Phone: 850 644-3361
These publications were made possible thanks to the hard-working students, postdocs and technicians in the Blaber Lab; as well as the opportunity to work with outstanding collaborators.
From 1994-2005 the Blaber Lab was located in the Institute of Molecular Biophysics at FSU. Since 2005, the lab has been in the Department of Biomedical Sciences in the FSU College of Medicine.
Blaber Lab Recent Publications - (Updated 5/7/18)
The following is a partial listing of the most recent publications from the Blaber lab.
A listing of all publications for Dr. Michael Blaber can be found in his cv.
A Mathematical Model for the Determination of Mouse Excisional Wound Healing Parameters from Photographic Data, Cogan, N.G., Mellers, A.P., Patel, B.N., Powell, B.D., Aggarwal, M., Harper, K.M. and Blaber, M. (Wound Repair and Regeneration, in press). Access can be found here.
We present a mathematical model to quantify parameters of mouse excisional wound healing from photographic data. The equation is a piecewise linear function in log scale that includes key parameters of initial wound radius (R0), an initial wound stasis phase (Ti), and time to wound closure (Tc); subsequently, these terms permit calculation of a latter active proliferative phase (Tp), and the healing rate (HR) during this active phase. A daily photographic record of wound healing (utilizing 6 mm diameter splinted excisional wounds) permits the necessary sampling for robust parameter refinement. When implemented with an automated nonlinear fitting routine, the healing parameters are determined in an operator-independent (i.e. unbiased) manner. The model was evaluated using photographic data from a splinted excisional surgical procedure involving several different mouse cohorts. Model fitting demonstrates excellent coefficients of determination (R2) in each case. The model thus permits quantitation of key parameters of excisional wound healing, from initial wounding through to wound closure, from photographic data.
Investigating the Dynamics and Polyanion Binding Sites of Fibroblast Growth Factor-1 Using Hydrogen-Deuterium Exchange Mass Spectrometry, Angalakurthi, S.K., Tenorio, C.A, Blaber, M., Middaugh, C.R.(Protein Science, in press). Access can be found here.
In this study, we examined the local dynamics of acidic fibroblast growth factor (FGF-1) as well as the binding sites of various polyanions including poly-sulfates (heparin and low MW heparin) and poly-phosphates (phytic acid and ATP) using hydrogen-deuterium exchange mass spectrometry (HX-MS). For local dynamics, results are analyzed at the peptide level as well as in terms of buried amides employing crystallographic B-factors and compared with a residue level heat map generated from HX-MS results. Results show that strand 4 and 5 and the turn between them to be the most flexible regions as was previously seen by NMR. On the other hand, the C-terminal strands 8, 9 and 10 appear to be more rigid which is also consistent with crystallographic B-factors as well as local dynamics studies conducted by NMR. Crystal structures of FGF-1 in complex with heparin have shown that heparin binds to N-terminal Asn18 and to C-terminal Lys105, Tryp107, Lys112, Lys113, Arg119, Pro121, Arg122, Gln127 and Lys128 indicating electrostatic forces as dominant interactions. Heparin binding as determined by HX-MS is consistent with crystallography data. Previous studies have also shown that other polyanions including low MW heparin, phytic acid and ATP dramatically increase the thermal stability of FGF-1. Using HX-MS, we find other poly anions tested bind in a similar manner to heparin, primarily targeting the turns in the lysine rich C-terminal region of FGF-1 along with two distinct N-terminal regions that contains lysines and arginines/ histidines. This confirms the interactions between FGF-1 and polyanions are primary directed by electrostatics.
The Folding Nucleus Structure Persists in Thermally-aggregated FGF-1, Longo, L.M., Gao, Y., Tenorio, C.A., Wang, G., Paravastu, A.K. and Blaber, M. (Protein Science 27, 431-440 (2018). First published: 27 October 2017
https://doi.org/10.1002/pro.3332. Access can be found here.
We compare the thermal unfolding of fibroblast growth factor-1 (FGF-1), a naturally-evolved beta-trefoil protein which thermally aggregates, and Phifoil, a designed purely-symmetric protein which exhibits reversible thermal denaturation. These two proteins share a folding nucleus sequence. Detailed thermodynamic analysis of FGF-1 indicates that thermal aggregation initiates early in the unfolding pathway. Solid-state NMR of the FGF-1 aggregate identifies regions of structure consistent with the folding nucleus. The results show that design of an efficient folding nucleus, and the avoidance of aggregation in the folding pathway, are potentially separable design criteria – the latter of which should focus upon the physicochemical properties of primary structure outside the folding nucleus.
An S116R Phosphorylation Site Mutation in Human FGF-1 Differentially Affects Mitogenic and Glucose Lowering Activities, Xia, X., Kumru, O.S., Blaber, S.I., Middaugh, C.R., Li, L., Ornitz, D.M., Suh, J.M., Atkins, A.R., Downes, M., Evans, R.M., Tenorio, C.A., Bienkiewicz, E. and Blaber, M., J. Pharm. Sci. 105, 3507-19. Access can be found here.
Human FGF-1 has recently been shown to be a novel insulin-sensitizer. Position Serine 116 in FGF-1 is a site of phosphorylation. We report the properties of an Arginine mutation at this position (S116R) that is naturally present in bovine FGF-1 and cannot be phosphorylated. The S116R mutant is shown to have a reduced mitogenic activity, but an increased duration of insulin sensitization in ob/ob hyperglycemic mice. Thus, the mitogenic and insulin sensitizing functions of FGF-1 can potentially be differentially targeted in therapeutic design.
Evolution of a Protein Folding Nucleus, Xia, X., Longo, L.M., Sutherland, M.A. and Blaber, M., Protein Science 25, 1227-1240 (2016). Access can be found here.
The folding nucleus (FN) is a cryptic element within protein primary structure that enables an efficient folding pathway - and is the postulated key heritable element in the evolution of protein architecture. However, almost nothing is known regarding how the FN structurally changes as complex protein architecture evolves from simpler peptide motifs. We report characterization of the FN of a designed purely symmetric ?-trefoil protein by ?-value analysis and compare it with the structure and folding properties of key foldable intermediates along the evolutionary trajectory of the ?-trefoil. The results show how novel turn structure created by gene fusion is subsequently incorporated into a larger and more efficient FN. Furthermore, the FN is adjusted by circular permutation in response to destabilizing functional mutation. This report provides the first insight into FN evolution. Selected for inclusion in the "Protein Evolution" special issue (June 2016).
Engineering a Cysteine-Free Form of Human Fibroblast Growth Factor-1 for “Second Generation” Therapeutic Application, Xia, X., Kumru, O.S., Blaber, S.I., Middaugh, C.R., Li, L., Ornitz, D.M., Sutherland, M.A., Tenorio, C.A. and Blaber, M., J. Pharm. Sci. 105, 1444-1453 (2016). Access can be found here.
Human FGF-1 has unique therapeutic potential in regenerative medicine and the treatment of metabolic disorder; however, biophysical properties intrinsic to FGF-1 are detrimental as regards drug development. FGF-1 contains three buried free thiols (cysteines) whose oxidation contributes to a short functional half-life. Simple mutational substitution without significant destablization is not possible. In this report a novel protein design strategy is employed whereby an additional cysteine is introduced that makes a stabilizing disulfide with one of the buried cysteines in FGF-1. The increase in stability of this novel disulfide almost exactly offsets the instability associated with mutational substitution of the remaining two cysteines - yielding a cysteine free form of FGF-1 with no overall stability perturbation. Cysteine free mutant forms of FGF-1 are shown to have substantially increased activity in functional assays of cell survival and mitogenicity.
Accelerated healing in NONcNZO10/LtJ type 2 diabetic mice by FGF 1, Blaber, S.I., Diaz, J. and Blaber, M. Wound Repair and Regeneration 23, 538-549 (2015) . Access can be found here.
This report describes a detailed animal study, using a genetic mouse model of type II diabetes, for excisional wound repair and its acceleration by topical delivery of FGF-1. There are several important results in this report. The NONcNZO10/LtJ mouse is a newly-developed polygenic model of type II diabetes; however, no detailed description of its dermal wound healing has been reported. We show a delay in wound healing due to the diabetic condition; thus, this mouse is an excellent model with which to study delayed healing of diabetic ulcers. We also show that the rate of healing of splinted excisional wounds can be acclerated by topical application of FGF-1 formulated with heparin. We go on to show that engineered forms of FGF-1 (eFGF-1) that are stabilized in the absence of heparin, can equivalently accelerate dermal healing. Finally, to perform these experiments we had to develop a novel mouse jacket, and the details of this design are included in the report.
Mutation Choice to Eliminate Buried Free Cysteines in Protein Therapeutics, Xia, X., Longo, L.M. and Blaber, M. J. Pharm. Sci. 104, 566–576 (2015). Access can be found here.
Protein therapeutics hold tremendous promise in treating human disease; however, proteins have unique problems as regards their choice as a drug. Principle among these is their ability to unfold and aggregate. A principle contributor to an irreversible unfolding pathway in proteins is the chemical modification of buried free thiols (i.e., cysteine residues). Mutation to eliminate such amino acids is an effective approach to removing one route of irreversible protein unfolding; however, exactly how to design such mutations is not clear. In this report we perform a comprehensive analysis of buried free cysteine mutations in FGF-1. We provide evidence for a key role in maintaining buried H-bonds that the wild-type cysteine engages in. There are two main structural effects in response to cysteine substitution - structural collapse and expansion. In the former case, serine substitution can maintain critical buried H-bonds despite its shorter H-bond distance compared to cysteine; in the latter case, alanine substitution can work best as it can permit room for inclusion of novel buried solvent that can similarly maintain critical buried H-bonds. The results show that simples rules for substitution of buried cysteines (e.g. substitution by the isosteric serine) are insufficient to deal with the structural complexity of the environment surrounding buried cysteines in proteins.
A Single Aromatic Core Mutation Converts a Designed "Primitive" Protein from Halophile to Mesophile Folding, Longo, L.M., Tenorio, C.A., Kumru, O.S., Middaugh, C.R. and Blaber, M., Protein Science 24, 27–37 (2015). Access can be found here.
This report builds upon prior work showing that design of a beta-trefoil protein using an amino acid alphabet enriched for the "pre-biotic" set yields a halophile protein. This suggested a halophile origin of foldable proteins, followed by adaptation into mesophile conditions. In this report we show that such adaptation occurs upon a single amino acid subsitition to the non-pre-biotic amino acid Phenylalanine. Thus, upon emergence of a biosynthetic pathway for aromatic amino acids, a single substititon mutation in the core can provide the stability increase necessary for halophile-to-mesophile adaptation.
Evolution and Design of Protein Structure by Folding Nucleus Symmetric Expansion, Longo, L.M., Kumru, O.S., Middaugh, C.R. and Blaber, M. Cell: Structure 22, 1377-84 (2014) . Access can be found here.
One of the most important papers to come out of the lab. This research report describes successful symmetric protein design by first identifying the folding nucleus by phi-value analysis, followed by expansion of this sequence by the intrinsic structural symmetry to regenerate an intact protein. Experimentally we show that this design strategy yields a stable, foldable polypeptide for a purely-symmetric beta-trefoil architecture. This result provides an explanation as to why Top-Down Symmetric Deconstruction is successful, and also achieves a marked improvement in the design cycle efficiency. The results lead us to postulate that purely-symmetric proteins have redundant folding nuclei, and this provides a potential basis for selective advantage of symmetric protein folds in evolutionary processess involving gene duplication and fusion.
Symmetric Protein Architecture in Protein Design: Top-Down Symmetric Deconstruction, Longo, L. and Blaber, M., Protein Design, Meth. Molec. Biol. 1216:161-82 (2014). Access can be found here.
In this invited book chapter we describe the Top-Down Symmetric Deconstruction method of symmetric protein design. An image of a symmetric solution to the beta-trefoil fold was selected as the cover art for the book.
Prebiotic Protein Design Supports a Halophile Origin of Foldable Proteins, Longo, L.M. and Blaber, M., Frontiers in Extreme Microbiology 4, 1-3 (2014). Access can be found here.
In this Opinion Article we argue in support of the halophile environment as the cradle of proteogenesis. The article covers: 1. Compelling data for a consensus prebiotic set of Ala, Asp, Glu, Gly, Ile, Leu, Pro, Ser, Thr and Val amino acids; 2. Favorable propensities for formation of helix, strand and reverse-turn secondary structure within this set; 3. Foldable potential of polypeptides with a prebiotic amino acid composition within the halophile environment; and 4. support for proteogenesis as a very early aspect of abiogenesis.
Alternative Folding Nuclei Definitions Facilitate the Evolution of a Symmetric Protein Fold from a Smaller Peptide Motif, Longo, L.M., Lee, J., Tenorio, C.A. and Blaber, M. Cell: Structure 21, 1-9 (2013). Access can be found here.
Protein 3° structure symmetry is a defining feature of nearly one-third of protein folds and is generally thought to result from a combination of gene duplication, fusion, and truncation events. Such events represent major replication errors, involving substantial alteration of protein 3° structure and causing regions of exact repeating 1° structure, both of which are generally considered deleterious to protein folding. Thus, the prevalence of symmetric protein folds is counterintuitive and suggests a specific, yet unexplained, robustness. Using a designed ?-trefoil protein, we show that purely symmetric 1° structure enables utilization of alternative definitions of the critical folding nucleus in response to gross structural rearrangement. Thus, major replication errors producing 1° structure symmetry can conserve foldability. The results of this study provide an explanation for the prevalence of symmetric protein folds and highlight a critical role for 1° structure symmetry in protein evolution.
Simplified Protein Design Biased for Pre-Biotic Amino Acids Yields a Foldable, Halophilic Protein, Longo, L., Lee, J. and Blaber, M., (2012) Proc. Natl. Acad. Sci. USA, 110, 2135-2139 (2013). (Kasha Award for best paper in molecular biophysics) (selected by Faculty of 1000). Access can be found here.
This report is an experimental test of our earlier hypothesis that the set of pre-biotic amino acids describes a foldable set, compatible with the halophile environment. In this study we enriched a de novo designed synthetic beta-trefoil protein (Symfoil)for the pre-biotic amino acid alphabet. This enrichment approached 80% pre-biotic composition. The resulting protein ("Primitive version #2")was fully foldable but only within a high salt (i.e., halophile) environment. Thus, the results suggest that proteogenesis was a potentially early abiogenic event, and likely occurred within a high-salt environment (e.g. evaporative lake). Both of these conclusions are counter to existing paradigms of hydrothermal vent origin, and RNA-first. This paper won the 2013 Kasha Award for best publication in molecular biophysics.
Activation Profiles of Human Kallikrein-related Peptidases by Matrix Metalloproteinases, Yoon, H., Blaber, S.I., Li, W., Scarisbrick, I.A. and Blaber, M., Biological Chemistry 394, 137-147 (2013). Access can be found here.
In this study we asked whether functional intersection is possible between
the kallikrein-related peptidases (i.e., KLKs) and members of the matrix metalloproteinase (MMP) family by evaluating the ability of the MMPs to activate pro-KLKs. The results identify MMP-20 as a broad activator of pro-KLKs, suggesting the potential for intersection of the KLK and MMP axes under pathological dysregulation of MMP-20 expression.
Protein Design: A Vast Unexploited Resource, Longo, L.M. and Blaber, M., J. Proteins and Proteomics, 3, 77-81. Access can be found here.
This review puts forth the argument that successful de novo protein design will bring about an economic windfall of a magnitude similar to that realized by the development of synthetic organic chemistry. Conversely, if such an economic benefit has not occurred, then the essential problem of de novo design has not yet been effectively solved. The review highlights developments in top-down protein design as a novel development in experimental de novo protein design.
Pharmacokinetic Properties of 2nd-generation Fibroblast Growth Factor-1 Mutants for Therapeutic Application, Xia, X., Babcock, J.P., Blaber, S.I., Harper, K.M. and Blaber, M., PLoS ONE 7(11): e48210. Access can be found here.
This report describes a pharmacokinetic study (in rabbits) of wild-type and mutant forms of human fibroblast growth factor-1. Through the use of a mutant form of FGF-1 that lacks heparin-binding functionality, we show that heparan sulfat proteoglycan sequestration is the basis of the peripheral compartment in the PK profile. FGF-1 is typically formulated in the presence of heparin; however, this study shows that a consequence of this is a high initial systemic concentration, and low overall mean residence time. Conversely, stabilized mutant forms of FGF-1 that do not require heparin in the formulation are sequested efficiently after administration and do not exhibit high circulating levels. Furthermore, stabilizing mutants exhibit reduced redistribution kinetics (i.e., release from heparan sulfate proteoglycan); thus, a longer mean residence time. The results show how engineered forms of FGF-1 (for application in wound healing) can achieve benefits of efficacy, safety as well as cost.
Experimental Support for the Foldability-Function Tradeoff Hypothesis: Segregation of the Folding Nucleus and Functional Regions in FGF-1, Longo, L., Lee, J. and Blaber, M., Prot. Sci. 21, 1911-1920. Access can be found here.
A "stability/function tradeoff" hypothesis in proteins has been supported by a number of studies over more than a decade; however, a "foldability/function tradeoff" hypothesis is a recent development with little direct experimental support. In this report we describe a phi-value analysis of the FGF-1 protein that identifies turn regions essential to formation of the critical folding transition state. Although the overall fold of FGF-1 is symmetric (the beta-trefoil fold) the distribution of regions forming the folding transition state is shown to be highly-asymmetric. Furthermore, these regions are segregated from regions known to be responsible for various functionality of FGF-1 (e.g., heparin-binding, receptor-binding, and nuclear localization). These results provide essentially the first experimental support for the "foldability/function tradeoff" hypothesis. Importantly for protein design, the results identify that only a fraction of the structure is essential for formation of the important folding transition state, and that functional residues are a form of deviation from ideal symmetry within the overall fold. This report received the 2012 "Best Paper Award" from the Protein Society.
Emergence of Symmetric Protein Architecture from a Simple Peptide Motif: Evolutionary Models, Blaber, M., Lee, J. and Longo, L., Cell. Molec. Life Sci. 69, 3999-4006. Access can be found here.
Symmetric elements within a protein fold argue for gene duplication and fusion in their evolution; however, purely-symmetric amino acid sequences are also reported by some investigators to contribute to folding frustration. This conundrum calls into question such evolutionary models as well as the utility of symmetry in de novo protein design (despite its potential to simplify the design problem substantially). This review covers this subject and argues that available data supports an evolutionary process involving a "conserved architecture" model - whereby, complex protein architecture was an early evolutionary accomplishment via oligomerization of simple peptide motifs. Subsequent duplication and fusion events produced complex proteins folds within a larger, single polypeptide chain. Some potential selective advantages of gene duplication and fusion events (i.e., resulting from what would otherwise be described as a major replication error) is discussed.
Protein Design at the Interface of the Pre-Biotic and Biotic Worlds, Longo, L.M. and Blaber, M. Arch. Biochem. Biophys. 526, 16-21. Access can be found here.
This review discusses the subject of proteogenesis (emergence of proteins from simple amino acids) within the larger context of abiogenesis (formation of living systems from simple non-living molecules). It is argued that a consensus is emerging that the alpha-amino acids Ala, Asp, Glu, Gly, Ile, Leu, Pro, Ser, Thr, and Val likely comprised the available pre-biotic amino acids, and thus, the building blocks for the earliest polypeptides. A key question is whether this set comprises a "foldable" set (i.e., can it provide a solution to the thermodynamic and kinetic requirements of protein folding)? If so, then complex protein architecture could have emerged early in abiogenesis (conversely, the emergence of complex protein architecture would have depended upon the prior emergence of (RNA-based?) biosynthetic pathways for the synthesis and accumulation of novel amino acids not present in this pre-biotic set). An analysis is presented that argues the pre-biotic set contains all fundamental properties required for foldable proteins; however, such proteins would likely be restricted to the halophile environment.
Designing Proteins from Simple Motifs: Opportunities in Top-Down Symmetric Deconstruction, Blaber, M. and Lee, J., Curr. Opin. Struct. Biol. 22, 442-450. Access can be found here.
This review covers recent developments in 'top-down' approaches to protein design. Common among such efforts is the exploitation of symmetry in protein design, and the power of such symmetry to simplify de novo protein design.
An Empirical Phase Diagram Approach to Investigate Conformational Stability of "Second-Generation" Functional Mutants of Acidic Fibroblast Growth Factor-1, Alsenaidy, M.A., Want, T., Kim, J.H., Joshi, S.B., Lee, J., Blaber, M., Volkin, D.B. and Middaugh, C.R., Prot. Sci. 21, 418-432. Access can be found here.
This report describes a comprehensive biophyscial characterization, utilizing "empirical phase diagrams" of a series of "second-generation" mutants of fibroblast growth factor-1 (FGF-1). FGF-1 for therapeutic use in pro-angiogenic or acclerated wound healing therapies utilizes heparin as an additive to combat issues of poor thermal stability. Several mutant forms were designed to increase the intrinsic stability of FGF-1, and this study compares the muational effects to the wild-type protein formulated with heparin. The results identify several mutant forms with stability features that may obviate the need for heparin in their formulation. Thus, stability-based protein engineering may achieve results formerly achieved by formulation additives.
A Polypeptide "Building Block" for the b-trefoil Fold Identified by "Top-Down Symmetric Deconstruction", Lee, J., Blaber, S.I., Dubey, V. and Blaber, M., Journal of Molecular Biology 407,744-63 (2011)PDF file 2,330KB
This report describes in detail the development of the novel "top-down symmetric deconstruction" method of protein design and its application in the successful creation of a 42-residue polypeptide able to symmetrically self-assemble into a b-trefoil protein architecture.
Experimental Support for the Evolution of Symmetric Protein Architecture from a Simple Peptide Motif, Lee, J. and Blaber, M., Proc. Natl. Acad. Sci. USA108, 126-130 (2011) PDF file 1,304KB
This report is the culmination of almost 18 years of effort to develop a strategy to successfully design purely-symmetric protein architecture. The method is termed "top-down symmetric deconstruction". Existing paradigm posits that pure symmetry of amino acid sequence is not compatible with foldability; thus, it is not tenable as a de novo design principle, and logically calls into question the feasibility of gene duplication and fusion in the evolution of symmetric protein architecture. Our report disproves this paradigm, as we successfully demonstrate excellent foldability, thermostability and solubility for a purely-3-fold symmetric form of the b-trefoil protein fold. The results support one of two competing models for the evolution of this symmetric architecture (i.e. the "conserved architecture model") and also opens the door to a new approach in protein design - one that takes maximum advantage of symmetry in simplifying the design process.
Increased Functional Half-life of Fibroblast Growth Factor-1 by Recovering a Vestigial Disulfide Bond, Lee, J. and Blaber, M., Journal of Proteins and Proteomics 1, 37-42 (2010) PDF File 429KB
This report describes how a novel disulfide bond mutant of FGF-1 increases the in vitro functional half-life of the protein by 14-fold. This mutant design approach may be applicable to "second-generation" forms of different members of the fibroblast growth factor family.
Functional Intersection of the Kallikrein-related Peptidases (KLKs) and Thrombostasis Axis, Blaber, M., Yoon, H., Juliano, M.A., Scarisbrick, I.A., and Blaber, S.I., Biological Chemistry 391, 311-320 (2010)(Due to publisher's restrictions, please email for PDF)
This is a review article describing the known interactions between the Kallikrein-related proteases and those proteases of the thrombogenesis/thrombolysis pathways.
Analysis of the Dynamics of Assembly and Structural Impact for a Histidine Tagged FGF-1–1.5 nm Au Nanoparticle Bioconjugate, Kogot, J.M., Parker, A.M., Lee, J., Blaber, M., Strouse, G.F. and Logan, T.M., Bioconjugate Chemistry 20, 2106-2113 (2009) PDF File 2,249KB .
Gold particle conjugates with proteins is a novel area of biotechnology with many unanswered questions. In particular, are such conjugates deleterious to protein conformation? This report shows that gold nanoparticle conjugates of FGF-1 are compatible with the native folded structure.
X-ray Structure and Biophysical Properties of Rabbit Fibroblast Growth Factor-1, Lee, J., Blaber, S.I., Irsigler, A., Aspinwall, E. and Blaber, M., Acta Cryst. F. 65, 1097-1104 (2009) PDF File 846KB.
The rabbit hind limb model is the de facto animal model for studies of ischemia; furthermore, FGF-1 is emerging as the leading growth factor for pro-angiogenic therapy. Despite its relevance to these types of investigations, the cDNA sequence, X-ray structure, biophysical properties (including thermostability and receptor-binding affinity) and mitogenic activity of rabbit FGF-1 have never been reported; this publication presents such data.
Engineering an Improved Crystal Contact across a Solvent-Mediated Interface of Human Fibroblast Growth Factor-1, Meher, A.K., Blaber, S.I., Lee, J., Honjo, E., Kuroki, R. and Blaber, M., Acta Cryst. F. 65, 1136-1140 (2009) PDF File 799KB.
Neutron diffraction studies of proteins requires large crystals; however, this is often difficult to achieve. FGF-1 crystals are large in two dimensions, but the third dimension is typically thin. This report describes mutations to the crystal contact in the thin phyisical dimension, whereby solvent-mediated contacts are replaced by direct protein-protein contacts. This approach improves the crystal growth along the thin dimension.
Structural Basis of the Conserved Cysteine in the Fibroblast Growth Factor Family: Evidence for a Vestigial Half-cystine, Lee, J. and Blaber, M., J. Mol. Biol. 393, 128-139 (2009) PDF File 907KB.
The FGF family of proteins contains an absolutely conserved Cys residue (at position 83 in the 140 amino acid form of FGF-1) that is present as a buried free-cysteine (in the majority of FGF proteins), or as a half-cystine involving adjacent position 66. We present evidence that mutation of position 66 to Cys can produce a stabilizing disulfide bond in FGF-1 (where Cys83 is a free cysteine), suggesting that the conserved free cysteine residue in the FGF family of proteins is actually a vestigial half-cystine.
The Interaction between Thermodynamic Stability and Buried Free Cysteines in Regulating the Functional Half-life of Fibroblast Growth Factor-1, Lee, J. and Blaber, M., J. Mol. Biol. 393, 113-127 (2009) PDF File 1529KB.
This study identifies a cooperative interaction between buried free cysteines and the thermostability of a protein that effectively regulates the functional half-life. A protein design principle is outlined whereby the functional half-life of a mutant protein may be modulated by several orders of magnitude; furthermore, the design principle suggests a means by which a desired half-life can be achieved with limited immunogenic potential.
A Completed KLK Activome Profile: Investigation of Activation Profiles of KLK9, 10 and 15, Yoon, H., Blaber, S.I., Debela, M., Goettig, P., Scarisbrick, I.A. and Blaber, M., Biological Chemistry 390, 373-377 (2009) PDF File 170KB
We complete our study of the KLK "activome" (i.e. the ability of mature KLK proteases to cleave the KLK pro-peptide and thereby activate the pro-KLKs) by reporting data for profiles of KLK9, 10 and 15. This report contains a now-complete matrix of the 215 possible pairwise activation combinations for the 15-member KLK family.
Activation Profiles of Human Kallikrein-related Peptidases by Proteases of the Thrombostasis Axis, Yoon, H., Blaber, S.I., Evans, D.M., Trim, J., Juliano, M.A., Scarisbrick, I.A. and Blaber, M., Prot. Sci. 17, 1998-2007 (2008) PDF File 427KB
The results of this study show that proteases of the thrombostasis family can efficiently activate specific pro-KLKs, demonstrating the potential for important regulatory interactions between these two major protease families.
A Logical OR Redundancy within the Asx-Pro-Asx-Gly Type I b-turn Motif, Lee, J., Dubey, V.K., Longo, L.M. and Blaber, M., J. Mol. Biol. 377, 1251-1264 (2008) PDF File 1579KB
Type 1 b-turns contain a consensus sequence of Asx-Pro-Asx-Gly, however, the details of the contribution of the residues in this motif to the turn structure, folding and stability are poorly understood. This study, involving 28 mutants of FGF-1, undertakes a comprehensive study of Asp, Asn and Ala mutations at each of the two canonical Asx positions. The results demonstrate a type of "Logical OR" interaction between the Asx residues that serve to stabilize the turn structure, and can even protect it from deleterious mutations.
Activation Profiles and Regulatory Cascades of the Human Kallikrein-related Peptidases, Yoon, H., Laxmikanthan, G., Lee, J., Blaber, S.I., Rodriguez, A., Kogot, J.M., Scarisbrick. I.A. and Blaber, M., J. Biol. Chem. 282, 31852-31864 (2007) PDF File 580KB
The ability of the different KLK proteases to cleave the 15 different pro-KLK peptide sequences was characterized using a fusion protein method. The results demonstrate the activation potential of the KLK's, and permit construction of hypothetical KLK activation cascades.
Spackling the Crack: Stabilizing Human Fibroblast Growth Factor-1 by Targeting the N- and C-terminus b-strand Interactions, Dubey, V.K., Lee, J., Somasundaram, T., Blaber, S. and Blaber, M., J. Mol. Biol. 371, 256-268 (2007) PDF File 779KB
The first and last beta-strands in FGF-1 form a beta-sheet and are known to be a key step in the folding pathway. Two positions in these regions, Lys12 and Pro134, are shown to destabilize the structure. Mutation to Val provides a substantial 6 kJ/mol of additional stability at each position. The results suggest that targeting the termini beta-strand interactions in a beta-barrel might be a generally useful approach to increase protein stability. Furthermore, although FGF-1 is characterized as a marginally-stable protein, the results suggest that a handful of mutations might shift the stability into the "thermophile" regime.
The Autolytic Regulation of Human Kallikrein-Related Peptidase 6, Blaber, S., Yoon, H., Scarisbrick, I.A., Juliano, M.A., and Blaber, M. Biochemistry 46, 5209-5217 (2007) PDF File 259KB
This study quantifies the ability of KLK6 to hydrolyze its pro-peptide activation sequence and internal autolytic inactivating sequence. The results show that KLK6 activity is two-orders of magnitude greater for autolytic inactivation as compared to pro-sequence activation. Furthermore, several other proteases are compared to KLK6 and shown to exhibit much greater efficiency in activating pro-KLK6. Overall, the results show that KLK6 is unlikely to effectively self-activate, and requires a distinct protease for this purpose (thus forming the basis for a proteolytic activation cascade).
A Comprehensive Nomenclature for Serine Proteases with Homology to the Tissue Kallikrein, Lundwall, A., Band, V., Blaber, M., Clements, J., Courty, Y., Diamandis, E.P., Lilja, H., Maltais, L.J., Olsson, Y., Petraki, C., Sotiropoulou, G., Stenman, U.-H., Stephan, C., Talieri, M. and Yousef, G. Biological Chemistry 387, 637-641(2006)
This paper describes a new nomenclature for the human kallikrein-related peptidases, supported by a consortium of investigators led by Dr. Ake Lundwall.
Conversion of Type I 4:6 to 3:5 b-turn Types in Human Acidic Fibroblast Growth Factor: Effects upon Structure, Stability, Folding and Mitogenic Function, Lee, J., Dubey, V.K., Somasundaram, T. and Blaber, M., Proteins 62, 686-697 (2006) PDF File 589KB
FGF-1 contains fundamentally two types of beta-turns: type I 3:5 and type I 4:6. The role of the type I 4:6 turn was probed by mutations designed to convert the turn into type I 3:5. The results showed that a type I 3:5 turn can be implemented at each turn location in FGF-1 and yield a stable folded polypeptide. However, the mitogenic activity of such a mutant is substantially decreased, due to the functional requirement of a specfic type I 4:6 turn for receptor interaction. Thus, functional rather than folding considerations have resulted in the presence of a type I 4:6 turn in FGF-1.
Solvent Structure in the Active Site of Human Kallikrein 1, Laxmikanthan, G., Blaber, S.I., Scarisbrick, I.A. and Blaber, M., Hydrogen- and Hydration-Sensitive Structural Biology, Niimura, N., Mizuno, H., Helliwell, J. and Westhof, E., Eds., Kubapro, Co. Ltd Publishers, Tokyo Japan (2005) Ch. 3-6 PDF File 564KB
This is a contributed book chapter, detailing the solvent structure in the active site of human kallikrein 1, as presented at the International Workshop on "Hydrogen and Hydration in Proteins and Nucleic Acids", January 17-18, 2005, The University of Tokyo.
Redesigning Symmetry-Related “Mini-Core” Regions of FGF-1 to Increase Primary Structure Symmetry: Thermodynamic and Functional Consequences of Structural Symmetry, Dubey, V.K., Lee, J. and Blaber, M., Protein Science 14, 2315-2323 (2005) PDF File 426KB
Symmetry-related hydrophobic packing groups in FGF-1 exhibit an asymmetric tolerance to side chain substitution. The packing environment of the positions in question is formed by specific surface loops. These loop regions vary in both sequence and length, and these differences dictate the asymmetric tolerance of the core substitutions. Thus, mutations in a specific surface loop region have "locked in" a requirement for a Cys residue at position 83. This residue cannot be mutated to a symmetrically-conserved Ile residue without first mutating the surrounding surface loop. Thus, this report highlights the co-evolutionary aspect of core and surface regions.
1.70Å X-ray Structure of Human Apo Kallikrein 1: Structural Changes upon Peptide Inhibitor/substrate Binding, Laxmikanthan, G., Blaber, S.I., Bernett, M.J., Scarisbrick, I.A., Juliano, M.A. and Blaber, M., Proteins: Structure, Function and Bioinformatics 58, 802-814 (2005) PDF File 502KB
Only the second example of a human kallikrein structure to be deposited in the structural databank, we describe the x-ray structure of human apo- kallikrein 1. We detail the structural features, including solvent structure, of the S2 to S2' binding pockets. In comparison to porcine K1 with bound peptide inhibitors, the apo- kallikrein 1 structure exhibits a variety of induced-fit conformational changes upon substrate binding. These induced-fit changes include the active site serine, and indicate that the binding energy is utilized to position the active site serine for efficient catalysis.
Sequence-swapping Does Not Result in Conformation-swapping for the b4/b5 and b8/b9 b-hairpin Turns in Human Acidic Fibroblast Growth Factor, Kim, J., Lee, J., Brych, S.R., Logan, T.M. and Blaber, M. Protein Science 14, 351 - 359 (2005) PDF File 241KB
Two b-hairpin structures in human fibroblast growth factor 1 are the same length but have different structures. Since they also differ in sequence, we thought this might be the reason for their different structures. However, sequence swapping mutations between the two turns did not result in a corresponding structural change. Thus, the turn structure is likely determined by the interaction of the turn residues with the local environment.
Structural and Energetic Consequences of Mutations in a Solvated Hydrophobic Cavity, Adamek, D.H., Guerrero, L., Blaber, M. and Caspar, D.L.D., J. Mol. Biol. 346, 307-318 (2005) PDF File 694KB
In this collaboration with Dr. Donald Caspar the structural and energetic consequences of mutations designed to alter the hydrophobic characteristics of the central cavity within interleukin 1-b are described. These mutations were designed to probe potential interactions with positionally-disordered solvent within the cavity (previously identified using low-resolution x-ray crystal data).
Symmetric Primary And Tertiary Structure Mutations Within A Symmetric Superfold: A Solution, Not A Constraint, To Achieve A Foldable Polypeptide, Brych, S.R., Dubey, V.K., Bienkiewicz, E., Lee, J., Logan, T.M. and Blaber, M., J. Mol. Biol. 344, 769-780 (2004) PDF File 460KB
This paper is really important for the lab. It shows that mutations designed to increase both the tertiary and primary structure symmetry of human fibroblast growth factor 1 also result in a mutant form that is dramatically more stable than the wild type protein. Thus, we believe that the b-trefoil (symmetric) superfold can be designed with a symmetric constraint upon the primary structure. There are several important ramifications of this work, related to protein evolution and de novo design. We hypothesize that a fundamental property of the symmetric protein superfolds is the ability to adopt a symmetric primary structure with an intrinsically high thermal stability.
Atomic Resolution Structure of Human Acidic Fibroblast Growth Factor, Bernett, M.J., Somasundaram, T. and Blaber, M. Proteins:Structure, Function and Bioinformatics 57:626-634 (2004) PDF File 395KB
This report describes a 1.10Å resolution x-ray structure of human fibroblast growth factor 1. The data was of such high-resolution and quality that we were able to refine anisotropic thermal factors (i.e. the three-dimensional positional displacement for each atom). Using this information we asked whether certain regions of the protein exhibit correlated anisotropic motion (i.e. rigid body motion). We identified a region involving b-strands 5-12 that act as a rigid body distinct from the rest of the molecule. This demarcation also delineates heparin-binding from receptor-binding functionalities within the protein. It was selected as the cover article for the November 2004 issue of Proteins (the second cover article for the lab).
Targeting Kallikrein 6 Proteolysis Attenuates CNS Inflammatory Demyelinating Disease, Blaber, S., Ciric, B., Bernett, M., Blaber, M., Rodriguez, M. and Scarisbrick, I.A., FASEB J. 18:920-922 (2004) PDF File 111KB, or Full text published online: PDF File 8038KB
This paper is breakthrough work that shows that if the activity of kallikrein 6 (K6) is reduced (using a pre-immunization strategy) then animals that have been treated so as to develop a Multiple Sclerosis type of disease exhibit a delayed onset, and reduced severity. Thus, K6 represents a novel target in the treatment of inflammatory neurological diseases, like MS.
Structural Alteration of Cofactor Specificity in Corynebacterium 2,5-diketo-D-Gluconic Acid Reductase, Sanli, G., Banta, S., Anderson, S. and Blaber, M. Protein Science 13, 504-512 (2004), PDF File 194KB
In this report we describe the x-ray structure of a mutant form of 2,5-diketo-D-gluconate reductase (the "vitamin C enzyme") which has an enhanced selectivity for NADH, versus NADPH, as a cofactor. The mutant was designed by our collaborator, Dr. Stephen Anderson at Rutgers University, and should prove useful in a new method for the industrial production of vitamin C.
Engineering Allostery via Tandem Duplication and Turn Energetics, Blaber, M., Trends in Biotechnology 22, 1-2 (2004), PDF File 307KB
With permission from Elsevier. Trends in Biotechnology Homepage at http://www.sciencedirect.com/science/journal/01677799
This is a short invited review of a very interesting protein engineering study by Dr. Brian Matthews that investigates a possible mechanism for the evolution of allostery.
The Role of the Turn Symmetry in the Folding and Stability of FGF-1, J., Kim, J., Blaber, M. and Logan, T. National High Magnetic Field Laboratory Reports 10 (5), 11-13 (2003) PDF File 1798KB
This is a report that details our NMR studies of turn mutants in human fibroblast growth factor 1 that were performed at the National High Magnetic Field Laboratory. Some mutants do not readily crystallize, and Jihun Lee and Jaewon Kim, two graduate students in the lab, are spearheading efforts to utilize NMR to obtain structural information for such mutants.
Accommodation of a Highly Symmetric Core within a Symmetric Protein Superfold, Brych, S.R., Kim, J., Logan, T.M. and Blaber, M., Protein Science 12, 2704-2718 (2003) PDF File 507KB
This is another very important paper for the lab. It details the ability to redesign the core region of human fibroblast growth factor 1 using a symmetric design constraint. Despite the imposed design constraint, the repacked core is among the most successful published and let to our hypothesis (developed in our later publications) that perhaps symmetric superfolds can be successfully designed using a symmetric primary structure. This article was selected as the cover for the Dec 2003 issue of Protein Science - the first cover article for the lab.
Identification of a Key Structural Element for Protein Folding within b-hairpin Turns, Kim, J., Brych, S.R., Lee, J., Logan, T.M. and Blaber, M., J. Mol. Biol. 328, 951-961 (2003) PDF File 405KB
This paper details one of the more successful discoveries of a design principle for turn structures. We show that a glycine amino acid at the i+3 position in a type I 3:5 and 4:6 b-turn plays a critical role in stabilizing such structures. This discovery allowed us to reconcile conflicting published reports regarding the thermodynamic consequences of glycine substitutions at positions that lie within the L-a region of the Ramachandran plot.
Structural Biology of the Aldo-keto Reductase Family of Enzymes: Catalysis and Cofactor Binding, Sanli, G., Dudley, J.I. and Blaber, M., Cell Biochem. Biophys. 38, 79-101 (2003) PDF File
This is an invited review article that arose from my graduate student, Gulsah Sanli, giving a poster at a scientific conference. In addition to presenting a review of known information, Gulsah also included a structural analysis of the apo- and holo-forms of 2,5-diketo-D-gluconate reductase and identified a hinge-bending motion associated with cofactor binding. The hinge-bending motion delineates two halves of the symmetric structure of the enzyme, and suggests that it may have evolved from an ancient homo-dimer.
Crystal Structure and Biochemical Characterization of Human Kallikrein 6 Reveals a Trypsin-like Kallikrein is Expressed in the Central Nervous System, Bernett, M.J., Blaber, S.I., Scarisbrick, I.A., Dhanarajan, P., Thompson, S.M., and Blaber, M., J. Biol. Chem 277, 24562-24570 (2002) PDF File 723KB
This report describes the x-ray structure determination for human kallikrein 6 (K6) - the first human kallikrein structure deposited into the Protein Data Bank. Unknown to us at the time, another group also solved a structure for K6 (the inactive pro-form). Their report came out only two weeks after ours, and in the same journal! K6 is shown to be structurally related to the digestive enzyme trypsin, although it is expressed primarily in the brain and spinal cord.
Alternative Type I and I' Turn Conformations in the b8/b9 b-hairpin of Human Acidic Fibroblast Growth Factor, Kim, J., Blaber, S.I. and Blaber, M. Prot. Sci. 11, 459-466 (2002) PDF File 143KB
In different crystal forms of human fibroblast growth factor a particular turn region exhibits distinctly different structures. Which one is correct, and which one may be a crystal packing artifact? This study describes a series of glycine mutations, in conjunction with stability studies, that identify one particular conformation as being likely populated in solution. The results also describe a statistical study of the structural data bank that suggests that a certain turn conformation may be present at higher than expected percentages due to crystal packing artifacts.
Enzymatic Properites of Rat Myelencephalon Specific Protease, Blaber, S.I., Scarisbrick, I.A., Bernett, M.J., Dhanarajan, P., Seavy, M.A., Jin, Y., Schwartz, M.A., Rodriguez, M. and Blaber, M. Biochemistry 41, 1165-1173 (2002) PDF File 452KB
Our lab was the first to express recombinant kallikrein 6 (from the rat) and to determine its enzymatic properties. In this report, K6 is identified as a digestive-type protease, with broad substrate specificity. We also show that K6 has a built-in mechanism of autolytic inactivation, and this likely plays a key regulatory role.
Structure and Stability Effects of Mutations Designed to Increase the Primary Sequence Symmetry within the Core Region of a b-Trefoil, Brych, S.R., Blaber, S.I., Logan, T.M. and Blaber, M., Protein Science 10, 2587-2599 (2001) PDF File 519KB
This is our first report on efforts to redesign the core region of fibroblast growth factor 1 using a symmetric design constraint. We introduce three mutations into the 15 amino acid core and show that the mutations are well-tolerated. This study paved the way for further symmetric redesign studies.
An Efficient, Flexible-Model Program for the Analysis of Differential Scanning Calorimetry Data, Grek, S., Davis, J. and Blaber, M., Prot. Pept. Letters 6, 429-436 (2001) PDF File 220KB
Two outstanding undergraduate students in the lab, Sasha Grek and John Davis, undertook the Herculean task of writing a Windows™-based software program to deconvoluted data from differential scanning calorimetry studies. The program was a success, and currently 15 copies are downloaded each month from the lab's website.
Active Site Organization in an Aldo-keto Reductase by NADPH Cofactor, Sanli, G. and Blaber, M., J. Mol. Biol. 309, 1209-1218 (2001) PDF File 1640KB
We solved the x-ray structures of 2,5-diketo-D-gluconate reductase (the "vitamin C enzyme") in both holo- (i.e. with NADPH cofactor) and apo- forms (i.e. without) and discovered that the active sites is in a non-catalytically competent conformation in the absence of bound cofactor. Thus, the NADPH cofactor not only provides a hydride for catalysis, but also serves to organize the active site. This was the first time that such a role for the cofactor in this family of enzymes had been reported.
Reduction of Wobble-position GC Bases in Corynebacteria Genes and Enhancement of PCR and Heterologous Expression, Sanli, G., Blaber, S.I. and Blaber, M., J. Molec. Microbiol. Biotech. 3, 123-126 (2001) PDF File
Genes from Corynebacterium are GC rich and prove problematic for DNA polymerase-based manipulations (i.e. PCR and sequencing). We designed a couple of synthetic genes that retained the amino acid sequence, but substantially reduced the GC content (and also improving E. coli codon bias). These genes were shown to be amenable to polymerase-based manipulations, and could be readily mutated, sequenced and expressed in E. coli. This study laid the groundwork for further mutagenesis studies to improve the stability and catalytic features of this enzyme.
Thermodynamic Characterization of Mutants of Human FGF-1 with an Increased Physiological Half-life, Culajay, J.F., Blaber, S.I., Khurana, A. and Blaber, M., Biochemistry 39, 7153-7158 (2000) PDF File 72KB
Previous reports suggested that cysteine to serine mutations in human fibroblast growth factor 1 (FGF1) resulted in substantially longer half-life in cell culture mitogenic assays. This observation was postulated to be due to either an increase in stability or elimination of thiol-based modifications. We had developed the methodology for determination of the DG unfolding for FGF1 and decided to test the stability hypothesis. Our results showed that the serine mutations destabilized the protein. Thus, the increase in half-life was due to elimination of thiol-based modifications. We postulate that a mechanism to limit protein half-life is to introduce free cysteine residues within the core region of a protein. Such proteins cannot refold if the cysteine forms thiol adducts or are chemically modified by oxidation.
Molecular Modeling of Substrate Binding in Wild Type and Mutant Corynebacteria 2,5-Diketo-D-Gluconate Reductase, Khurana, S., Sanli, G., Powers, D.B., Anderson, S.A. and Blaber, M., Proteins: Structure, Function and Genetics 39, 68-75 (2000) PDF File 359KB
Using bound solvent as a guide, this report details the modeling of substrate into the active site of 2,5-DKG-reducates, the "vitamin C enzyme". Although this enzyme was originally identified from screening of soil bacteria capable of reducing 2,5-diketo-D-gluconate, this study shows that this compound is unlikely to be the natural substrate.
Reversible Thermal Denaturation of Human FGF-1 Induced by Low Concentrations of Guanidine Hydrochloride, Blaber, S.I., Culajay, J.F., Khurana, A. and Blaber, M., Biophys. J. 77, 470-477 (1999) PDF File 106KB
This was an important publication for the lab. We detail the development of the methodology by which the reversible thermal denaturation of human fibroblast growth factor 1 can be achieved. The results allowed us to demonstrate the generally good agreement between isothermal equilibrium denaturation and differential scanning calorimetry, and showed that the 2-state models being invoked to analyze the data were valid.
Disordered Water within a Hydrophobic Protein Cavity Visualized by X-ray Crystallography, Yu, B., Blaber, M., Clore, M., Gronenborn, M. and Caspar, D.L.D., Proc. Natl. Acad. Sci. USA 96, 103-108 (1999) PDF File 329KB
This report was part of a collaboration with Dr. Don Caspar at Florida State University. Interleukin-1b has a large central cavity and there were conflicting reports in the literature on whether this cavity contained disordered solvent. By collecting low resolution data and scaling the electron density on an absolute scale, we were able to show that the central cavity contains approximately 1.8 water molecules worth of disordered electron density - suggesting that solvent is indeed rattling around within the core.
Crystal Structure of 2,5-Diketo-D-Gluconic Acid Reductase A Complexed with NADPH at 2.1-Å Resolution, Khurana, S., Powers, D.B., Anderson, S. and Blaber, M., Proc. Natl. Acad. Sci. USA 95, 6768-6773 (1998) PDF File 485KB
This was the first x-ray structure for a prokaryotic member of the aldo-keto reductase family. The structure provided details of the structural "seat belt", a pair of loops that covered the cofactor binding site and contributed to the kinetics of cofactor binding.
X-ray Crystal Structure of Human Acidic Fibroblast Growth Factor, Blaber, M., DiSalvo, J. and Thomas, K.A., Biochemistry 35, 2086-2094 (1996) PDF File 455KB
This is the first x-ray structure reported for human acidic fibroblast growth factor. A single crystal was used for both spacegroup determination (using precession photography) and data collection (using a multiwire detector). This was in the good old days when you needed to know the space group and cell dimensions prior to data collection. This is also the first crystal structure reported by our laboratory.