Peter E. Wright Home Page

We are using multidimensional nuclear magnetic resonance (NMR) spectroscopy to investigate the structures and dynamics of proteins in solution. Such studies are essential for understanding the mechanisms of action of these proteins and for elucidating structure-function relationships.

Protein Structure Determination in Solution

Solution 3-dimensional structures have been determined for a number of proteins and protein complexes of molecular weight up to more than proteins20kD. We are using heteronuclear 3- and 4-dimensional NMR and proteins labeled with deuterium, carbon 13, or nitrogen 15 to extend the NMR structure determination method to even larger proteins and protein complexes.

In addition, new computational methods are being developed to facilitate structure determination and refinement, including the use of template-assisted assignment methods for NOESY spectra obtained with multidimensional nuclear Overhauser effect spectrometry. These methods utilize existing three-dimensional structures of homologous proteins and should greatly increase the speed of structure determination.

Transcription Factor-DNA Complexes

NMR methods are being used to determine the 3-dimensional structures and intramolecular dynamics of various DNA-binding motifs from eukaryotic transcriptional regulatory proteins, both free and complexed with the target DNA. Structures determined include proteins containing zinc finger motifs, the HMG box transcription factor LEF-1 (lymphoid factor, lymphoid enhancer-binding factor 1, and the runt domain of the polyoma virus enhancer-binding protein 2 (PEBP2).

Three-dimensional structures have been determined for the complex of zf1-3, a protein containing the N-terminal 3 of the 9 zinc fingers of transcription factor IIIA (TFIIIA), with the cognate 15-bp oligonucleotide duplex. The 3 zinc fingers bind in the DNA major groove, thus validating parts of our earlier model of the TFIIIA-DNA complex deduced from biochemical experiments. The structures contain several novel features and indicate that prevailing models of DNA recognition, which assume that zinc fingers are independent modules that contact bases through a limited set of amino acids, are outmoded.

In addition to its role in binding to and regulating the 5S RNA gene, TFIIIA also forms a complex with the 5S RNA transcript. The minimal set of zinc fingers required for high-affinity 5S RNA recognition consists of fingers 4-6, and the minimal region of the 5S RNA needed to bind these fingers has been mapped in J. Gottesfeld’s laboratory, Department of Molecular Biology. NMR studies of the protein-RNA complex are in progress and should give important insights into the structural basis for 5S RNA recognition.

NMR studies of 2 alternate splice variants of the zinc finger protein of Wilms tumor are in progress. These proteins differ only through insertion of 3 additional amino acids (lysine, threonine, and serine) in the linker between fingers 3 and 4, yet have marked differences in their DNA-binding properties and subcellular localization. NMR studies indicate that the insertion increases the flexibility of the linker between fingers 3 and 4 and abrogates binding of the fourth zinc finger to its cognate site in the DNA major groove, thereby modulating DNA-binding activity.

We are studying DNA-binding domains from 2 important transcription thefactors: PEBP2 and the human estrogen-related nuclear receptor 2. PEBP2 participates in the normal functioning of T cells and in the onset of certain types of leukemia. Using NMR, we determined the fold of the DNA-binding domain of PEBP2, termed the runt domain, and mapped the binding surface for DNA. We also determined the structure of the DNA-binding domain of human estrogen-related nuclear receptor 2 bound to its cognate DNA recognition element. The estrogen receptor differs from most other hormone receptors in that it binds DNA as a monomer. The structure reveals novel minor groove contacts stabilized by intramolecular protein-protein interactions and provides new insights into the evolution of the nuclear hormone receptors. We also completed structure determination for an RNA recognition motif of the nuclear protein ALY, which plays an essential role in posttranscriptional regulation and RNA export. We are now mapping its interactions with RNA.

Protein-Protein Interactions in Transcriptional Regulation

Transcriptional regulation in eukaryotes relies on protein-protein interactions between DNA-bound factors and coactivators that, in turn, interact with the basal transcription machinery. The transcriptional coactivator CREB-binding protein (CBP) and its homolog p300 play an essential role in cell growth, differentiation, and development. Understanding the molecular mechanisms by which CBP and p300 recognize their various target proteins is of fundamental biomedical importance. CBP and p300 have been implicated in diseases such as leukemia, cancer, and mental retardation and are novel targets for therapeutic intervention.

In recent years, we determined the structure of the kinase-inducible activation domain of the transcription factor CREB bound to its target domain (the KIX domain) in CBP and the structures of the PHD, bromo, and CH3 domains of CBP. Ongoing work is directed toward mapping the interactions between KIX and the transcriptional activation domain of the proto-oncogene c-Myb, and understanding of the thermodynamics of the coupled folding and binding processes involved in these interactions.

In the past year, we completed the structure determination of the complex consisting of CBP and a p160 nuclear receptor coactivator termed ACTR. Molecular interactions between these 2 proteins are central to regulation of gene expression by the nuclear hormone receptors and are essential for proper control of the cell cycle, differentiation, and apoptosis. Because ACTR is overexpressed in many breast and ovarian cancers, perturbing CBP-mediated signal transduction, this complex is an attractive target for the design of antitumor drugs.

In addition, we determined the structure of the complex between the hypoxia-inducible factor Hif-1a and the CH1 domain of CBP. The interaction between Hif-1a and CBP/p300 is of major therapeutic interest because of the central role Hif-1a plays in tumor progression and metastasis; disruption of this interaction leads to attenuation of tumor growth. We are continuing to map the multiplicity of interactions between CBP/p300 domains and their biological targets to understand the complex interplay of interactions that mediate key biological processes in health and disease.

Folding of Proteins and Protein Fragments

The molecular mechanism by which proteins fold into their 3-dimensional structures remains one of the most important unsolved problems in structural biology. Nuclear magnetic resonance (NMR) spectroscopy is uniquely suited to provide information on the structure of transient intermediates formed during protein folding. We used NMR methods to show that many peptide fragments of proteins have a tendency to adopt folded conformations in water solution. The presence of transiently populated folded structures, including reverse turns, helices, nascent helices, and hydrophobic clusters, in water solutions of short peptides has important implications for initiation of protein folding. Formation of elements of secondary structure probably plays an important role in the initiation of protein folding by reducing the number of conformations that must be explored by the polypeptide chain and by directing subsequent folding pathways.

Apomyoglobin Folding Pathway

A major program in our laboratory is directed toward a structural and mechanistic description of the apomyoglobin folding pathway. We used quenched-flow pulse labeling methods in conjunction with 2-dimensional NMR spectroscopy to map the kinetic folding pathway of the wild type protein. With these methods, we showed that an intermediate in which the A, G, and H helices adopt hydrogen-bonded secondary structure is formed within 6 ms of the initiation of refolding. Folding then proceeds by stabilization of structure in the B helix and then in the C and E helices. We are now using carefully selected myoglobin mutants and both optical stopped flow spectroscopy and NMR methods to further probe the kinetic folding pathway. For at least 2 of the mutants studied, the changes in amino acid sequence have resulted in changes in the folding pathway of the protein. In addition, these experiments are provided important insights into the local interactions that stabilize the kinetic folding intermediate and revealed significant heterogeneity in the folding process.

The structure of unfolded and partly folded forms of the protein. Apomyoglobin provides a unique opportunity for detailed characterization of the structure and dynamics of a protein-folding intermediate. Conditions were identified under which the apomyoglobin molten globule intermediate is sufficiently stable for acquisition of multidimensional heteronuclear NMR spectra. Analysis of carbon 13 and other chemical shifts and measurements of polypeptide dynamics provided unprecedented insights into the structure of this state. The A, G, and H helices and part of the B helix are folded and form the core of the molten globule. This core is stabilized by relatively nonspecific hydrophobic interactions which restrict the motions of the polypeptide chain. Fluctuating helical structure is formed in regions outside the core, although the population of helix is low and the chain retains considerable flexibility. The F helix acts as a gate for heme binding and only adopts stable structure in the fully folded holoprotein.

The acid-denatured state of apomyoglobin is an excellent model for the fluctuating local interactions that lead to the transient formation of unstable elements of secondary structure and local hydrophobic clusters during the earliest stages of folding. We fully assigned the polypeptide backbone resonances in the acid-denatured unfolded states of apomyoglobin and in the urea-denatured unfolded state. The NMR data indicated substantial formation of helical secondary structure in the acid-denatured state in regions that form the A and H helices in the folded protein and also reveal nonnative structure in the D- and E-helix regions.

Because the A and H regions adopt stabilized helical structure in the earliest detectable folding intermediate, these results lend strong support to folding models in which spontaneous formation of local elements of secondary structure plays a role in initiating formation of the A-[B]-G-H molten globule folding intermediate. In addition to formation of transient helical structure, local formation of hydrophobic cluster has been detected by using nitrogen 15 relaxation measurements. Significantly, these clusters are formed in regions where the average surface area buried upon folding is large. In contrast to acid-denatured unfolded apomyoglobin, the urea-denatured state is largely devoid of structure, although residual hydrophobic interactions have been detected by using relaxation measurements.

To probe long-range interactions in unfolded and partially folded states of apomyoglobin, we introduced spin label probes at several sites throughout the polypeptide chain. These experiments led to the surprising discovery that structures with nativelike topology exist within the ensemble of conformations formed by the acid-denatured unfolded state of apomyoglobin. They also indicate that the packing of helices in the molten globule state is similar to that in the native folded protein.

The view of protein folding that resulted from our work on apomyoglobin is one in which collapse of the polypeptide chain to form increasingly compact states leads to progressive accumulation of secondary structure and increasing restriction of fluctuations in the polypeptide backbone. Chain flexibility is greatest at the earliest stages of folding, in which transient elements of secondary structure and local hydrophobic clusters are formed. As the folding protein becomes increasingly compact, backbone motions become more restricted, the hydrophobic core is formed and extended, and nascent elements of secondary structure are progressively stabilized. The ordered tertiary structure characteristic of the native protein, with well-packed side chains and relatively low-amplitude local dynamics, appears to form rather late in folding.

Apoplastocyanin Folding

Structural characterization of an unfolded state and the kinetic folding pathways of the b-sheet protein apoplastocyanin are also in progress. Apoplastocyanin offers a unique opportunity to study an unfolded protein under nondenaturing conditions, because it forms an unfolded state at neutral pH under low-salt conditions. Extensive NMR analysis reveals that the unfolded polypeptide has a propensity to populate the b-region of dihedral angle space in regions of the chain that form b-strands in the folded protein. A region of nonnative helical structure is also present. Relaxation measurements indicated that the polypeptide chain is highly flexible throughout its length, with large-amplitude fluctuations on a timescale shorter than 1 ns. We are using real-time NMR experiments to probe the kinetic folding of apoplastocyanin.

Fragments of Prion Proteins

A number of protein systems are unfolded or only partly folded until they bind ligand or substrate. Although scientists have known for many years that this fact applies to peptide hormones, we now recognize that certain transcription factors, cyclin-dependent kinase inhibitors, and other proteins are unfolded in the absence of their natural receptors. In addition, folding has been implicated in a number of disease states, such as Alzheimer’s disease and the prion diseases such as kuru and bovine spongiform encephalopathy (mad cow disease).

We are characterizing the structure and dynamics of some of the fragments of the prion protein. Relaxation measurements revealed that the dynamics of the N-terminal part of the molecule differ substantially from the dynamics of the C-terminal folded domain, indicating that the N-terminal part of the molecule is unfolded in solution. Binding of copper to the octapeptide repeats was examined with both full-length protein and peptide fragments. We found that copper binds cooperatively and induces structure formation. Increasingly, evidence indicates that the physiological function of the prion protein may involve copper storage or transport.

Biographical Information

Education

University of Auckland, New Zealand, B.Sc., 1964-68, Chemistry

University of Auckland, New Zealand, M.Sc., 1968-69, Chemistry

University of Auckland, New Zealand, Ph.D., 1969-72, Chemistry

University of Oxford, England, postdoctoral, 1972-76, Chemistry/Biochemistry

Professional Record

1972-1973	New Zealand University Grants Committee Postdoctoral Fellow, University of Oxford, England
1973-1976	Research Associate, Inorganic Chemistry Laboratory, University of Oxford, England
1976-1980	Lecturer, Department of Inorganic Chemistry, University of Sydney, Australia
1980-1984	Senior Lecturer, Department of Inorganic Chemistry, University of Sydney, Australia
1984-present	Member/Professor, Department of Molecular Biology, The Scripps Research Institute.
1987-present	Chairman, Department of Molecular Biology, The Scripps Research Institute
1987-present	Cecil H. and Ida M. Green Investigator in Biomedical Research, The Scripps Research Institute

Awards and Activities - 2002

Editor-in-Chief, Journal of Molecular Biology;

Editorial Boards: Biochemistry, Current Opinion in Structural Biology, Journal of Biomolecular NMR, Encyclopedia of Molecular Biology.

Last revised: June 18, 2009

Peter E. Wright Home Page

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Nuclear Magnetic Resonance Investigations of the Three-Dimensional Structure and Dynamics of Proteins in Solution