Daniel Raleigh

Structural Biology

The research in our group is centered upon studies of protein folding, protein structure and the mechanism of amyloid formation. We want to understand how proteins fold to their final three dimensional structure and we want to understand why the folded conformation of proteins is stable. Our work on amyloid formation is directed towards understanding the physical basis for the pathological aggregation of polypeptides in certain diseases. These projects involve a wide range of techniques, including but not limited to: high resolution multidimensional NMR, protein design, stop flow methods, peptide synthesis and protein chemistry.

Protein Folding

Elucidating how the amino acid sequence determines structure, the protein folding problem, is a central issue in modern structural biology. Our laboratory is currently studying the folding of two proteins, the ribosomal protein L9 and the E3/E1p-binding domain. A second area of interest is the nature of partially folded states of proteins. In recent years it has become clear that many proteins can exist in a partially folded state, known as the molten globule state, which contains a high degree of secondary structure but which lacks fixed tertiary interactions. We are studying the molten globule state formed by a small calcium binding protein, a-lactalbumin. Our studies of natural proteins are complemented by efforts in de novo protein design.

Amyloid Formation

In certain diseases normally soluble proteins aggregate to form a dense structure called the amyloid fibril. Amyloid deposits are present in individuals suffering from type-II diabetes, Alzheimer's disease and in patients undergoing kidney dialysis. Little is known about the molecular mechanism of amyloid formation. We are studying amyloid formation by the polypeptides amylin and calcitonin. Amylinis a 37 residue peptide which forms amyloid deposits in type-II diabetes while calcitonin forms amyloid deposits in the thyroid. Our work involves the preparation of the peptides via solid phase peptide synthesis, the characterization of the structure of these molecules and studies of the kinetics of amyloid formation.

• National Science Foundation Career Award 1996-2000.

pH-dependent stability and folding kinetics of a protein with an unusual alpha-beta topology: The C-terminal domain of the ribosomal protein L9. Sato S, Raleigh DP.JOURNAL OF MOLECULAR BIOLOGY, 318 (2): 571-582 APR 26 2002

Low levels of asparagine deamidation can have a dramatic effect on aggregation of amyloidogenic peptides: Implications for the study of amyloid formation. Nilsson MR, Driscoll M, Raleigh DP.PROTEIN SCIENCE, 11 (2): 342-349 FEB 2002

On the relationship between protein stability and folding kinetics: A comparative study of the N-terminal domains of RNase HI, E-coli and Bacillus stearothermophilus L9. Sato S, Xiang S, Raleigh DP.JOURNAL OF MOLECULAR BIOLOGY, 312 (3): 569-577 SEP 21 2001

Rescuing a destabilized protein fold through backbone cyclization. Camarero JA, Fushman D, Sato S, Giriat I, Cowburn D, Raleigh DP, Muir TW. JOURNAL OF MOLECULAR BIOLOGY, 308 (5): 1045-1062 MAY 18 2001

A comparative study of peptide models of the alpha-domain of alpha-lactalbumin, lysozyme, and alpha-lactalbumin/lysozyme chimeras allows the elucidation of critical factors that contribute to the ability to form stable partially folded states. Demarest SJ, Zhou SQ, Robblee J, Fairman R, Chu B, Raleigh DP.BIOCHEMISTRY, 40 (7): 2138-2147 FEB 20 2001

Synthesis and purification of amyloidogenic peptides. Nilsson MR, Nguyen LL, Raleigh DP.ANALYTICAL BIOCHEMISTRY, 288 (1): 76-82 JAN 1 2001

A protein dissection study demonstrates that two specific hydrphobic clusters play a key role in stabilizing the core structure of the molten globule state of human alpha-lactalbumin. Demarest SJ, Horng JC, Raleigh DP.PROTEINS-STRUCTURE FUNCTION AND GENETICS, 42 (2): 237-242 FEB 1 2001

De novo design of helical bundles as models for understanding protein folding and function. Hill RB, Raleigh DP, Lombardi A, Degrado NF. ACCOUNTS OF CHEMICAL RESEARCH, 33 (11): 745-754 NOV 2000

The failure of simple empirical relationships to predict the viscosity of mixed aqueous solutions of guanidine hydrochloride and glucose has important implications for the study of protein folding. Sato S, Sayid CJ, Raleigh DP.PROTEIN SCIENCE, 9 (8): 1601-1603 AUG 2000

pH-dependent interactions and the stability and folding kinetics of the n-terminal domain of L9. Electrostatic interactions are only weakly formed in the transition state for folding. Luisi DL, Raleigh DP.JOURNAL OF MOLECULAR BIOLOGY, 299 (4): 1091-1100 JUN 16 2000

N-15 R-1 rho measurements allow the determination of ultrafast protein folding rates. Vugmeyster L, Kroenke CD, Picart F, Palmer AG, Raleigh DP.JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 122 (22): 5387-5388 JUN 7 2000

Stereospecificity of the reaction catalyzed by enoyl-CoA hydratase. Wu WJ, Feng YG, He X, Hofstein HA Raleigh DP, Tonge PJ. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 122 (17): 3987-3994 MAY 3 2000

pH jump studies of the folding of the multidomain ribosomal protein L9: The structural organization of the N-terminal domain does not affect the anomalously slow folding of the C-terminal domain. Sato S, Luisi DL, Raleigh DP.BIOCHEMISTRY, 39 (16): 4955-4962 APR 25 2000

Rational modification of protein stability by the mutation of charged surface residues. Spector S, Wang MH, Carp SA, Robblee J, Hendsch ZS, Fairman R, Tidor B, Raleigh DP.BIOCHEMISTRY, 39 (5): 872-879 FEB 8 2000

Solution structure of a peptide model of a region important for the folding of alpha-lactalbumin provides evidence for the formation of nonnative structure in the denatured state. Demarest SJ, Raleigh DP.PROTEINS-STRUCTURE FUNCTION AND GENETICS, 38 (2): 189-196 FEB 1 2000

Analysis of amylin cleavage products provides new insights into the amyloidogenic region of human amylin. Nilsson MR, Raleigh DP.JOURNAL OF MOLECULAR BIOLOGY, 294 (5): 1375-1385 DEC 17 1999

Local interactions and the role of the 6-120 disulfide bond in alpha-lactalbumin: implications for formation of the molten globule state. Moriarty DF, Demarest SJ, Robblee J, Fairman R, Raleigh DP.BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY, 1476 (1): 9-19 JAN 3 2000

Defining the core structure of the alpha-lactalbumin molten globule state. Demarest SJ, Boice JA, Fairman R, Raleigh DP.JOURNAL OF MOLECULAR BIOLOGY, 294 (1): 213-221 NOV 19 1999

Submillisecond folding of the peripheral subunit-binding domain. Spector S, Raleigh DP.JOURNAL OF MOLECULAR BIOLOGY, 293 (4): 763-768 NOV 5 1999

Rethinking the "amyloidogenic" region of human amylin. Nilsson MR, Raleigh DP.BIOPHYSICAL JOURNAL, 76 (1): A418-A418 Part 2 JAN 1999

Effects of varying the local propensity to form secondary structure on the stability and folding kinetics of a rapid folding mixed alpha/beta protein: Characterization of a truncation mutant of the N-terminal domain of the ribosomal protein L9. Luisi DL, Kuhlman B, Sideras K, Evans PA, Raleigh DP.JOURNAL OF MOLECULAR BIOLOGY, 289 (1): 167-174 MAY 28 1999

Local interactions drive the formation of nonnative structure in the denatured state of human alpha-lactalbumin: A high resolution structural characterization of a peptide model in aqueous solution. Demarest SJ, Hua YX, Raleigh DP.BIOCHEMISTRY, 38 (22): 7380-7387 JUN 1 1999

Folding of the multidomain ribosomal protein L9: The two domains fold independently with remarkably different rates. Sato S, Kuhlman B, Wu WJ, Raleigh DP.BIOCHEMISTRY, 38 (17): 5643-5650 APR 27 1999

pK(a) values and the pH dependent stability of the N-terminal domain of L9 as probes of electrostatic interactions in the denatured state. Differentiation between local and nonlocal interactions. Kuhlman B, Luisi DL, Young P, Raleigh DP.BIOCHEMISTRY, 38 (15): 4896-4903 APR 13 1999

Nativelike structure and stability in a truncation mutant of a protein minidomain: The peripheral subunit-binding domain. Spector S, Young P, Raleigh DP.BIOCHEMISTRY, 38 (13): 4128-4136 MAR 30 1999

Conformational analysis of a set of peptides corresponding to the entire primary sequence of the n-terminal domain of the ribosomal protein L9: Evidence for stable native-like secondary structure in the unfolded state. Luisi DL, Wu WJ, Raleigh DP.JOURNAL OF MOLECULAR BIOLOGY, 287 (2): 395-407 MAR 26 1999

Effects of sequential proline substitutions on amyloid formation by human amylin(20-29). Moriarty DF, Raleigh DP.BIOCHEMISTRY, 38 (6): 1811-1818 FEB 9 1999

Conformational analysis of peptide fragments derived from the peripheral subunit binding domain from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus: Evidence for nonrandom structure in the unfolded state. Spector S, Rosconi M, Raleigh DP. BIOPOLYMERS, 49 (1): 29-40 JAN 1999

Global analysis of the effects of temperature and denaturant on the folding and unfolding kinetics of the N-terminal domain of the protein L9. Kuhlman B, Luisi DL, Evans PA, Raleigh DP.JOURNAL OF MOLECULAR BIOLOGY, 284 (5): 1661-1670 DEC 18 1998

Global analysis of the thermal and chemical denaturation of the N-terminal domain of the ribosomal protein L9 in H2O and D2O. Determination of the thermodynamic parameters, Delta H degrees, Delta S degrees, and Delta C degrees(p), and evaluation of solvent isotope effects. Kuhlman B, Raleigh DP. PROTEIN SCIENCE, 7 (11): 2405-2412 NOV 1998

Stereospecific H-1 and C-13 NMR assignments of crotonyl CoA and hexadienoyl CoA: Conformational analysis and comparison with protein-CoA complexes. Wu WJ, Tonge PJ, Raleigh DP.JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 120 (39): 9988-9994 OCT 7 1998

Peptide models of local and long-range interactions in the molten globule state of human alpha-lactalbumin. Demarest SJ, Fairman R, Raleigh DP.JOURNAL OF MOLECULAR BIOLOGY, 283 (1): 279-291 OCT 16 1998

Conformational heterogeneity about pipecolic acid peptide bonds: Conformational, thermodynamic, and kinetic aspects. Wu WJ, Raleigh DP.JOURNAL OF ORGANIC CHEMISTRY, 63 (19): 6689-6698 SEP 18 1998

Amide proton exchange measurements as a probe of the stability and dynamics of the N-terminal domain of the ribosomal protein L9: Comparison with the intact protein. Vugmeyster L, Kuhlman B, Raleigh DP.PROTEIN SCIENCE, 7 (9): 1994-1997 SEP 1998

Conformational analysis of the interdomain linker of the central homology region of chloroplast initiation factor IF3 supports a structural model of two compact domains connected by a flexible tether. Hua YX, Raleigh DP.FEBS LETTERS, 433 (1-2): 153-156 AUG 14 1998

Submillisecond folding of the N-terminal domain of the ribosomal protein L9. Kuhlman B, Evans PA, Raleigh DP. BIOPHYSICAL JOURNAL, 74 (2): A128-A128 Part 2 FEB 1998

On the global architecture of initiation factor IF3: A comparative study of the linker regions from the Escherichia coli protein and the Bacillus stearothermophilus protein. Hua YX, Raleigh DP. JOURNAL OF MOLECULAR BIOLOGY,A role for the C-terminus of calcitonin in aggregation and gel formation: A comparative study of C-terminal fragments of human and salmon calcitonin. Moriarty DF, Vagts S, Raleigh DP.BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 245 (2): 344-348 APR 17 1998

A Cooperative folding of a protein mini domain: The peripheral subunit-binding domain of the pyruvate dehydrogenase multienzyme complex. Spector S, Kuhlman B, Fairman R, Wong E, Boice JA, Raleigh DP.JOURNAL OF MOLECULAR BIOLOGY, 276 (2): 479-489 FEB 20 1998

Structure and stability of the N-terminal domain of the ribosomal protein L9: Evidence for rapid two-state folding. Kuhlman B, Boice JA, Fairman R, Raleigh DP.BIOCHEMISTRY, 37 (4): 1025-1032 JAN 27 1998

Identification of the epitopes of calcitonin gene-related peptide (CGRP) for two anti-CGRP monoclonal antibodies by 2D NMR. Hubbard JAM, Raleigh DP, Bonnerjea JR, Dobson CM. PROTEIN SCIENCE, 6 (9): 1945-1952 SEP 1997

An exceptionally stable helix from the ribosomal protein L9: Implications for protein folding and stability. Kuhlman B, Yang HY, Boice JA, Fairman R, Raleigh DP. JOURNAL OF MOLECULAR BIOLOGY, 270 (5): 640-647 AUG 1 1997

Calcium binding peptides from alpha-lactalbumin: Implications for protein folding and stability. Kuhlman B, Boice JA, Wu WJ, Fairman R, Raleigh DP.BIOCHEMISTRY, 36 (15): 4607-4615 APR 15 1997

Structure of hexadienoyl-CoA bound to enoyl-CoA hydratase determined by transferred nuclear Overhauser effect measurements: Mechanistic predictions based on the X-ray structure of 4-(chlorobenzoyl)-CoA dehalogenase. Wu WJ, Anderson VE, Raleigh DP, Tonge PJ. BIOCHEMISTRY, 36 (8): 2211-2220 FEB 25 1997

Crystallization of a designed peptide from a molten globule ensemble. Betz SF, Raleigh DP, DeGrado WF, Lovejoy B, Anderson D, Ogihara N, Eisenberg D. FOLDING & DESIGN, 1 (1): 57-64 1996

Protein folding: From basic science to biotechnology. Miller WT, Raleigh DP.GENETIC ANALYSIS BIOMOLECULAR ENGINEERING, 12 (5-6): 169-172 MAR 1996

A De-novo designed protein mimics the native-state of natural proteins. Raleigh DP, Betz SF, Degrado WF. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 117 (28): 7558-7559 JUL 19 1995

Thermodynamic genetics of the folding of the b1 immunoglobulin-binding domain from streptococcal protein-g. Oneil KT, Hoess RH, Raleigh DP, Degrado WF. PROTEINS-STRUCTURE FUNCTION AND GENETICS, 21 (1): 11-21 JAN 1995