selection of different nitroxides and a typical structure and spin labeling reaction 3 is shown in Fig. 1. The unpaired electron density is mainly located in the oxygen and nitrogen orbitals. This paramagnetic site gives rise to a magnetic resonance signal that is detected, for example, by an X-band ESR spectrometer that operates at a frequency near 9.0 GHz. Coupling between the electron spin and the spin of the ~4N nucleus usually gives rise to a three-line ESR speqtrum~ Because of a fortuitous combination the local magnetic interactions from the nitroxide structure, along with the 9.0 GHz frequency, ESR spectra are extremely sensitive to rotational motion of the nitroxide group. Rotational correlation times (ZR) are readily determined from Conventional G. L. Millhauser is at the Departmentof Chemistryand Biochemistry,Universityof California, Santa Cruz, CA 95064, USA.
448
Selective placement of electron spin resonance spin labels: new structural methods for peptides and proteins
Electron spin resonance (ESR) is more powerful than ever as a technique for solving biochemical and biophysical problems. Part of the great utility of ESR arises from the use of modern biochemical methods to place spin labels at important positions along the primary sequence of a peptide or protein.
ESR spectra over the range from 0.1 ~s to 10 ps. This range has been greatly extended by modern methods including saturation transfer 4 and various electron-spin echo experiments ~,6. Spinlabel ESR can now detect motions from milliseconds to picoseconds, a range that covers nine orders of magnitude! Spin-label ESR spectra are also quite sensitive. For example, it is routine to collec t spectra from 10 pl samples containing micromolar concentrations and nanomolar concentrations can be detected with signal averaging. There are also new developments in resonator design that are greatly increasing these sensitivity factors 7. Thus, in modern biochemical research, where molecular details are the key to understanding biological function, spin label ESR is a method o f high sensitivity that offers the ready determination of local structure and local dynamics over a wide range of time scales. Over the past few years ESR has proved essential in providing insights into a range of biochemical and biophysical findings including: the flexi-
bility of DNA8, the motion of myosin head groups during muscle movement 9,1°, the impact of membrane fluidity on the rotational dynamics of membrane proteins u, the mechanism of oxygen movement through membranes ~2, the mechanism of ion channel formation from peptides ~3 and the aggregation state of peptides ~4. In concert with this, a host of new techniques are emerging and are being refined for biological applications. In addition to those mentioned above, these include: highfield ESR (250 GHz)~5, novel applications of electron-electron double resonance 16,~7 and ESE envelope modulation (ESEEM)18,19, which reveals fine details about metalcoordination sites. Regretfully, space limitations do not allow for a detailed discussi6n of these many new and exciting methods and applications. This review will therefore focus on a subject of present particular interest - the use of peptide and protein sequence manipulation along with site-specific attachment of spin labels to map structure and local dynamic processes. Recent experiments suggest that spin-label ESR,
0
0
+
~>
6
+ 6
Figure 1 A commonly used spin-label reaction. This methanethiosulfonate spin label (MTSSL) attaches only to free sulfhydrylgroups. © 1992,ElsevierSciencePublishers, (UK)
TIBS 17 - NOVEMBER 1992
(a)
(b) (i) API/2, Oxygen
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Figure 2 (a) A schematic structure of bacteriorhodopsin where the cylinders A-G represent the seven transmembrane c~-helices and retinal is the branched structure nestled within the helices. The numbered residues indicate the sequential locations of the single X ~ Cys replacements in the 18 mutants. (b) The accessibility parameter API/2 as seen for bacteriorhodopsin in lipid vesicles upon exposure to oxygen (i) and CROX (ii) at each spin-labeled position. Note the helical periodicity for residues 131 to 138 in (i), OG refers to the detergent octyl glucoside which is used to solubilize the protein. At 10% OG, the concentration of CROX near bacteriorhodopsin is increased and this highlights the region of the interhelical loop in (ii).
along With sequence manipulation, can reveal critical details that are simply not available to other measurement techniques. Below we cover two recent spinlabel studies: one that has revealed the detailed structure of a membrane protein and another that is re-evaluating our understandihg of peptides in solution.
lab at MIT, the technique was applied to bacteriorhodopsin, a light-driven proton pump, which has served as a model membrane protein in recent years. Bacteriorhodopsin houses the chromophore retinal within seven transmembrane co-helices. Although the gross structure of bacteriorhodopsin is known, the detailed orientation of the transmembrane s-helices with .respect Membrane protein structure Membrane proteins are very difficult to each other and the exact-location of to crystallize and consequently offer an inter-helical loops remain unclear. Spin-label relaximetry is used to enormous challenge in protein structure determination. Indeed, the photo- determine the membrane protein topsynthetic reaction center is the only ex- ography, that is, whether a particular ample of a membrane protein structure residue site is either buried within the solved by crystallographic methods 2°. protein matrix, exposed to the memTo address this, spin-label relaximetry brane interior or exposed to the extrawas recently developed and combined cellular solution. Classifying each memwith site-directed mutagenesis as a brane protein residue along a primary means for mapping secondary and ter- sequence into one of these three cattiary structure in membrane proteins 21. egories reveals insights into both the In a collaborative effort between local secondary structure and the terHubbell's lab at UCLA and Khorana's tiary contacts; This classification is per-
formed by measuring the spin relaxation of nitroxide spin labels selectively placed at each position. Different side chain environments are determined with the use of medium-specific spinrelaxing agents. As an example, consider a residue that is on the exterior of the protein and buried in the membrane. Oxygen (O2), an effective spin-relaxing agent, readily dissolves into both membranes and aqueous solution. Upon exposure to 02, the residue in the membrane will exhibit an enhanced relaxation rate. By contrast, a spin-relaxing agent that dissolves only in the aqueous solution, such as chromium oxalate (CROX), will not affect the relaxation rate. Following the same reasoning, a residue exposed to the aqueous solution will show sensitivity to both 02 and CROX whereas a residue buried within the protein will show sensitivity to neither. Consequently, if a particular amino acid residue in the sequence of a
449
TIBS 17 - NOVEMBER 1992
2000. •
•
1800,
1600"
O& a
[]
0
Aet 6
I Ao
[] G
Ao
G
>~ 1400 []
•
1200 •
1000
0 M
I=1
• []
800 5000
!
!
15000
25000
-[8]222
(deg cm 2 dmol 1)
Figure 3 A view of local molecular dynamics (VL, see text) vs peptide helicity (-[0]222) for the 3K-8 peptide (e) and the 3K4 peptide (D). Note that position 4, which is closer to the amino terminus of the peptide, shows greater nanosecond dynamics only near the middle of the helix-to-coil transition. Data are also shown for the 3K-8 at pH 2 (A).
membrane protein is replaced by a nitroxide, one can readily determine whether the label is within the protein interior or exposed to either membrane or aqueous solvent. Eighteen site-directed mutants of bacteriorhodopsin were prepared each with a single X--*Cys substitution sequentially placed from positions 125 to 142 (Fig. 2). The Cys was spin labeled with the reaction shown in Fig. 1. These residues are in a particularly interesting region of the protein, which starts near the end of one transmembrane helix, passes through an interhelical loop and then stretches well into the next helical segment. Extreme care was taken to characterize the folding and functionality of each spin-labeled mutant and the researchers found that only three of the 18 mutants exhibited any measurable perturbation from the native protein. Power-saturation studies involve monitoring the ESR signal as the 9 GHz power is increased, and serve as a convenient method for measuring spin relaxation. Relaxation data were gathered for each mutant and the results expressed in terms of an accessibility parameter (APl~2) to both CROX and Oz as a function of-sequence position (Fig. 2). The interhelical loop, which is identified as the only region exposed to the CROX-containing aqueous solution, stretches from residues
450
129 to 131. Beyond the loop, the 02 experiments reveal a region that smoothly alternates from exposure to membrane to exposure to protein with a period of 3.6. This is dramatic evidence for an co-helix and leaves no ambiguity as to which residues are buried within the protein interior. These methods provide a recipe for solving the transmembrane structure of virtually any membrane protein. The same methods have been used for gaining structural information on the solution structure of colicin E122. The main prerequisite for spin-label relaximetry is that one must be able to prepare a series of mutants, which is not unreasonable given the modern capabilities of molecular biology. It appears, however, that solving structure is only the initial result of combining spin-label ESR with site-directed mutagenesis. Spin labels may be used to monitor long-lived conformational changes. For instance, if a transmembrane helix twists or partially unfolds, this will certainly be revealed by the accessibility parameter profile. Spin4abel relaximetry may provide the first structural clues as to the mechanism behind ion channel gating and receptor response, for example. Peptides in solution
In our laboratory we have been investigating the folding of helical
peptides. We h a v e been particularly interested in the series of peptides discovered by R. L. Baldwin and coworkers 23'24. Whereas helices are extremely common in folded proteins, previous theory and experiments suggested that peptide oligomers would not form isolated stable helices in aqueous solution. However, a series of papers starting in 198723 dramatically demonstrated that short peptides (16mers and 17-mers) containing a large percentage of Ala formed remarkably stable helices. The physical basis behind this stability remains unclear. Nevertheless, these peptides provide an exciting opportunity to study protein-like helices in isolation. It is our belief that, if the protein folding problem is ever to be solved, we should first be able to deeply understand the folding of isolated domains of secondary structure. In addition to containing Ala, these peptides also contain Lys to render them soluble in aqueous solution. For Our experiments we introduce a single Ala~Cys substitution for the attachment of a spin label 25. Two of the peptides we have synthesized and examined are Ac-AAACKAAAAKAAAAKA- NH2 3K-4 Ac-AAAAKAACAKAAAAKA- NH2 3K-8
where the single letter symbols are Ala, A; Lys, K; Cys, C 26. We attach a spin label specifically to the Cys using the reaction in Fig. 1. Circular dichroism (CD) is the main diagnostic tool for the detection of helices and we have shown that the CD spectra of our spin-labeled peptides is nearly identical, to the parent peptide (which does not have the Ala-*Cys substitution). These peptides undergo a thermal helix-to-coil unfolding transition as the temperature is raised from 0*C to 60°C and they also unfold in the presence of guanidine and other denaturants. We have compared the unfolding characteristics of the 3K-4 and 3K-8 peptides in order to reveal the position dependence of helix unraveling. Our ESR data are interpreted in terms of a parameter we call the local volume (VL), which is the effective hydrodynamic volume detected by the spin label. Therefore, for comparing two peptides of the same sequence but different label attachment points, the peptide with the higher VL has greater rigidity at the label position. On the other hand, a lower VL indicates an increase in nanosecond dynamics of the peptide backbone in
TIBS 17 - NOVEMBER 1992
(a)
(c)
7
/ ~
(b)
3K-(4,7)
3K-8
j/
6
4
7
Figure 4 Helical wheels for 16-mer peptides in both (a) the
/ii! i
0
TECHNI UES =
ELECTRON SPIN RESONANCE (ESR) has been one of the core techniques in biochemistry and biophysics for nearly three decades. It directly detects the presence of free radicals and reveals information on structure and motional dynamics at the site of one or more unpaired electrons. The field of ESR is presently undergoing considerable rejuvenation in terms of novel new techniques as well as inventive new applications. In particular, nitroxide spin labels are proving to be remarkably useful in solving m o d e r n problems. This resurgence of the use of spin labels is fueled by modern chemical and molecular biological approaches that allow the researcher to specifically place a nitroxide at points of interest in biomolecules. This review will focus on just a few of these modern uses of nitroxide spin labels. (For more detail on experimental techniques and labeling methods see Refs 1,2.) Nitroxides are stable, organic freeradical reporter groups that can be chemically attached to strategic points on biomolecules to provide detailed information on the local molecular environment. They can be used to probe local dynamics, exposure to solvent, exposure to oxygen, molecular collisions, local ordering a n d local structure. There is a large selection of different nitroxides and a typical structure and spin labeling reaction 3 is shown in Fig. 1. The unpaired electron density is mainly located in the oxygen and nitrogen orbitals. This paramagnetic site gives rise to a magnetic resonance signal that is detected, for example, by an X-band ESR spectrometer that operates at a frequency near 9.0 GHz. Coupling between the electron spin and the spin of the ~4N nucleus usually gives rise to a three-line ESR speqtrum~ Because of a fortuitous combination the local magnetic interactions from the nitroxide structure, along with the 9.0 GHz frequency, ESR spectra are extremely sensitive to rotational motion of the nitroxide group. Rotational correlation times (ZR) are readily determined from Conventional G. L. Millhauser is at the Departmentof Chemistryand Biochemistry,Universityof California, Santa Cruz, CA 95064, USA.
448
Selective placement of electron spin resonance spin labels: new structural methods for peptides and proteins
Electron spin resonance (ESR) is more powerful than ever as a technique for solving biochemical and biophysical problems. Part of the great utility of ESR arises from the use of modern biochemical methods to place spin labels at important positions along the primary sequence of a peptide or protein.
ESR spectra over the range from 0.1 ~s to 10 ps. This range has been greatly extended by modern methods including saturation transfer 4 and various electron-spin echo experiments ~,6. Spinlabel ESR can now detect motions from milliseconds to picoseconds, a range that covers nine orders of magnitude! Spin-label ESR spectra are also quite sensitive. For example, it is routine to collec t spectra from 10 pl samples containing micromolar concentrations and nanomolar concentrations can be detected with signal averaging. There are also new developments in resonator design that are greatly increasing these sensitivity factors 7. Thus, in modern biochemical research, where molecular details are the key to understanding biological function, spin label ESR is a method o f high sensitivity that offers the ready determination of local structure and local dynamics over a wide range of time scales. Over the past few years ESR has proved essential in providing insights into a range of biochemical and biophysical findings including: the flexi-
bility of DNA8, the motion of myosin head groups during muscle movement 9,1°, the impact of membrane fluidity on the rotational dynamics of membrane proteins u, the mechanism of oxygen movement through membranes ~2, the mechanism of ion channel formation from peptides ~3 and the aggregation state of peptides ~4. In concert with this, a host of new techniques are emerging and are being refined for biological applications. In addition to those mentioned above, these include: highfield ESR (250 GHz)~5, novel applications of electron-electron double resonance 16,~7 and ESE envelope modulation (ESEEM)18,19, which reveals fine details about metalcoordination sites. Regretfully, space limitations do not allow for a detailed discussi6n of these many new and exciting methods and applications. This review will therefore focus on a subject of present particular interest - the use of peptide and protein sequence manipulation along with site-specific attachment of spin labels to map structure and local dynamic processes. Recent experiments suggest that spin-label ESR,
0
0
+
~>
6
+ 6
Figure 1 A commonly used spin-label reaction. This methanethiosulfonate spin label (MTSSL) attaches only to free sulfhydrylgroups. © 1992,ElsevierSciencePublishers, (UK)
TIBS 17 - NOVEMBER 1992
(a)
(b) (i) API/2, Oxygen
•
• Vesicles
/~/
&1%OG
FI
~
A
o,oo o / / / i 0
'
l'
0
/
2.5
(ii)
AP,/,, 50rnM C h r o m i u m Oxalclte
2.0 ~ I I
1.5
1.0 /
\
II I i I
\
\
•-~-o \
~..
A .
&
Vesicles &I~OG
~ - - o - - e 10% OG
/
0.5
/
0.0 I
I
I
I
I
1
1
1
1
1
1
1
1
1
1
1
1
1
Figure 2 (a) A schematic structure of bacteriorhodopsin where the cylinders A-G represent the seven transmembrane c~-helices and retinal is the branched structure nestled within the helices. The numbered residues indicate the sequential locations of the single X ~ Cys replacements in the 18 mutants. (b) The accessibility parameter API/2 as seen for bacteriorhodopsin in lipid vesicles upon exposure to oxygen (i) and CROX (ii) at each spin-labeled position. Note the helical periodicity for residues 131 to 138 in (i), OG refers to the detergent octyl glucoside which is used to solubilize the protein. At 10% OG, the concentration of CROX near bacteriorhodopsin is increased and this highlights the region of the interhelical loop in (ii).
along With sequence manipulation, can reveal critical details that are simply not available to other measurement techniques. Below we cover two recent spinlabel studies: one that has revealed the detailed structure of a membrane protein and another that is re-evaluating our understandihg of peptides in solution.
lab at MIT, the technique was applied to bacteriorhodopsin, a light-driven proton pump, which has served as a model membrane protein in recent years. Bacteriorhodopsin houses the chromophore retinal within seven transmembrane co-helices. Although the gross structure of bacteriorhodopsin is known, the detailed orientation of the transmembrane s-helices with .respect Membrane protein structure Membrane proteins are very difficult to each other and the exact-location of to crystallize and consequently offer an inter-helical loops remain unclear. Spin-label relaximetry is used to enormous challenge in protein structure determination. Indeed, the photo- determine the membrane protein topsynthetic reaction center is the only ex- ography, that is, whether a particular ample of a membrane protein structure residue site is either buried within the solved by crystallographic methods 2°. protein matrix, exposed to the memTo address this, spin-label relaximetry brane interior or exposed to the extrawas recently developed and combined cellular solution. Classifying each memwith site-directed mutagenesis as a brane protein residue along a primary means for mapping secondary and ter- sequence into one of these three cattiary structure in membrane proteins 21. egories reveals insights into both the In a collaborative effort between local secondary structure and the terHubbell's lab at UCLA and Khorana's tiary contacts; This classification is per-
formed by measuring the spin relaxation of nitroxide spin labels selectively placed at each position. Different side chain environments are determined with the use of medium-specific spinrelaxing agents. As an example, consider a residue that is on the exterior of the protein and buried in the membrane. Oxygen (O2), an effective spin-relaxing agent, readily dissolves into both membranes and aqueous solution. Upon exposure to 02, the residue in the membrane will exhibit an enhanced relaxation rate. By contrast, a spin-relaxing agent that dissolves only in the aqueous solution, such as chromium oxalate (CROX), will not affect the relaxation rate. Following the same reasoning, a residue exposed to the aqueous solution will show sensitivity to both 02 and CROX whereas a residue buried within the protein will show sensitivity to neither. Consequently, if a particular amino acid residue in the sequence of a
449
TIBS 17 - NOVEMBER 1992
2000. •
•
1800,
1600"
O& a
[]
0
Aet 6
I Ao
[] G
Ao
G
>~ 1400 []
•
1200 •
1000
0 M
I=1
• []
800 5000
!
!
15000
25000
-[8]222
(deg cm 2 dmol 1)
Figure 3 A view of local molecular dynamics (VL, see text) vs peptide helicity (-[0]222) for the 3K-8 peptide (e) and the 3K4 peptide (D). Note that position 4, which is closer to the amino terminus of the peptide, shows greater nanosecond dynamics only near the middle of the helix-to-coil transition. Data are also shown for the 3K-8 at pH 2 (A).
membrane protein is replaced by a nitroxide, one can readily determine whether the label is within the protein interior or exposed to either membrane or aqueous solvent. Eighteen site-directed mutants of bacteriorhodopsin were prepared each with a single X--*Cys substitution sequentially placed from positions 125 to 142 (Fig. 2). The Cys was spin labeled with the reaction shown in Fig. 1. These residues are in a particularly interesting region of the protein, which starts near the end of one transmembrane helix, passes through an interhelical loop and then stretches well into the next helical segment. Extreme care was taken to characterize the folding and functionality of each spin-labeled mutant and the researchers found that only three of the 18 mutants exhibited any measurable perturbation from the native protein. Power-saturation studies involve monitoring the ESR signal as the 9 GHz power is increased, and serve as a convenient method for measuring spin relaxation. Relaxation data were gathered for each mutant and the results expressed in terms of an accessibility parameter (APl~2) to both CROX and Oz as a function of-sequence position (Fig. 2). The interhelical loop, which is identified as the only region exposed to the CROX-containing aqueous solution, stretches from residues
450
129 to 131. Beyond the loop, the 02 experiments reveal a region that smoothly alternates from exposure to membrane to exposure to protein with a period of 3.6. This is dramatic evidence for an co-helix and leaves no ambiguity as to which residues are buried within the protein interior. These methods provide a recipe for solving the transmembrane structure of virtually any membrane protein. The same methods have been used for gaining structural information on the solution structure of colicin E122. The main prerequisite for spin-label relaximetry is that one must be able to prepare a series of mutants, which is not unreasonable given the modern capabilities of molecular biology. It appears, however, that solving structure is only the initial result of combining spin-label ESR with site-directed mutagenesis. Spin labels may be used to monitor long-lived conformational changes. For instance, if a transmembrane helix twists or partially unfolds, this will certainly be revealed by the accessibility parameter profile. Spin4abel relaximetry may provide the first structural clues as to the mechanism behind ion channel gating and receptor response, for example. Peptides in solution
In our laboratory we have been investigating the folding of helical
peptides. We h a v e been particularly interested in the series of peptides discovered by R. L. Baldwin and coworkers 23'24. Whereas helices are extremely common in folded proteins, previous theory and experiments suggested that peptide oligomers would not form isolated stable helices in aqueous solution. However, a series of papers starting in 198723 dramatically demonstrated that short peptides (16mers and 17-mers) containing a large percentage of Ala formed remarkably stable helices. The physical basis behind this stability remains unclear. Nevertheless, these peptides provide an exciting opportunity to study protein-like helices in isolation. It is our belief that, if the protein folding problem is ever to be solved, we should first be able to deeply understand the folding of isolated domains of secondary structure. In addition to containing Ala, these peptides also contain Lys to render them soluble in aqueous solution. For Our experiments we introduce a single Ala~Cys substitution for the attachment of a spin label 25. Two of the peptides we have synthesized and examined are Ac-AAACKAAAAKAAAAKA- NH2 3K-4 Ac-AAAAKAACAKAAAAKA- NH2 3K-8
where the single letter symbols are Ala, A; Lys, K; Cys, C 26. We attach a spin label specifically to the Cys using the reaction in Fig. 1. Circular dichroism (CD) is the main diagnostic tool for the detection of helices and we have shown that the CD spectra of our spin-labeled peptides is nearly identical, to the parent peptide (which does not have the Ala-*Cys substitution). These peptides undergo a thermal helix-to-coil unfolding transition as the temperature is raised from 0*C to 60°C and they also unfold in the presence of guanidine and other denaturants. We have compared the unfolding characteristics of the 3K-4 and 3K-8 peptides in order to reveal the position dependence of helix unraveling. Our ESR data are interpreted in terms of a parameter we call the local volume (VL), which is the effective hydrodynamic volume detected by the spin label. Therefore, for comparing two peptides of the same sequence but different label attachment points, the peptide with the higher VL has greater rigidity at the label position. On the other hand, a lower VL indicates an increase in nanosecond dynamics of the peptide backbone in
TIBS 17 - NOVEMBER 1992
(a)
(c)
7
/ ~
(b)
3K-(4,7)
3K-8
j/
6
4
7
Figure 4 Helical wheels for 16-mer peptides in both (a) the
