Abstract:
Spin labeled ice binding compounds (IBCs) including ice binding proteins (IBPs), also called antifreeze proteins (AFPs) and their analogs are exploited to carry the paramagnetic centers for dynamic nuclear polarization (DNP), for enhancing nuclear magnetic resonance (NMR) signal intensities. Use of spin labeled IBCs to perform DNP exploits the IBCs&#39; ability to homogeneously distribute the paramagnetic centers in frozen water solution at low temperature, leading to high DNP efficiency. Other advantages of using spin labeled IBCs include: (1) ability to cryo-protect biological samples; (2) the relative positions and orientations of the spin labeling groups in an IBC may also be cryo-preserved; (3) positions and orientations of spin labeling groups to an IBC can be selected with great freedom and without technical barrier to making multiple spin labels in an IBC; and (4) water solubilities of spin labeled IBCs are potentially high, enabling use of a solvent that is primarily water for DNP at low temperatures.

Description:
RELATED APPLICATIONS/PRIORITY CLAIMS 
       [0001]    This application claims priority to U.S. Provisional Patent No. 61/509,107, filed on Jul. 18, 2011, which is incorporated herein by reference in its entirety. 
     
    
     FEDERAL FUNDING STATEMENT 
       [0002]    This work was supported by NIH Grant 5SC1 GM083826-04. 
     
    
     BACKGROUND 
       [0003]    The present invention relates generally to methods and compositions for performing dynamic nuclear polarization (DNP), and for further performing Nuclear Magnetic Resonance (NMR) and Magnetic Resonance Imaging (MRI) using the compounds so produced. NMR has become an irreplaceable tool in the studies of macromolecular structures, dynamics, and interactions, such as in the studies of proteins, DNA, their complexes, and also in imaging tissue structures. However, a major bottle neck of NMR for high throughput applications is its low sensitivity. The intrinsic reason for low sensitivity arises from the low equilibrium polarization of Boltzmann populations at different energy levels of nuclear spins in an external magnetic field. The equilibrium polarization is proportional to the strength of the external magnetic field and inversely proportional to the absolute temperature of the sample. Thus, to increase the polarization, one can either increase the strength of the external magnetic field, decrease the sample temperature, or do both. 
         [0004]    To date, the highest field of commercially available magnet is ˜23.5 Tesla (corresponding to proton magnetic resonance frequency ˜1 GHz). To increase the magnetic field strength of the highest commercial magnet even by two-fold (to 47 Tesla) cannot currently be technologically and/or financially foreseen. Another way to handle the problem of low sensitivity is to develop more sensitive instruments to detect NMR signals. For example, CryoProbe™ devices developed in recent years have been able to increase NMR sensitivity up to four-fold. CryoProbe™ device technology deals with suppressing the instrumental thermal noises by reducing the temperature of the NMR coil and preamplifier using cold helium gas at ˜20K. In order to tremendously increase the NMR signal intensities, other technologies and methods have yet to be developed. 
         [0005]    A promising approach to increasing NMR sensitivity is to dynamically polarize the nuclear spins of the samples. If a sample contains either exogenous or endogenous paramagnetic centers, such as paramagnetic metal ions (including transition, actinide, and lanthanide metal ions), nitroxide radicals, and trityl radicals, for example, irradiation of the paramagnetic centers in a magnetic field can lead to dynamic polarization of the nuclear spins. In principle, this technique can enhance the proton signal by 660-fold and that of  13 C by 2600-fold due to the gyromagnetic ratios of electron to nuclei (γ e /γ n ) of proton and  13 C, respectively. Use of dinitroxide-type biradical compounds for dynamic nuclear polarization (DNP) previously has been described. A factor of signal enhancement of 175-fold was achieved. The DNP experiments are carried out at temperatures ≦100° K in order to suppress the electronic and nuclear spin-lattice relaxation rates to favor the polarization transfer. At such a low temperature, a solvent is required for homogenous distribution of the paramagnetic centers in the frozen solution for effective polarization distribution throughout the sample. The most commonly used solution is a 40:60 v/v mixture of water:glycerol, which forms a glassy matrix at low temperature regardless of the cooling rate. Biological samples are also expected to be cryo-protected using this solvent. 
         [0006]    Despite the achievement of the DNP experiments, one drawback is the use of the mixture of water and glycerol as the solvent. This solvent requirement may limit many biological applications because in almost all of the studies of biomolecules, water represents the sole environment to mimic the behaviors and structures of biomolecules as in living cells. This invention addresses the issue of using water as the primary solvent for DNP at low temperatures through the use of electron spin labeled ice binding compounds (IBCs). 
       SUMMARY OF THE INVENTION 
       [0007]    It has now been discovered that electron spin, or “spin” (including single spin, multiple spin, and paramagnetic ion) labeled ice binding compounds (IBCs), including ice binding proteins (IBPs) and antifreeze proteins (AFPs) may be used to homogeneously distribute paramagnetic centers (the assemblies of the electron spins) in frozen water solution for DNP. For the purposes of this disclosure, the terms “IBP” and “AFP” will be used interchangeably, and include naturally occurring and synthetic forms, as well as complexes having other compounds chemically connected to IPBs or AFPs, for example, peptides, proteins and other nonproteinaceous compounds. These terms are meant to also include any other organic and/or inorganic compounds that have similar ability to bind to ice surfaces (ice binding compounds, “IBCs”). Also included are multiple complexes, for example, dimers. 
         [0008]    IBPs provide protection for organisms that express AFPs living through subzero winters from freezing damage due to their property to bind to specific ice surfaces, thereby inhibiting growth of seed ice crystals and the recrystallization of ice crystals. The ice binding property allows the homogeneous dispersion of IBPs in frozen aqueous solutions, primarily aqueous solutions, and also other solutions that form glassy solids at low temperature. The terms “primarily aqueous,” “primarily water,” and the like terms are meant to refer to solutions in which the majority constituent is water. Therefore, after electron spins are labeled to the non-ice-binding side chains of IBPs, the paramagnetic centers can be homogeneously distributed in frozen solution which can serve for effectively polarizing the nuclear spins in the frozen solution. The term “frozen solution” is meant to include aqueous solutions, primarily aqueous solutions, and other solutions wherein glassy solids form at low temperatures. This approach may allow broad applications of DNP, especially for the study of biomolecular structures and tissue structures where the solution is water. However, it is contemplated that the spin labeled IBPs will also have application for analyses of non-biological compounds. 
         [0009]    Other underlying rationales for using spin labeled IBPs to perform DNP include: (1) IBPs have the ability to cryo-protect biological samples; (2) the structures of IBPs are naturally cryo-protected at low temperature enabling the relative positions and orientations of the spin labeling groups in an IBP to also be cryo-preserved; (3) using site-directed mutagenesis techniques, the positions and orientations of the spin labeling groups to an IBP can be selected with great freedom and there is no technical barrier to making multiple spin labels in an IBP; and (4) water solubilities of the spin labeled IBPs are potentially high. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0010]      FIGS. 1A-1D  show structures of exemplary spin labels and resulting chemical modifications of cysteine side chains.  FIG. 1(A)  shows MTSL, (1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl) methanethiosulfonate;  FIG. 1(B)  shows MSL, 4-maleimido-TEMPO;  FIG. 1(C)  IAP, 3-(2-iodoacetamido)-PROXYL; and  FIG. 1(D)  IASL, 4-(2-iodoacetamido)-TEMPO. 
           [0011]      FIG. 2A  shows the general structures of several types of AFPs. 
           [0012]      FIG. 2B  shows the typical structural data of several types of AFPs. 
           [0013]      FIG. 3(A)-3(F)  show the structural models of the HPLC6 AFP in relation to various spin labeled HPLC6 AFPs.  FIG. 3(A)  shows a structural model for HPLC6 AFP.  FIG. 3(B)  shows a structural model for a single spin labeled HPLC6_L23C.  FIG. 3(C)  shows a structural model for a double spin labeled HPLC6_A19C_L23C.  FIG. 3(D)  shows a structural model for a triple spin labeled HPLC6_A20C_L23C.  FIG. 3(E)  shows a structural model for a triple spin labeled HPLC6_L12C_A19C_L23C.  FIG. 3(F)  shows a structural model for a triple spin labeled HPLC6_L12C_A20C_L23C. 
           [0014]      FIG. 4  shows examples of free radical groups. 
           [0015]      FIG. 5  shows structures of TEMPO-based biradicals. (Adapted from Mak-Jurkauskas M L, Griffin R G: High-frequency dynamic nuclear polarization. In: 2010: John Wiley &amp; Sons Ltd.; 2010: 159-174.) 
           [0016]      FIG. 6  shows functional groups in a peptide or protein that can be used to perform spin labeling. 
           [0017]      FIG. 7  shows EPR spectra of single spin labeled HPLC6_L12 and double spin labeled HPLC6 A20C_L23C water solutions at different temperatures. 
           [0018]      FIG. 8(A)  shows the structure of 3-(2-iodoacetamide) DTPA. 
           [0019]      FIG. 8(B)  shows the structure of 3-(2-iodoacetamide) DTPA and its Gd 3+  complex. 
       
    
    
     DETAILED DESCRIPTION 
       [0020]    Throughout this specification, the terms “a” and “an” and variations thereof represent the phrase “at least one.” In all cases, the terms “comprising”, “comprises” and any variations thereof should not be interpreted as being limitative to the elements listed thereafter. Unless otherwise specified in the description, all words used herein carry their common meaning as understood by a person having ordinary skill in the art. In cases where examples are listed, it is to be understood that combinations of any of the alternative examples are also envisioned. The scope of the invention is not to be limited to the particular embodiments disclosed herein, which serve merely as examples representative of the limitations recited in the issued claims resulting from this application, and the equivalents of those limitations. 
         [0021]    Site Directed Spin Labeling (SDSL) 
         [0022]    Site Directed Spin Labeling (SDSL) is often accomplished by cysteine-substitution mutagenesis, followed by modification of the unique sulfhydryl group with selective nitroxide or other reagents.  FIGS. 1(A)-1(D)  give examples of spin labeling reagents and their chemical reactions with a sulfhydryl group. In  FIGS. 1(A)-1(D) , Cys-substitution mutants of AFPs are used to provide examples of spin labeled AFPs. 
         [0023]    Presently, five types of fish AFPs have been discovered, including antifreeze glycoprotein (AFGP) and type I, II, III and IV AFPs. AFPs also have been found in insects such as  Tenebrio molitor , Spruce budworm, and Snow flea, and in plants such as Winter Rye ( Secale cereale  L.) and ryegrass ( Lolium perenne ). Although AFPs have different structures and were found in diversified species, they all display similar antifreeze function by binding to specific surfaces, and preventing the growth of seed ice crystals at subzero environment. Thus, other compounds with similar functional capabilities are contemplated as similarly useful for the methods disclosed herein and they are also included within the scope of IBCs. A summary of typical structures of several AFPs are given in  FIG. 2A  and  FIG. 2B . 
         [0024]    Single Spin Labeled AFPs 
         [0025]    As an example, the HPLC6 isoform of type I AFP has an α-helical secondary structure (See  FIG. 3(A) ) and contains three 11-residue repeat units commencing with Thr residues. It has the following sequence: Asp- Thr -Ala-Ser-Asp- Ala -Ala-Ala-Ala- Ala -Ala-Leu(12)- Thr -Ala-Ala(15)-Asn- Ala -Lys-Ala-Ala(20)-Ala-Glu-Leu(23)- Thr -Ala-Ala-Asn- Ala -Ala-Ala-Ala- Ala -Ala-Ala-Thr-Ala-Arg, where the ice binding residues are underlined and the residues preferred for substitution with Cys are bolded and numbered (according to the chronological order of the sequence). The corresponding single Cys-substitution mutants are named as: (1) HPLC6_L12C; (2) HPLC6_A15C; (3) HPLC6_A20C; and (4) HPLC6_L23C, where, for example, the L23C denotes that the L residue at position 23 is replaced by a C residue. These Cys substituted residues are not in the ice binding surface (IBS) and located around the mid section of the α-helical structure. The reason for replacing the L12 or L23 residues with a Cys residue is that Leu side chains are bulky and hydrophobic. Thus, the spin labeling group at these positions may cause less perturbation to the secondary structure and to the function of the AFP. The side chain of the C23 residue directly points to the opposite direction with respect to the IBS (ice binding surface). The side chains of C20 and C15 point to the two opposite side directions. Other choices of residues for making spin labeled AFPs are also possible and would be readily apparent in view of the disclosures herein. 
         [0026]    Spin labels may be attached to Cys side chains according to standard procedures ( FIGS. 1(A)-1(D) ). Briefly, AFPs are buffer-exchanged (HiPrep 26/10 Desalting Column, Amersham) into 10 mM Tris, pH 6.9, for the MTSL spin-labeling reaction, or into 10 mM HEPES, pH 7.6, for IAP, IASL, and MSL reactions. Spin labels are dissolved into 100% ethanol and added to the AFP solution at a 100:1 label/Cys-site concentration to ensure complete labeling. The labeling reaction proceeds overnight in darkness at 4° C. After the reactions, excess spin labels are separated using a size-exclusion chromatography equilibrated in acetate buffer (2 mM NaOAc, pH 5.0), or by using dialysis method. The purified spin labeled AFP is stored at −20° C. A structural model using the IAP reagent ( FIG. 1(C) ) to make a single spin labeled AFP at the C23 side chain is given in  FIG. 3(B)  as an example. 
         [0027]    The Overhauser Effect (OE) was originally proposed to enhance nuclear spin&#39;s polarization from that of electron spins. OE involves the simultaneous transitions of an electron spin and a nuclear spin driven by relaxation processes induced by time-dependent dipolar and/or scalar electron-nuclear couplings. However, the efficiency of the OE is lowered dramatically at higher magnetic fields because ω 0 τ c &lt;1 must be satisfied, where ω 0  is the electron Larmor frequency and τ c  is the rotational correlation time of the molecules. Thus, it is necessary to find other DNP mechanisms to enhance the nuclear spin&#39;s polarization at high magnetic fields for enhancing the signals of modern NMR and MRI. 
         [0028]    One of the DNP mechanisms is the Solid Effect (SE), which is a two-spin process. Single spin labeled IBPs facilitate this mechanism. Microwave irradiation at ω 0S ±ω 0I , where ω 0S  and ω 0I  respectively denote the electronic and nuclear Larmor frequencies, can excite the partially allowed electron-nucleus transitions. For the SE to be effective, the inhomogeneous breadth (Δ) and the homogeneous linewidth (δ) of the Electron Paramagnetic Resonance (EPR) spectrum should be much smaller than the nuclear Larmor frequency (±ω 0I &gt;Δ, δ) so that only one of the two forbidden transitions can be excited at a time to avoid cancellation. Additionally, the transition probability has a frequency dependence as 1/(ω 0I ) 2 . Thus, the SE&#39;s efficiency will decrease quickly with the increase of the magnetic field, resulting in poorer efficiency at high field. Two readily available radicals that have sufficiently narrow linewidths are bisdiphenylene-beta-phenylallyl (BDPA) and trityl ( FIG. 4 ). At 5 T they have spectral breadth of ˜20 and ˜90 MHz, respectively, which are smaller than the  1 H Larmor frequency ω 0I =211 MHz. Conjugation of a single radical of one of these two radicals to an IBP is anticipated to function better than the nitroxide radicals through the SE mechanism for DNP in frozen solutions. 
         [0029]    Double Spin Labeled AFPs 
         [0030]    SDSL can also be used to make double spin labeled AFPs. In the following HPLC6 sequence: Asp- Thr -Ala-Ser-Asp- Ala -Ala-Ala-Ala- Ala -Ala-Leu- Thr -Ala-Ala-Asn- Ala -Lys-Ala(19)-Ala(20)- Ala -Glu-Leu(23)-Thr-Ala-Ala-Asn- Ala -Ala-Ala-Ala- Ala -Ala-Ala-Thr-Ala-Arg, the preferred residues selected for double Cys substitutions are bolded and numbered. These residues are not in the IBS and located around the mid section of the α-helix. For example, the following double Cys-substitution mutants can be designed: (1) HPLC6_A19C_L23C; (2) HPLC6_A20C_L23C; and (3) HPLC6_A19C_A20C. 
         [0031]    To make double spin labeled IBPs, spin labels are attached to the Cys side chains according to the standard procedures ( FIGS. 1(A)-1(D) ). In the foregoing mutants, the side chain of the C23 residue directly points to the opposite direction with respect to the ice surface, that of the C20 points to a side direction, and that of the C19 points roughly to a direction above the ice surface. The distance between the two β-carbons (C β ) in C20 and C23 is ˜5.7 Å, between those in C19 and C23 is ˜6.5 Å, and between the C19 and the C20 is ˜5.3 Å. All these conditions may result in favored radical-to-radical distances and orthogonal relative orientations of the two π systems in the pairs of nitroxide groups as those of the TOTAPOL as described in the art. Examples of structural models using the IAP reagent ( FIG. 1(C) ) to make double spin labeled HPLC6_A19C_L23C and HPLC6_A20C_L23C IBPs are given in  FIGS. 3(C) and 3(D) . 
         [0032]    Another DNP mechanism is the Cross Effect (CE). The double spin labeled IBPs facilitate the CE, which involves two electrons that are in close physical proximity in order to be effectively dipolarly coupled. To have the CE, the difference of the two EPR frequencies should match the nuclear frequency (ω 0S1 −ω 0S2 =ω 0I ). The CE is the dominant mechanism in the regime Δ&gt;ω 0I &gt;δ. When two transitions matching the condition above, one from an inhomogeneous line broadening packet Δ 1  and the other from the other Δ 2 , the frequency difference of the two transitions goes into polarizing the nuclear spins. This mechanism is a three-spin process and is based on allowed EPR transitions which exhibit a 1/(ω 0I ) field dependence. Therefore, the CE is more efficient than the SE. Examples of biradicals that support CE are shown in  FIG. 5 . An enhancement of ˜175 was obtained at electron concentration of 10 mM. To match the condition (ω 0S1 −ω 0S2 =ω 0I ), the relative orientations of the nodal planes of the π systems of the two nitroxide groups should be nearly orthogonal. 
         [0033]    Multiple Spin Labeled AFPs 
         [0034]    SDSL can also be used to make triple and multiple spin labeled AFPs. In the following HPLC6 sequence: Asp-Thr-Ala-Ser-Asp-Ala-Ala-Ala-Ala-Ala-Ala-Leu(12)-Thr-Ala-Ala-Asn-Ala-Lys-Ala(19)-Ala(20)-Ala-Glu-Leu(23)-Thr-Ala-Ala-Asn-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Thr-Ala-Arg, the preferred residues selected for Cys substitutions are bolded and numbered. These residues are not in the IBS and located around the mid-section of the α-helix. For example, the following Cys-substitution mutants can be designed: 
         [0000]    (1) Triple Cys-substitution mutants: HPLC6_L12C_A19C_L23C; (2) Triple Cys-substitution mutants: HPLC6_L12C_A20C_L23C; (3) Triple Cys-substitution mutants: HPLC6_L19C_A20C_L23C; and (4) Quadruple Cys-substitution mutants: HPLC6_L12C_A19C_A20C_L23C. 
         [0035]    In addition to the distances between the pairs of β-carbons in the double Cys substituted mutants, the distance between the C12 C β  and C23 C β  is ˜16.6 Å, that between the C12 C β  and C20 C β  is ˜12.8 Å, and that between the C12 C β  and C19 C β  is ˜10.5 A. To make the multiple spin labeled IBPs, the spin labels are attached to the Cys side chains according to the standard procedures ( FIGS. 1(A)-1(D) ). Structural models using the IAP reagent ( FIG. 1(C) ) to make the triple spin labeled HPLC6_L12C_A19C_L23C and HPLC6_L12C_A20C_L23C IBPs are given in  FIG. 3(E)  and  FIG. 3(F)  as examples. Similar strategy can be applied to make other triple and multiple spin labeled IBPs. To perform multiple spin labeling, larger AFPs than the type I AFP have the advantage of being able to be attached with more nitroxide radicals without significant perturbation to the structures and functions. 
         [0036]    Another DNP mechanism is Thermal Mixing (TM). Multiple spin labeled AFPs facilitate the (TM) effect, which involves multiple electron spins and the EPR spectrum is homogeneously broadened. The conditions under which the (TM) is the dominant mechanism are Δ&gt;ω 0I , δ. 
         [0037]    Using the Native Functional Side Chains of Proteins to Make Spin Labeled IBPs 
         [0038]    The native functional side chains in IBPs may also be used for spin labeling. This method does not require Cys-substitution mutagenesis. However, the choices for selecting the spin labeled sites may be more limited by the available functional side chains. Examples of functional groups in the side chains in peptides or proteins that can be used to perform spin labeling are listed in  FIG. 6 . They contain (but are not limited to) the N-terminus and C-terminus, and the side chains of Cystein, Serine, Threonine, Tyrosine, Lysine, Aspartic Acid, Glutamic Acid, Histidine, Tryptophan and Arginine. A summary of spin labeling chemistry for spin labeling these residues can be found in  Biological Magnetic Resonance  8 : Spin Labeling Theory and Applications , Chapter 9 Chemistry of Spin-Labeled Amino Acids and Peptides (K. Hideg and O. H. Hankovszky), Plenum Press, New York and London, pp. 427-487, which is incorporated by reference herein in its entirety. It should be appreciated that departures from the methods described therein would be considered routine and within the ordinary skill of the art. 
         [0039]    Other Spin Labeled Compounds Having the Ability to Bind to Ice Surfaces for DNP 
         [0040]    Other compounds that are not peptides or proteins but that may be the complexes of peptides or proteins, and organic and/or inorganic molecules, any of which possess ice binding properties and have functional side chains as described in the sections “Site Directed Spin Labeling (SDSL)” and  FIGS. 1(A)-1(D) , and in the section “Strategy to use the native functional side chains of proteins to make spin labeled IBPs” and  FIG. 6 , can be spin labeled using the methods described herein. These spin labeled compounds can be used as the paramagnetic centers for DNP, and may be especially useful in primarily water solutions. 
         [0041]    Using Free Radicals for Synthesizing Paramagnetic Centers for DNP 
         [0042]    Compounds containing stable free radicals (which contain unpaired electron spins) that can be used to perform spin labeling of IBPs include, but are not limited to those given in  FIG. 4 . A single compound can also contain more than one radical or spin. These structures with chemical bonding to extra functional groups, for instance, as those described in the section “Site Directed Spin Labeling (SDSL)” and  FIGS. 1(A)-1(D) , and in section “Strategy to use the native functional side chains of proteins to make spin labeled IBPs” and  FIG. 6 , can be conjugated to specific side chains in IBPs and other ice binding compounds. It is possible to use these methods for spin labeling any compound having similar properties as those disclosed herein. 
         [0043]    Labeling Paramagnetic Ions to IBPs for DNP 
         [0044]    Paramagnetic metal ions including those of transition, actinide, and lanthanide metals contain unpaired electron spins which can also be used to polarize nuclear spins through DNP. These ions may be attached to IBPs through chelating ligands. Chelating ligands are known to use more than one atom to bind to a metal ion to form an ion complex with high stability. For example, chelating agents like DTPA (Pentetic acid or diethylene triamine pentaacetic acid) and EDTA (Ethylenediaminetetraacetic acid) or their derivatives, can easily complex Gd 3+ . These ligands with chemical bonding to extra functional groups such as those described in  FIGS. 1(A)-1(D)  can be conjugated to specific side chains in IBPs and other ice binding compounds, resulting in paramagnetic ion labeled IBPs and compounds which can be used for DNP of frozen solutions. 
         [0045]    The paramagnetic metal ions that can be used for DNP include, for example, lanthanide metals of atomic numbers 58-70, transition metals of atomic numbers 21-29, 42 and 44. Suitable paramagnetic ions include, for example, Cr 3+ , Mn 2+ , Fe 3+ , Co 2+ , Ni 2+ , Cu 2+ , Nd 3+ , Sm 3+ , Gd 3+ , Tb 3+ , Dy 3+ , Ho 3+ , Er 3+ , and Yb 3+ . 
         [0046]    A variety of chelating agents is known to form ion complexes. Generally, chelating agents often containing heteroatoms as electron pair donors, such as N and/or O, and may also contain P and/or S. DTPA, for example, is an often used acyclic chelating agent. Its functional derivatives include DTPA-BMA, DTPA-BP, DTPA-BMEA, EOB-DTPA, BOPTA and MS-325. EDTA is another commonly used acyclic chelating agent. Its functional derivatives include EDTA-BMA, DPDP, PLED, HPTA, amides or diamides, such as TOGDA, sulphonates and phosphonates. Cyclic chelating agents may also be used for this purpose, and include, for example, cryptands, PCTA-[12], PCTP-[12], PCTP-[13], DOTA, DO3A and derivatives thereof, such as HP-DO3A and DO3A-butriol. 
         [0047]    Paramagnetic metal ions of one or different metals may be used to make paramagnetic ionic spin labeled IBPs and ice binding compounds (IBCs). The combinations of the organic radicals as discussed herein can also be attached to the same IBPs. The combination of the free radical spin labeled IBPs and the paramagnetic ion labeled IBPs can also be used in a single solution for DNP. 
         [0048]    Storage and shipment of the spin labeled IBCs prepared according to the general methods disclosed herein would be readily apparent to any person having ordinary skill within the art. For example, the spin labeled IBCs could be freeze dried and stored in airtight containers, preferably at ≦−20° C. for prolonged shelf life. 
         [0049]    DNP NMR may be performed according to standard procedures that would be readily apparent to any person having skill within the art. For example, a sample for analysis may be dissolved in, or otherwise homogeneously mixed with a spin labeled IBC solution. After being frozen at low temperature, the DNP process for the sample may be performed followed by optional temperature variation and NMR procedure. 
         [0050]    Likewise, imaging tissue structures, including cells, may also be performed according to standard procedures. For example,  1 H of water of a spin labeled IBC solution may be polarized through DNP at low temperature. Thereafter, the frozen solution may be promptly thawed to liquid state. Finally, the polarized solution may be injected into the tissue sample for analysis followed by an MRI procedure. The spin labeled IBC can also be filtered out leaving only water to be injected into the tissue. 
         [0051]    For tissues, including cells, where low temperature may not cause serious damage to the structures, or where the structures at low temperature will be studied, tissues soaked with Spin labeled IBC, for example, through injection or diffusion, can be frozen directly at low temperature for DNP, which can be followed by optional temperature variation and then by MRI procedure. 
       Example 1 
     EPR Experimental Results of Single and Double Spin Labeled HPLC6 Peptides Using the IAP Reagent 
       [0052]    We have synthesized the single spin labeled HPLC6_L12 and double spin labeled HPLC6_A20C_L23C using the IAP reagent. The EPR spectra of their water solutions at different temperatures are shown in  FIG. 7 . The lineshapes at the temperatures above the freezing points (298° K and 273° K) showed the typical triplet patterns of the nitroxide radicals. When the temperature was lowered below the freezing point, slow motion patterns of the lineshapes began to appear showing that the motions of the spin labeled side chains are severely restricted after the spin labeled IBPs were frozen in the ice matrices. The lineshapes continued to become broader down to 90° K, indicating increased restriction of motions of the spin labeling groups. Then, the lineshapes stayed similarly at lower temperatures. These experiments indicate that 90° K could be sufficiently low enough to perform DNP experiments. It is important to note that the lineshapes of the double spin labeled IBP were broader than those of the single spin labeled IBP at the same temperatures below the freezing point. This phenomenon shows the intramolecular spin-spin dipolar interactions of the double spin labeled IBPs. As discussed above, these dipolar interactions facilitate the DNP mechanism of Cross Effect. 
       Example 2 
     Using the Native Functional Groups of HPLC6 Isoform of Type I AFP 
       [0053]    This AFP has the following sequence: Asp-Thr-Ala-Ser-Asp- Ala -Ala-Ala-Ala- Ala -Ala-Leu- Thr -Ala-Ala-Asn- Ala -Lys-Ala-Ala- Ala -Glu-Leu- Thr -Ala-Ala-Asn- Ala -Ala-Ala- Ala -Ala-Ala-Ala-Thr-Ala-Arg, where the ice binding residues are underlined and the residues preferred for performing spin labeling are bolded. The bolded residues include lysine, arginine, glutamic acid and aspartic acid residues. Using these residues, single spin labeled through spin labeling Lys or Arg side chain, double spin labeled through spin labeling Lys and Arg side chain, and triple spin labeled through spin labeling Asp, Asp and Glu side chains, etc., type I AFPs can be synthesized. 
       Example 3 
     Using the Native Functional Groups of the QAEop12 Isoform of Type III AFP 
       [0054]    This protein has the following sequence: 
         [0055]    MNQASVVAN QL IP INTA L TLVM MRSEVVTPVGIPAEDIPRL VS M Q VNRAVPLGTTLMP DMVKGYAA where the residues involved in the putative ice binding surfaces are underlined and the available non-ice binding residues for performing spin labeling are bolded. They include lysine (K), arginine (R), glutamic acid (E), aspartic acid (D) and Tyrosine (Y) residues. Using these side chains, single spin labeled (using K or Y), double spin labeled (using K and Y) and multiple spin labeled (using R, R, R for making triple spin labeled, using E, E, D, D for making quadruple spin labeled, etc.) type III AFPs can be synthesized. 
       Example 4 
     Functionalized DTPA Gd 3+  Complex for Conjugation to IBPs 
       [0056]    As an example, we can synthesize 3-(2-iodoacetamido)-DTPA as shown in  FIG. 8(A) . The structure of its Gd 3+  complex is shown in  FIG. 8(B) . 3-(2-iodoacetamido)-DTPA can be easily conjugated to the Cys side chains of Cys substitution mutants of IBPs according to the chemistry described in section “Site Directed Spin Labeling” and shown in  FIGS. 1(A)-1(D) . Other functional groups as shown in  FIG. 1(A)  and  FIG. 1  (B) can also be chemically bonded to DTPA. Functional groups that can conjugate other functional side chains as exemplified in  FIG. 6  may also be chemically bonded to DTPA.